Chlorophyll a Fluorescence Predicts Total Photosynthetic Electron Flow to CO(2) or NO(3)/NO(2) under Transient Conditions

A model which predicts total photosynthetic electron flow from a linear regression of the relationship between corrected steady-state quantum yield and nonphotochemical quenching (E Weis, JA Berry [1987] Biochem Biophys Acta 894: 198-208) was formulated for N-limited cells of the green alga Selenastrum minutum. Unlike other models based on net CO₂ fixation, our model is based on total photosynthetic electron flow measured as gross O₂ evolution. This allowed for the prediction of total photosynthetic electron flow from water to both CO₂ fixation and NO₃⁻/NO₂⁻ reduction. The linear regression equation predicting electron flow is of the form: J = I · Q_q[0.4777-0.3282 Q_NP] (where J = gross photosynthetic electron flow, I = incident PAR, Q_q = photochemical quenching, Q_NP = nonphotochemical quenching). During steady-state photosynthesis, over a range of irradiance, the model predicted a photosynthetic light saturation curve which was well correlated with that observed. Although developed under steady-state conditions, the model was tested during nonsteady-state photosynthesis induced by transient nitrogen assimilation. The model predicted transient rates of gross O₂ evolution which were in excellent agreement with the rates observed under a variety of conditions regardless of whether CO₂ or NO₃⁻/NO₂⁻ served as the physiological electron acceptor. The fluorescence transients resulting from ammonium and nitrate assimilation are discussed with respect to metabolic demands for reductant and ATP.     

Limitations of photosynthesis in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> under drought stress: gas exchange,chlorophyll fluorescence and Calvin cycle enzymes

In this article, the effects of drought stress (DS) on gas exchange, chlorophyll (Chl) a fluorescence and Calvin cycle enzymes in Phaseolus vulgaris are evaluated. Three-week-old plants were exposed to DS by receiving only so much water every evening to ensure 30% field capacity water content overnight. After three days under these conditions, we observed that DS induced a decline of the CO₂ assimilation. Gas-exchange data showed that the closure of stomata during DS did not lead to a concomitant decline in calculated intercellular CO₂ concentration. Moreover, DS plants showed a reduction of the photochemical Chl fluorescence quenching, photosystem II quantum yield and electron transport rate and a higher pH gradient and more heat dissipation as compared to controls. The activity of Calvin cycle enzymes, Rubisco, sFBPase, and Ru5PK, decreased strongly in DS plants as compared to controls. Data analysis suggest that the decrease of CO₂ assimilation under drought conditions is not related to a diminished capacity of the use of NADPH and ATP but probably to the decline of enzyme activity involved in RuBP regeneration (Ru5PK).     

Fluorescence Quenching and Gas Exchange in a Water Stressed C(3) Plant, Digitalis lanata

A leaf cuvette has been adapted for use with a pulse-modulation fluorometer and an open gas exchange system. Leaf water potential (ψ) was decreased by withholding watering from Digitalis lanata EHRH. plants. At different stages of water deficiency the photochemical (q_Q) and nonphotochemical (q_E) fluorescence quenching was determined during the transition between darkness and light-induced steady state photosynthesis of the attached leaves. In addition, the steady state CO₂ and H₂O gas exchange was recorded. Following a decrease of leaf water potential with increasing water deficiency, the transition of photochemical quenching was almost unaffected, whereas nonphotochemical quenching increased. This is indicative of an enhanced thylakoid membrane energization during the transition and is interpreted as a partial inhibition of either the ATP generating or the ATP consuming reaction sequences. Complete reversion of the stress induced changes was achieved within 6 hours after rewatering. In contrast to the variations during transition, the final steady state values of q_Q and q_E remained unchanged over the entire stress range from −0.7 to −2.5 megapascals. From these results we conclude that, once established, electron transport via photosystem II and the transmembrane proton gradient remain unaffected by water stress. These data are indicative of a protective mechanism against photoinhibition during stress, when net CO₂ uptake is limited.     

