Agu Laisk

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.

A mathematical model of C4 photosynthesis: The mechanism of concentrating CO2 in NADP-malic enzyme type species

A computer model comprising light reactions in PS II and PS I, electron-proton transport reactions in mesophyll and bundle sheath chloroplasts, all enzymatic reactions and most of the known regulatory functions of NADP-ME type C4 photosynthesis has been developed as a system of differential budget equations for intermediate compounds. Rate-equations were designed on principles of multisubstrate-multiproduct enzyme kinetics. Some of the 275 constants needed (ΔG0′ and Km values) were available from literature and others (Vm) were estimated from reported rates and pool sizes. The model provided good simulations for rates of photosynthesis and pool sizes of intermediates under varying light, CO2 and O2. A basic novelty of the model is coupling of NADPH production via NADP-ME with ATP production and regulation of the C3 cycle in bundle sheath chloroplasts. The functional range of the ATP/NADPH ratio in bundle sheath chloroplasts extends from 1.5 to 2.1, being energetically most efficient around 2. In the presence of such stoichiometry, the CO2 concentrating function can be explained on the basis of two processes: (a) extra ATP consumption for starch and protein synthesis in bundle sheath leads to a faster NADPH and CO2 import compared with CO2 fixation in bundle sheath, and (b) the residual photorespiratory activity consumes RuBP by oxygenation, NADPH and ATP and causes the imported CO2 to accumulate in bundle sheath cells. As a wider application, the model may be used for predicting results of genetic engineering of plants.

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.

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 22u2009°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:

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

Abstract. The photosynthetic linear electron transport rate in excess of that used for CO2 reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C4 plant], Amaranthus cruentus L. (NAD-ME-type C4 plant) and Helianthus annuus L. (C3 plant) leaves at different CO2 and O2 concentrations. The electron transport rate (JF) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500u2009μmol CO2u2009·u2009mol−1 and 2% O2. Under high light intensities there was a large excess of JF/4u2009at 10–100% O2 in the C3 plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C4 photosynthesis is limited by supply of atmospheric CO2 to the C4 cycle, the C3 cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO2 fixation in C4 plants was very sensitive to the presence of O2 in the gas phase, rapidly increasing between 0.01 and 0.1% O2, and at 2% O2 it was about two-thirds of that at 21% O2. This shows the importance of the Mehler O2 reduction as an electron sink, compared with photorespiration in C4 plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C4 photosynthesis.

Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere. II: Consequences of SO2 uptake as revealed by computer analysis

A computer model is used to analyze fluxes of SO2 from polluted air into leaves and the intracellular distribution of sulfur species derived from SO2. The analysis considers only effects of acidification and of anion accumulation. (i) The SO2 flux into leaves is practically exclusively controlled by the boundary-layer resistance of leaves to gas diffusion and by stomatal opening. At constant stomatal opening, flux is proportional to the concentration of SO2 in air. (ii) The sink capacity of cellular compartments for SO2 depends on intracellular pH and the intracellular localization of reactions capable of oxidizing or reducing SO2. In the mesophyll of illuminated leaves, the chloroplasts possess the highest trapping potential for SO2. (iii) If intracellular ion transport were insignificant, and if bisulfite and sulfite could not be oxidized or reduced, leaves with opened stomata would rapidly be killed both by the accumulation of sulfites and by acidification of chloroplasts and cytosol even if SO2 levels in air did not exceed concentrations thought to be permissible. Acidification and sulfite accumulation would remain confined largely to the chloroplasts and to the cytosol under these conditions. (iv) Transport of bisulfite and protons produced by hydration of SO2 into the vacuole cannot solve the problem of cytoplasmic accumulation of bisulfite and sulfite and of cytoplasmic acidification, because SO2 generated in the acidic vacuole from the bisulfite anion would diffuse back into the cytoplasm. (v) Oxidation to sulfate which is known to occur mainly in the chloroplasts can solve the problem of cytoplasmic sulfite and bisulfite accumulation, but aggravates the problem of chloroplastic and cytosolic acidification. (vi) A temporary solution to the problem of acidification requires the transfer of H+ and sulfate into the vacuole. This transport needs to be energized. The storage capacity of the vacuole for protons and sulfate defines the extent to which SO2 can be detoxified by oxidation and removal of the resulting protons and sulfate anions from the cytoplasm. Calculations show that even at atmospheric levels of SO2 thought to be tolerable, known vacuolar buffer capacities are insufficient to cope with proton production during oxidation of SO2 to sulfate within a vegetation period. (vii) A permanent solution to the problem of acidification is the removal of protons. Protons are consumed during the reduction of sulfate to sulfide. Proteins and peptides contain sulfur at the level of sulfide. During photosynthesis in the presence of the permissible concentration of 0.05μl·l-1 SO2, sulfur may be deposited in plants at a ratio not far from 1/500 in relation to carbon. The content of reduced sulfur to carbon is similar to that ratio only in fast-growing, protein-rich plants. Such plants may experience little difficulty in detoxifying SO2. In contrast, many trees may contain reduced sulfur at a ratio as low as 1/10 000 in relation to carbon. Excess sulfur deposited in such trees during photosynthesis in polluted air gives rise to sulfate and protons. If detoxification of SO2 by reduction is inadequate, and if the storage capacity of the vacuoles for protons and sulfate is exhausted, damage is unavoidable. Calculations indicate that trees with a low ratio of reduced S to C cannot tolerate long-term exposure to concentrations of SO2 as low as 0.02 or 0.03 μl·l-1 which so far have been considered to be non-toxic to sensitive plant species.

Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere : I. Computer analysis.

Using experimental information obtained in earlier studies on cellular buffering and SO2 uptake into leaves (Pfanz and Heber, 1986, Plant Physiol. 81, 597–602; Pfanz et al., 1987 a, b, Plant Physiol.), a mathematical model is presented which permits computer analysis of the transport of SO2 from the atmosphere into the mesophyll of leaves and describes the intracellular distribution of hydration products of SO2. Oxidation of sulfite and metabolization of sulfate can also be included. Although the model does not attempt to incorporate all available information on the intracellular transport of sulfur species, it permits general conclusions in regard to cellular responses to SO2. The model can be extended and modified for gases other than SO2. Examples are presented to illustrate the information the model is able to give. Times required for SO2 equilibration are long. Equilibrium relationships between SO2 in the atmosphere and cellular SO2 show that in order to survive in even slightly contaminated air, leaves must prevent equilibration between external and internal SO2.

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.

Oxygen yield from single turnover flashes in leaves: non-photochemical excitation quenching and the number of active PSII

O(2) evolution from single turnover flashes of up to 96 micromol absorbed quanta m(-2) and from multiple turnover pulses of 8.6 and 38.6 ms duration and 12800 and 850 micromol absorbed quanta m(-2) s(-1) intensity, respectively, was measured in sunflower leaves with the help of zirconium O(2) analyser. O(2) evolution from one flash could be measured with 1% accuracy on the background of 10-50 micromol O(2) mol(-1). Before the measurements leaves were pre-adapted either at 30-60 or 1700 micromol quanta m(-2) s(-1) to induce different non-photochemical excitation quenching (q(N)). Short (1 min) exposures at the high light that created only energy-dependent, q(E) type quenching, caused no changes in the O(2) yield from saturating flashes or pulses that could be related to the q(E) quenching, but the yield from low intensity flashes and pulses decreased considerably. Long 30-60-min exposures at the high light induced a reversible inhibitory, q(I) type quenching that decreased the O(2) yield from both, saturating and limiting flashes and pulses (but more from the limiting ones), which reversed within 15 min under the low light. The results are in agreement with the notion that q(E) is caused by a quenching process in the PSII antenna and no changes occur in the PSII centres, but the reversible (15-30 min) q(I) quenching is accompanied by inactivation of a part of PSII centres.

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase, but not ferredoxin-NADP reductase, controls electron flow to phosphoglycerate in the dark-light transition

P700 is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P700 is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)−1 electrons in geranium leaves, 16 nmol · (mg chlorophyll)−1 in sunflower and 22 nmol · (mg chlorophyll)−1 in oleander. Reduction of NADP during the initial phase of P700 oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.