Vello Oja

Quantum Yields and Rate Constants of Photochemical and Nonphotochemical Excitation Quenching (Experiment and Model)

Sunflower (Helianthus annuus L.), cotton (Gossypium hirsutum L.), tobacco (Nicotiana tabacum L.), sorghum (Sorghum bicolor Moench.), amaranth (Amaranthus cruentus L.), and cytochrome b6f complex-deficient transgenic tobacco leaves were used to test the response of plants exposed to differnt light intensities and CO2 concentrations before and after photoinhibition at 4000 [mu]mol photons m-2 s-1 and to thermoinhibition up to 45[deg]C. Quantum yields of photochemical and nonphotochemical excitation quenching (YP and YN) and the corresponding relative rate constants for excitation capture from the antenna-primary radical pair equilibrium system (k[prime]P and k[prime]N) were calculated from measured fluorescence parameters. The above treatments resulted in decreases in YP and K[prime]P and in approximately complementary increases in YN and K[prime]N under normal and inhibitory conditions. The results were reproduced by a mathematical model of electron/proton transport and O2 evolution/CO2 assimilation in photosynthesis based on budget equations for the intermediates of photosynthesis. Quantitative differences between model predictions and experiments are explainable, assuming that electron transport is organized into domains that contain relatively complete electron and proton transport chains (e.g. thylakoids). With the complementation that occurs between the photochemical and nonphotochemical excitation quenching, the regulatory system can constantly maintain the shortest lifetime of excitation necessary to avoid the formation of chlorophyll triplet states and singlet oxygen.

Chlorophyll fluorescence at 680 and 730 nm and leaf photosynthesis

Chlorophyll fluorescence constitutes a simple, rapid, and non-invasive means to assess light utilization in Photosystem II (PS II). This study examines aspects relating to the accuracy and applicability of fluorescence for measurement of PS II photochemical quantum yield in intact leaves. A known source of error is fluorescence emission at 730 nm that arises from Photosystem I (PS I). We measured this PS I offset using a dual channel detection system that allows measurement of fluorescence yield in the red (660 nm < F < 710 nm) or far red (F > 710 nm) region of the fluorescence emission spectrum. The magnitude of the PS I offset was equivalent to 30% and 48% of the dark level fluorescence F0 in the far red region for Helianthus annuus and Sorghum bicolor, respectively. The PS I offset was therefore subtracted from fluorescence yields measured in the far red spectral window prior to calculation of PS II quantum yield. Resulting values of PS II quantum yield were consistently higher than corresponding values based on emission in the red region. The basis for this discrepancy lies in the finite optical thickness of the leaf that leads to selective reabsorption by chlorophyll of red fluorescence emission originating in deeper cell layers. Consequently, red fluorescence measurements preferentially sense emission from chloroplasts in the uppermost layer of the leaf where levels of photoprotective nonphotochemical quenching are higher due to increased photon density. It is suggested that far red fluorescence, corrected for the PS I offset, provides the most reliable quantitative basis for calculation of PS II quantum yield because of reduced sensitivity of these measurements to gradients in leaf transmittance and quenching capacity.

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO2. It was found that at the first maximum, light-and CO2-saturated photosynthesis can be much faster than steady-state photosynthesis. Both QA in the reaction center of PS II and P700 in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P700 oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force FA, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(Pi), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO2, the assimilatory force FA declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P700. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.

pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves.

Abstract. After opening the stomata in CO2-free air, darkened leaves of several plant species were titrated with CO2 at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO2 for times ranging between 4.4 and 300 s, fast CO2 release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO2 was released with time constants of about 5, 15 and approximately 300 s. With CO2 as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit−1 in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit−1, i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO2 reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO2-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO2 minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO2-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO2 titration with and without added buffer was somewhat below 4 mM pH-unit−1. Thus the apoplastic and cytosolic pH responses to additions of CO2 indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast.

Deciphering the 820 nm signal: redox state of donor side and quantum yield of Photosystem I in leaves.