Glycolaldehyde Inhibits CO(2) Fixation in the Cyanobacterium Synechococcus UTEX 625 without Inhibiting the Accumulation of Inorganic Carbon or the Associated Quenching of Chlorophyll a Fluorescence

When studying active CO₂ and HCO₃⁻ transport by cyanobacteria, it is often useful to be able to inhibit concomitant CO₂ fixation. We have found that glycolaldehyde was an efficient inhibitor of photosynthetic CO₂ fixation in Synechococcus UTEX 625. Glycolaldehyde did not inhibit inorganic carbon accumulation due to either active CO₂ or HCO₃⁻ transport. When glycolaldehyde (10 millimolar) was added to rapidly photosynthesizing cells, CO₂ fixation was stopped within 15 seconds. The quenching of chlorophyll a fluorescence remained high (≤ 82% control) when CO₂ fixation was completely blocked by glycolaldehyde. This quenching was relieved upon the addition of a glucose oxidase oxygentrap. This is consistent with our previous finding that q-quenching in the absence of CO₂ fixation was due to O₂ photoreduction. Photosynthetic CO₂ fixation was also inhibited by d,l,-glyceraldehyde but a sixfold higher concentration was required. Glycolaldehyde acted much more rapidly than iodoacetamide (15 seconds versus 300 seconds) and did not cause the onset of net O₂ evolution often observed with iodoacetamide. Glycolaldehyde will be a useful inhibitor when it is required to study CO₂ and HCO₃⁻ transport without the complication of concomitant CO₂ fixation.     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.     

Thermal phase and excitonic connectivity in fluorescence induction

Chl fluorescence induction (FI) was recorded in sunflower leaves pre-adapted to darkness or low preferentially PSI light, or inhibited by DCMU. For analysis the FI curves were plotted against the cumulative number of excitations quenched by PSII, n _q, calculated as the cumulative complementary area above the FI curve. In the +DCMU leaves n _q was <1 per PSII, suggesting pre-reduction of Q _A during the dark pre-exposure. A strongly sigmoidal FI curve was constructed by complementing (shifting) the recorded FI curves to n _q = 1 excitation per PSII. The full FI curve in +DCMU leaves was well fitted by a model assuming PSII antennae are excitonically connected in domains of four PSII. This result, obtained by gradually reducing Q _A in PSII with pre-blocked Q _B (by DCMU or PQH₂), differs from that obtained by gradually blocking the Q _B site (by increasing DCMU or PQH₂ level) in leaves during (quasi)steady-state e^? transport (Oja and Laisk, Photosynth Res 114, 15–28, 2012). Explanations are discussed. Donor side quenching was characterized by comparison of the total n _q in one and the same dark-adapted leaf, which apparently increased with increasing PFD during FI. An explanation for the donor side quenching is proposed, based on electron transfer from excited P680* to oxidized tyrosine Z (TyrZ^ox). At high PFDs the donor side quenching at the J inflection of FI is due mainly to photochemical quenching by TyrZ^ox. This quenching remains active for subsequent photons while TyrZ remains oxidized, following charge transfer to Q _A. During further induction this quenching disappears as soon as PQ and Q _A become reduced, charge separation becomes impossible and TyrZ is reduced by the water oxidizing complex.     

In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate-deficient sunflower and maize leaves

The effects of extreme phosphate (Pi) deficiency during growth on the contents of adenylates and pyridine nucleotides and the in vivo photochemical activity of photosystem II (PSII) were determined in leaves of Helianthus annuus and Zea mays grown under controlled environmental conditions. Phosphate deficiency decreased the amounts of ATP and ADP per unit leaf area and the adenylate energy charge of leaves. The amounts of oxidized pyridine nucleotides per unit leaf area decreased with Pi deficiency, but not those of reduced pyridine nucleotides. This resulted in an increase in the ratio of reduced to oxidized pyridine nucleotides in Pi-deficient leaves. Analysis of chlorophyll a fluorescence at room temperature showed that Pi deficiency decreased the efficiency of excitation capture by open PSII reaction centres (φ_e), the in vivo quantum yield of PSII photochemistry (φ_PSII) and the photochemical quenching co-efficient (q_P), and increased the non-photochemical quenching co-efficient (q_N) indicating possible photoinhibitory damage to PSII. Supplying Pi to Pi-deficient sunflower leaves reversed the long-term effects of Pi-deficiency on PSII photochemistry. Feeding Pi-sufficient sunflower leaves with mannose or FCCP rapidly produced effects on chlorophyll a fluorescence similar to long-term Pi-deficiency. Our results suggest a direct role of Pi and photophosphorylation on PSII photochemistry in both long-and short-term responses of photosynthetic machinery to Pi deficiency. The relationship between φ_PSII and the apparent quantum yield of CO₂ assimilation determined at varying light intensity and 21 kPa O₂ and 35 Pa CO₂ partial pressures in the ambient air was linear in Pi-sufficient and Pi-deficient leaves of sunflower and maize. Calculations show that there was relatively more PSII activity per mole of CO₂ assimilated by the Pi-deficient leaves. This indicates that in these leaves a greater proportion of photosynthetic electrons transported across PSII was used for processes other than CO₂ reduction. Therefore, we conclude that in vivo photosynthetic electron transport through PSII did not limit photosynthesis in Pi-deficient leaves of sunflower and maize and that the decreased CO₂ assimilation was a consequence of a smaller ATP content and lower energy charge which restricted production of ribulose, 1-5, bisphosphate, the acceptor for CO₂.     

