Hillar Eichelmann

Developmental changes in mesophyll diffusion conductance and photosynthetic capacity under different light and water availabilities in Populus tremula: how structure constrains function.

Finite mesophyll diffusion conductance (g(m) ) significantly constrains net assimilation rate (A(n) ), but g(m) variations and variation sources in response to environmental stresses during leaf development are imperfectly known. The combined effects of light and water limitations on g(m) and diffusion limitations of photosynthesis were studied in saplings of Populus tremula L. An one-dimensional diffusion model was used to gain insight into the importance of key anatomical traits in determining g(m) . Leaf development was associated with increases in dry mass per unit area, thickness, density, exposed mesophyll (S(mes) /S) and chloroplast (S(c) /S) to leaf area ratio, internal air space (f(ias) ), cell wall thickness and chloroplast dimensions. Development of S(mes) /S and S(c) /S was delayed under low light. Reduction in light availability was associated with lower S(c) /S, but with larger f(ias) and chloroplast thickness. Water stress reduced S(c) /S and increased cell wall thickness under high light. In all treatments, g(m) and A(n) increased and CO(2) drawdown because of g(m) , C(i) -C(c) , decreased with increasing leaf age. Low light and drought resulted in reduced g(m) and A(n) and increased C(i) -C(c) . These results emphasize the importance of g(m) and its components in determining A(n) variations during leaf development and in response to stress.

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.

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 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:

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.

The rate of nitrite reduction in leaves as indicated by O2 and CO2 exchange during photosynthesis

Light response (at 300 ppm CO2 and 10–50 ppm O2 in N2) and CO2 response curves [at absorbed photon fluence rate (PAD) of 550 μmol m−2 s−1] of O2 evolution and CO2 uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO3− or NH4+ as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH4NO3. Photosynthetic O2 evolution in excess of CO2 uptake was measured with a stabilized zirconia O2 electrode and an infrared CO2 analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO2, mainly NO2−, SO42−, and oxaloacetate. In NO3−-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O2–CO2 flux difference rapidly increased to about 1 μmol m−2 s−1 at very low PADs and the process was saturated at 50 μmol quanta m−2 s−1. At higher PADs the O2–CO2 flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m−2 s−1. In NH4+-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O2 evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO2− which successfully competes with CO2 reduction and saturates at a rate of about 1 μmol O2 m−2 s−1 (9% of the maximum O2 evolution rate). The high-PAD component of about 1 μmol O2 m−2 s−1, superimposed on NO2− reduction, may represent oxaloacetate reduction. The roles of NO2−, oxaloacetate, and O2 reduction in the regulation of ATP/NADPH balance are discussed.

CO2 Uptake and Electron Transport Rates in Wild-Type and a Starchless Mutant of Nicotiana sylvestris (The Role and Regulation of Starch Synthesis at Saturating CO2 Concentrations)

CO2 uptake rate, chlorophyll fluorescence, and 830-nm absorbance were measured in wild-type (wt) Nicotiana sylvestris (Speg. et Comes) and starchless mutant NS 458 leaves at different light intensities and CO2 concentrations. Initial slopes of the relationships between CO2 uptake and light and CO2 were similar, but the maximum rate at CO2 and light saturation was only 30% in the mutant compared with the wt. O2 enhancement of photosynthesis at CO2 and light saturation was relatively much greater in the mutant than in the wt. In 21% O2, the electron transport rate (ETR) calculated from fluorescence peaked near the beginning of the CO2 saturation of photosynthesis. With the further increase of CO2 concentration ETR remained nearly constant or declined a little in the wt but drastically declined in the mutant. Absorbance measurements at 830 nm indicated photosystem I acceptor side reduction in both plants at saturating CO2 and light. Assimilatory charge (postillumination CO2 uptake) measurements indicated trapping of chloroplast inorganic phosphate, supposedly in hexose phosphates, in the mutant. It is concluded that starch synthesis gradually substitutes for photorespiration as electron acceptor with increasing CO2 concentration in the wt but not in the mutant. It is suggested that starch synthesis is co-controlled by the activity of the chloroplast fructose bisphosphatase.

Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C3 plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO2 concentrations varying from 1 to 20% in air. When they were transferred back into CO2-free air, CO2 was rapidly released. It originated from dissolved CO2 and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO2 from bicarbonate. The data were fitted to a model which accounts for the CO2/bicarbonate equilibrium in buffers with different CO2 concentrations and initial pH values. From this, pH values and CO2-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO2 concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO2 between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H+ per pH unit. The work shows that measurements of solubilized CO2 are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.