By recording leaf transmittance at 820 nm and quantifying the photon flux density of far red light (FRL) absorbed by long-wavelength chlorophylls of Photosystem I (PS I), the oxidation kinetics of electron carriers on the PS I donor side was mathematically analyzed in sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.) and birch (Betula pendula Roth.) leaves. PS I donor side carriers were first oxidized under FRL, electrons were then allowed to accumulate on the PS I donor side during dark intervals of increasing length. After each dark interval the electrons were removed (titrated) by FRL. The kinetics of the 820 nm signal during the oxidation of the PS I donor side was modeled assuming redox equilibrium among the PS I donor pigment (P700), plastocyanin (PC), and cytochrome f plus Rieske FeS (Cyt f + FeS) pools, considering that the 820 nm signal originates from P700+ and PC+. The analysis yielded the pool sizes of P700, PC and (Cyt f + FeS) and associated redox equilibrium constants. PS I density varied between 0.6 and 1.4 μmol m−2. PS II density (measured as O2 evolution from a saturating single-turnover flash) ranged from 0.64 to 2.14 μmol m−2. The average electron storage capacity was 1.96 (range 1.25 to 2.4) and 1.16 (range 0.6 to 1.7) for PC and (Cyt f + FeS), respectively, per P700. The best-fit electrochemical midpoint potential differences were 80 mV for the P700/PC and 25 mV for the PC/Cyt f equilibria at 22 °C. An algorithm relating the measured 820 nm signal to the redox states of individual PS I donor side electron carriers in leaves is presented. Applying this algorithm to the analysis of steady-state light response curves of net CO2 fixation rate and 820 nm signal shows that the quantum yield of PS I decreases by about half due to acceptor side reduction at limiting light intensities before the donor side becomes oxidized at saturating intensities. Footnote:

The state of the photosynthetic apparatus in leaves as analyzed by rapid gas exchange and optical methods: the pH of the chloroplast stroma and activation of enzymes in vivo.

The exchange of CO2 and O2 was measured in leaves using specially constructed equipment capable of responding to rapid transients. Optical measurements provided information on cytochrome f and P 700 oxidation in the light. The following results were obtained: i) The solubilization of CO2 was used to calculate the pH of the chloroplast stroma in darkened leaves. Values ranged from pH 7.8 to pH 8.0 in different C3 plants. ii) Illumination of predarkened leaves of Helianthus annuus L. resulted in three distinct phases of O2 evolution that illustrate the complexity of light activation of the photosynthetic apparatus. A first burst of O2 is attributed to the reduction of electron carriers of the electron-transport chain. While plastoquinone was reduced, cytochrome f was oxidized. Appreciable oxidation of P 700 became possible only during the second O2 burst, which indicates the reduction of the phosphoglycerate pool. Extensive oxidation required the opening of an electron gate on the reducing side of photosystem I. The subsequent slow rise in O2 evolution towards a steady state reflects activation of the Calvin cycle and is the result of CO2 assimilation. iii) Light-dependent CO2 uptake by predarkened leaves occurred in four phases, three of them based on pH changes in the chloroplast stroma. Initial CO2 uptake was small and probably caused by protonation of reduced plastoquinone. In the second phase, which coincided with the reduction of the pool of phosphoglycerate, the initial alkalization of the chloroplast stroma was substantially increased. In the third phase, the stroma alkalization decreased, and the fourth phase was dominated by CO2 assimilation. iv) Respiratory CO2 production was partially suppressed in the light during the second phase of O2 evolution while phosphoglycerate was being reduced.

Range of photosynthetic control of postillumination P700+ reduction rate in sunflower leaves

The kinetics of the postillumination reduction of P700+ which reflects the rate constant for plastoquinol (PQH2) oxidation was recorded in sunflower leaves at different photon absorption densities (PAD), CO2 and O2 concentrations. The P700 oxidation state was calculated from the leaf transmittance at 830 nm logged at 50 μs intervals. The P700+ dark reduction kinetics were fitted with two exponents with time constants of 6.5 and about 45 ms at atmospheric CO2 and O2 concentrations. The time constant of the fast component, which is the major contributor to the linear electron transport rate (ETR), did not change over the range of PADs of 14.5 to 134 nmol cm-2 s-1 in 21% O2, but it increased up to 40 ms under severe limitation of ETR at low O2 and CO2. The acceptor side of Photosystem I (PS I) became reduced in correlation with the downregulation of the PQH2 oxidation rate constant. It is concluded that thylakoid pH-related downregulation of the PQH2 oxidation rate constant (photosynthetic control) is not present under normal atmospheric conditions but appears under severe limitation of the availability of electron acceptors. The measured range of photosynthetic control fits with the maximum variation of ETR under natural stress in C3 plants. Increasing the carboxylase/oxygenase specificity would lead to higher reduction of the PS I acceptor side under stress.