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C₃/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO₂ uptake typical of C₃ plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q_n), with the largest differences being observed during the middle of the day. Cycling plants had lower q_n than CAM plants, indicating altered photosynthetic regulation processes. This q_n difference was believed to be related to reduced internal CO₂ concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO₂ given to plants in the CAM mode resulted in a decline in q_n in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO₂ was added.     

Dynamic modelling of limitations on improving leaf CO2 assimilation under fluctuating irradiance

A dynamic model of leaf CO₂ assimilation was developed as an extension of the canonical steady‐state model, by adding the effects of energy‐dependent non‐photochemical quenching (qE), chloroplast movement, photoinhibition, regulation of enzyme activity in the Calvin cycle, metabolite concentrations, and dynamic CO₂ diffusion. The model was calibrated and tested successfully using published measurements of gas exchange and chlorophyll fluorescence on Arabidopsis thaliana ecotype Col‐0 and several photosynthetic mutants and transformants affecting the regulation of Rubisco activity (rca‐2 and rwt43), non‐photochemical quenching (npq4‐1 and npq1‐2), and sucrose synthesis (spsa1). The potential improvements on CO₂ assimilation under fluctuating irradiance that can be achieved by removing the kinetic limitations on the regulation of enzyme activities, electron transport, and stomatal conductance were calculated in silico for different scenarios. The model predicted that the rates of activation of enzymes in the Calvin cycle and stomatal opening were the most limiting (up to 17% improvement) and that effects varied with the frequency of fluctuations. On the other hand, relaxation of qE and chloroplast movement had a strong effect on average low‐irradiance CO₂ assimilation (up to 10% improvement). Strong synergies among processes were found, such that removing all kinetic limitations simultaneously resulted in improvements of up to 32%.     

Regulation of Photosynthesis in Triazine-Resistant and -Susceptible Brassica napus

The response of photosynthetic carbon assimilation and chlorophyll fluorescence quenching to changes in intercellular CO₂ partial pressure (C_i), O₂ partial pressure, and leaf temperature (15-35°C) in triazine-resistant and -susceptible biotypes of Brassica napus were examined to determine the effects of the changes in the resistant biotype on the overall process of photosynthesis in intact leaves. Three categories of photosynthetic regulation were observed. The first category of photosynthetic response, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-limited photosynthesis, was observed at 15, 25, and 35°C leaf temperatures with low C_i. When the carbon assimilation rate was Rubisco-limited, there was little difference between the resistant and susceptible biotypes, and Rubisco activity parameters were similar between the two biotypes. A second category, called feedback-limited photosynthesis, was evident at 15 and 25°C above 300 microbars C_i. The third category, photosynthetic electron transport-limited photosynthesis, was evident at 25 and 35°C at moderate to high CO₂. At low temperature, when the response curves of carbon assimilation to C_i indicated little or no electron transport limitation, the carbon assimilation rate was similar in the resistant and susceptible biotypes. With increasing temperature, more electron transport-limited carbon assimilation was observed, and a greater difference between resistant and susceptible biotypes was observed. These observations reveal the increasing importance of photosynthetic electron transport in controlling the overall rate of photosynthesis in the resistant biotype as temperature increases. Photochemical quenching of chlorophyll fluorescence (q_P) in the resistant biotype never exceeded 60%, and triazine resistance effects were more evident when the susceptible biotype had greater than 60% q_P, but not when it had less than 60% q_P.     

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of ¹⁴C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.Abbreviations 9-AA 9-aminoacridine - J _e flux of photosynthetic electron transport - PC photosynthetic control - pH₁ H⁺ concentration in the thylakoid lumen - pmf proton motive force - _P potential quantum yield of photochemistry of photosystem II (with open reaction centers) - Q _A primary quinone-type electron acceptor of photosystem II - q _Q photochemical quenching of chlorophyll fluorescence - q _E energy-dependent quenching of chlorophyll fluorescence - q _AA light-induced quenching of 9-amino-acridine fluorescence     

Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces

In spinach (Spinacia oleracea) and barley (Hordeum vulgare) leaves, chlorophyll a fluorescence and O₂ evolution have been measured simultaneously following re-illumination after a dark interval or when steady state photosynthesis has been perturbed by changes in the gas phase. In high CO₂ concentrations, both O₂ and fluorescence can display marked dampening oscillations that are antiparallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O₂ by about 10 to 15 seconds). Infrared gas analysis measurements showed that CO₂ uptake behaved like O₂ evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence. In the steady state, oscillations were initiated by increases in CO₂ or by increases or decreases in O₂. Oscillations in O₂ or CO₂ did not occur without associated oscillations in fluorescence and the latter were a sensitive indicator of the former. The relationship between such oscillations in photosynthetic carbon assimilation and chlorophyl a fluorescence is discussed in the context of the effect of ATP or NADPH consumption on known quenching mechanisms.     

Effects of frost hardening, dehardening and freezing stress on in vivo chlorophyll fluorescence of seedlings of Scots pine (Pinus sylvestris L.)

Abstract. The kinetics of in vivo chlorophyll fluorescence of photosystem II (PS II) was measured at room temperature and 77 K during frost hardening of seedlings of Scots pine (Pinus sylvestris L.), and after exposure of frost-hardened shoots to sub-freezing temperatures. A more pronounced decrease in variable fluorescence yield for the upper exposed than for the lower shaded surface of the needles suggested that some photoinhibition occurred during prolonged frost hardening at 50 μmol photons m^?2 s^?1 and 4°C. Reversible inhibition of photosynthesis after exposure to sub-freezing temperatures was initially manifested as an increase of steady-state energy-dependent fluorescence quenching (q_E) and a reduction in the rate of O₂ evolution. Further inhibition after treatment at still lower temperatures caused a progressive decline of steady-state photochemical quenching (q_Q) and the rate of O₂ evolution, whereas q_E remained high. This implies an inactivation of enzymes in the photosynthetic carbon reduction cycle decreasing the consumption of ATP and NADPH, which is likely to cause an increase of membrane energization and a reduction of the primary electron acceptor (Q_A) of PS II. Alternatively, the changes in q_Q and q_E might be attributed to an inhibition of photophosphorylation. Severe, irreversible damage to photosynthesis resulted in a suppression of q_E and of variable fluorescence yield, probably because the photochemical efficiency of PS II was impaired. Changes in the fast fluorescence kinetics at room temperature after severe freezing damage were interpreted as an inhibition of the electron flow from Q_A to the plastoquinone pool. It is suggested that irreversible freezing injury to needles of frost-hardened P. sylvestris causes damage to the Q_B,-protein.     

CARBON SKELETON SOURCES FOR AMMONIUM ASSIMILATION IN N-SUFFICIENT AND N-LIMITED CELLS OF CYANIDIUM CALDARIUM (RHODOPHYTA)1

The possible origin of carbon skeletons for ammonium assimilation in Cyanidium caldarium (Tilden) Geitler was investigated. N-sufficient cells assimilated ammonium at a rate of 182 ± 18 μmol·mL packed cell volume (pcv)^-1· h^-1. Removal of CO₂ or darkening almost immediately prevented ammonium assimilation. N-limited cells in light assimilated ammonium at a rate of 493 ± 45 μmol · mL pcv^-1· h^-1 in the presence of CO₂ and at a lower rate of 168 ± 17 μmol · mL pcv^-1· h^-1 in the absence of CO₂. In darkness they assimilated ammonium at a rate of 293 ± 29 μmol · mL pcv^-1 h^-1 in the presence of CO₂, only 60% of the assimilation rate in light. In the absence of CO₂, ammonium was assimilated at a similar rate of 325 ± 14 μmol · mL pcv^-1· h^-1. Under the latter conditions, however, assimilation was inhibited after 40 min and ceased after 70 min; it resumed upon resupply of CO₂. We suggest that N-sufficient cells of C. caldarium obtain carbon skeletons for ammonium assimilation exclusively by photosynthetic reactions. Upon N-limitation they develop the ability, apparently through derepression or activation of regulatory enzyme system(s), to obtain a consistent quantity of additional carbon skeletons and ATP from mobilization of carbon reserves. This enables the N-limited cell to assimilate ammonium not only in light but also in darkness, and at a higher rate than N-sufficient cells. The fact that ammonium assimilation in light occurs at a higher rate than in darkness suggests that ammonium assimilation in light is the sum of both light and dark ammonium assimilation, which implies separate metabolic reactions for the two processes. These results suggest the existence of two distinct and differently controlled pathways in N-limited cells, but not in N-sufficient cells, through which carbon skeletons for ammonium assimilation originate. An important role for dark CO₂ fixation in dark or light ammonium assimilation is also indicated.     