Stimulation by Light of Rapid pH Regulation in the Chloroplast Stroma in Vivo as Indicated by CO2 Solubilization in Leaves

Leaves of Brassica oleracea, Helianthus annuus, and Nicotiana rustica were exposed for 20 s to high concentrations of CO2. CO2 uptake by the leaf, which was very fast, was measured as a transient increase in the concentration of oxygen. Rapid solubilization of CO2 in excess of that which is physically dissolved in aqueous phases is proposed to be caused by bicarbonate formation in the stroma of chloroplasts, which contain carbonic anhydrase. On this basis, pH values and bicarbonate accumulation in the chloroplast stroma were calculated. Buffer capacities were far higher than expected on the basis of known concentrations in the chloroplast stroma. Moreover, apparent buffer capacities increased with the time of exposure to high CO2, and they were higher when the measurements were performed in the light than in the dark. During prolonged exposure of leaves to 16% CO2, calculated bicarbonate concentrations in the chloroplast stroma exceeded 90 mM in the dark and 120 mM in the light. The observations are interpreted as indicating that under acid stress protons are rapidly exported from the chloroplasts in exchange for cations, which are imported. The data are discussed in terms of effective metabolic pH control by ion transport, first across the chloroplast envelope and, then, across the tonoplast of leaf mesophyll cells. The direct involvement of the vacuole in the regulation of the chloroplast pH in leaf cells is suggested.

Rubisco in planta kcat is regulated in balance with photosynthetic electron transport

Site turnover rate (kcat) of Rubisco was measured in intact leaves of different plants. Potato (Solanum tuberosum L.) and birch (Betula pendula Roth.) leaves were taken from field-growing plants. Sunflower (Helianthus annuus L.), wild type (wt), Rubisco-deficient (–RBC), FNR-deficient (–FNR), and Cyt b6f deficient (–CBF) transgenic tobacco (Nicotiana tabacum L.) were grown in a growth chamber. Rubisco protein was measured with quantitative SDS-PAGE and FNR protein content with quantitative immunoblotting. The Cyt b6f level was measured in planta by maximum electron transport rate and the photosystem I (PSI) content was assessed by titration with far-red light. The CO2 response of Rubisco was measured in planta with a fast-response gas exchange system at maximum ribulose 1,5-bisphosphate concentration. Reaction site kcat was calculated from Vm and Rubisco content. Biological variation of kcat was significant, ranging from 1.5 to 4 s−1 in wt, but was >6 s−1 at 23 °C in –RBC leaves. The lowest kcat of 0.5 s−1 was measured in –FNR and –CBF plants containing sufficient Rubisco but having slow electron transport rates. Plotting kcat against PSI per Rubisco site resulted in a hyperbolic relationship where wt plants are on the initial slope. A model is suggested in which Rubisco Activase is converted into an active ATP-form on thylakoid membranes with the help of a factor related to electron transport. The activation of Rubisco is accompanied by the conversion of the ATP-form into an inactive ADP-form. The ATP and ADP forms of Activase shuttle between thylakoid membranes and stromally-located Rubisco. In normal wt plants the electron transport-related activation of Activase is rate-limiting, maintaining 50–70% Rubisco sites in the inactive state.

Action spectra of photosystems II and I and quantum yield of photosynthesis in leaves in State 1.

The spectral global quantum yield (YII, electrons/photons absorbed) of photosystem II (PSII) was measured in sunflower leaves in State 1 using monochromatic light. The global quantum yield of PSI (YI) was measured using low-intensity monochromatic light flashes and the associated transmittance change at 810nm. The 810-nm signal change was calibrated based on the number of electrons generated by PSII during the flash (4·O2 evolution) which arrived at the PSI donor side after a delay of 2ms. The intrinsic quantum yield of PSI (yI, electrons per photon absorbed by PSI) was measured at 712nm, where photon absorption by PSII was small. The results were used to resolve the individual spectra of the excitation partitioning coefficients between PSI (aI) and PSII (aII) in leaves. For comparison, pigment-protein complexes for PSII and PSI were isolated, separated by sucrose density ultracentrifugation, and their optical density was measured. A good correlation was obtained for the spectral excitation partitioning coefficients measured by these different methods. The intrinsic yield of PSI was high (yI=0.88), but it absorbed only about 1/3 of quanta; consequently, about 2/3 of quanta were absorbed by PSII, but processed with the low intrinsic yield yII=0.63. In PSII, the quantum yield of charge separation was 0.89 as detected by variable fluorescence Fv/Fm, but 29% of separated charges recombined (Laisk A, Eichelmann H and Oja V, Photosynth. Res. 113, 145-155). At wavelengths less than 580nm about 30% of excitation is absorbed by pigments poorly connected to either photosystem, most likely carotenoids bound in pigment-protein complexes.