Effects of water vapor pressure deficit on photochemical and fluorescence yields in tobacco leaf tissue

The relationship between photochemical quantum yield (_s) and fluorescence yield have been investigated in leaf tissue from Nicotiana tabacum using CO₂ exchange and a modulated fluorescence measuring system. The quantum yield of CO₂ fixation at 1.6% (v/v) O₂ and limiting irradiance was reduced 20% by increasing the mean H₂O vapor pressure deficit (VPD) from 9.2 to 18.6 mbars. As [CO₂] and irradiance were varied, the intrinsic quantum yield of open photosystem II units (_s/q_Q where q_Q is the photochemical fluorescence quenching coefficient) declined linearly with the degree of nonphotochemical fluorescence quenching. The slope and y-intercept values for this function were significantly reduced when the mean VPD was 18.4 millibars relative to 8.9 millibars. Susceptibility of the leaf tissue to photoinhibition was unaffected by VPD. Elevated O₂ concentrations (20.5% v/v) reduced the intrinsic quantum yield of net CO₂ uptake due to the occurrence of O₂-reducing processes. However, the relative effect of high VPD compared to low VPD on intrinsic quantum yield was not dependent on the O₂ level. This suggests that the Mehler reaction does not mediate the response of quantum yield to elevated VPD. The results are discussed with regard to the possible role of transpiration stress in regulating dissipation of excitation by electron transport pathways other than noncyclic electron flow supporting reduction of CO₂ and/or O₂.     

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH₄⁺ addition we found that: (a) N accumulated in glutamine while glutamate and α-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH₄⁺ addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH₄⁺ addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.     

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO₂ uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q_NP), and the rate constant of radiationless energy dissipation (k_D). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO₂ uptake differed widely and light saturation of CO₂ uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO₂ uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q_NP, increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO₂ uptake was approached, q_NP showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k_D, which opens the possibility that zeaxanthin is related to the rapidly relaxing “high-energy-state quenching” in leaves.     

Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves

A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (q_E) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of CO₂) not only variable fluorescence, F_V, but also dark-level fluorescence, F_O, was quenched, leading to definition of a quenching coefficient, q_O. Information on q_O was shown to be essential for correct determination of photochemical (q_Q) and energy dependent quenching (q_E) by the saturation pulse method. The relationship between q_E and q_O was analysed over a range of light intensities at steady state conditions. q_E was found to consist of two components, the second of which is linearly correlated with q_O. q_O and the second component of q_E are interpreted to reflect the state 1 — state 2 shift caused by LHC II phosphorylation.     

Effect of nitrogen application and elevated CO2 on photosynthetic gas exchange and electron transport in wheat leaves

Nitrogen (N) availability is a critical factor affecting photosynthetic acclimation of C₃ plants under elevated atmospheric CO₂ concentration ([CO₂]_e). However, current understanding of N effects on photosynthetic electron transport rate and partitioning, as well as its impact on photosynthesis under [CO₂]_e, is inadequate. Using controlled environment open-top chambers, wheat (Triticum aestivum L.) was grown at two N levels (0 and 200 mg(N) kg^?1 soil) and two atmospheric CO₂ concentrations of 400 ([CO₂]_a) and 760 μmol mol^?1([CO₂]_e) during 2009 and 2010. Under [CO₂]_e high N availability increased stomatal conductance and transpiration rate, reduced limitations on the activity of triose phosphate isomerase, a Calvin cycle enzyme, and increased the rate of net photosynthesis (P _N). Considering photosynthetic electron transport rate and partitioning aspects, we suggest that greater N availability increased P _N under [CO₂]_e due to four following reasons: (1) higher N availability enhanced foliar N and chlorophyll concentrations, and the actual photochemical efficiency of photosystem (PS) II reaction centers under irradiance increased, (2) increase of total electron transport rate and proportion of open PSII reaction centers, (3) enhancement of the electron transport rate of the photochemical and carboxylation processes, and (4) reduced limitations of the Calvin cycle enzymes on the photosynthetic electron transport rate. Consequently, sufficient N improved light energy utilization in wheat flag leaves under [CO₂]_e, thus benefiting to photosynthetic assimilation.     

ATP and NADPH as the driving force of carbon reduction in leaves in relation to thylakoid energization by light

Carbon assimilation of spinach (Spinacia oleracea L.) leaves was measured in the presence of 2000l· l^–1CO₂ and 2% O₂ in the gas phase to suppress photorespiratory reactions and to reduce stomatal diffusion resistance. Simultaneously, membrane parameters such as modulated chlorophyll fluorescence, oxidation of P₇₀₀ in the reaction centre of photosystem I, and apparent changes in absorbance at 535 nm were recorded. After light-regulated enzymes were activated at a high irradiance, illumination was changed. About 3 min later (to maintain the previous activation state of enzymes), leaves were shock-frozen and freeze-dried. Chloroplasts were isolated nonaqueously and analysed for ATP, ADP, inorganic phosphate, NADPH and NADP. Observations made under the chosen conditions differed in some important aspects from those commonly observed when leaves are illuminated in air. (i) Not only assimilation, but also the phosphorylation potential [ATP]/([ADP]·[Pi]) increased hyperbolically with irradiance towards saturation. In contrast, the ratio of NADPH to NADP did not change much as irradiances increased from low to high photon flux densities. When ATP, the phosphorylation potential and the assimilatory force, F_A (the product of phosphorylation potential and NADPH/NADP ratio), were plotted against assimilation, ATP increased relatively less than assimilation, whereas the phosphorylation potential increased somewhat more steeply than assimilation did. A linear relationship existed between assimilation and F_A at lower irradiances. The assimilatory force F_A increased more than assimilation did when irradiances were very high. Differences from previous observations, where F_A was under some conditions higher at low than at high rates of carbon assimilation, are explained by differences in flux resistances caused not only by stomatal diffusion resistance but also by differences in the activity of light-regulated enzymes, (ii) The relationship between P₇₀₀ oxidation and a fast absorption change with a maximum close to 520 nm on one hand and carbon assimilation on the other hand was largely linear under the specific conditions of the experiments. A similar linear relationship existed also between the quantum efficiency of electron flow through photosystem II and the quantum efficiency of photosystem I electron transport. (iii) Whereas the increase in non-photochemical fluorescence quenching, q_N, was similar to the increase in assimilation, the relationship between light scattering and assimilation was distinctly sigmoidal. Light scattering appeared to be a better indicator of control of photosystem II activity under excessive irradiation than q_N. (iv) The results are discussed in relation to the relative significance of chloroplast levels of ATP and NADPH and of the assimilatory force F_A in driving carbon assimilation. From the observations, the proton/electron (H⁺/e^–) ratio of linear electron transport is suggested to be 3 and the H⁺/ATP ratio to be 4 in leaves. An H⁺/e^– ratio of 3 implies the existence of an obligatory Q-cycle in leaves.Abbreviations F_A assimilatory force - F_o fluorescence after long dark adaptation - F_m maximum fluorescence level - F_s steady-state fluorescence - PGA 3-phosphoglycerate - PFD photon flux density - P₇₀₀ (P₇₀₀+) electron-donor pigment in the reaction center of PSI (its oxidized form) - Q_A primary quinone acceptor of PSII - q_P photochemical quenching - q_N non-photochemical quenching - _PSII relative quantum efficiency of energy conversation at the level of photosystem II - _PSI relative quantum efficiency of photosystem II This research was supported by the Sonderforschungsbereich 251 of the University of Würzburg and the Stiftung Volkswagenwerk. U.G. is a member of the Graduate College of the Julius-von-Sachs Institut für Biowissenschaften, University of Würzburg, being on leave from Tartu University, Tartu, Estonia. The authors are grateful to Prof. A. Laisk, Chair of Plant Physiology, Tartu University, for stimulating discussions.