Excitation transfer and quenching in photosystem II, enlightened by carotenoid triplet state in leaves.

Accumulation of carotenoid (Car) triplet states was investigated by singlet-triplet annihilation, measured as chlorophyll (Chl) fluorescence quenching in sunflower and lettuce leaves. The leaves were illuminated by Xe flashes of 4 µs length at half-height and 525-565 or 410-490 nm spectral band, maximum intensity 2 mol quanta m-2 s-1, flash photon dose up to 10 µmol m-2 or 4-10 PSII excitations. Superimposed upon the non-photochemically unquenched Fmd state, fluorescence was strongly quenched near the flash maximum (minimum yield Fe), but returned to the Fmd level after 30-50 µs. The fraction of PSII containing a 3Car in equilibrium with singlet excitation was calculated as Te = (Fmd-Fe)/Fmd. Light dependence of Te was a rectangular hyperbola, whose initial slope and plateau were determined by the quantum yields of triplet formation and annihilation and by the triplet lifetime. The intrinsic lifetime was 9 µs, but it was strongly shortened by the presence of O2. The triplet yield was 0.66 without nonphotochemical quenching (NPQ) but approached zero when NP-Quenched fluorescence approached 0.2 Fmd. The results show that in the Fmd state a light-adapted charge-separated PSIIL state is formed (Sipka et al., The Plant Cell 33:1286-1302, 2021) in which Pheo-P680+ radical pair formation is hindered, and excitation is terminated in the antenna by 3Car formation. The results confirm that there is no excitonic connectivity between PSII units. In the PSIIL state each PSII is individually turned into the NPQ state, where excess excitation is quenched in the antenna without 3Car formation.

VHL-deficiency leads to reductive stress in renal cells.

Heritable renal cancer syndromes (RCS) are associated with numerous chromosomal alterations including inactivating mutations in von Hippel-Lindau (VHL) gene. Here we identify a novel aspect of the phenotype in VHL-deficient human renal cells. We call it reductive stress as it is characterised by increased NADH/NAD+ ratio that is associated with impaired cellular respiration, impaired CAC activity, upregulation of reductive carboxylation of glutamine and accumulation of lipid droplets in VHL-deficient cells. Reductive stress was mitigated by glucose depletion and supplementation with pyruvate or resazurin, a redox-reactive agent. This study demonstrates for the first time that reductive stress is a part of the phenotype associated with VHL-deficiency in renal cells and indicates that the reversal of reductive stress can augment respiratory activity and CAC activity, suggesting a strategy for altering the metabolic profile of VHL-deficient tumours.

Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury.

Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 °C to 22 °C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 °C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury.

Time- and reduction-dependent rise of photosystem II fluorescence during microseconds-long inductions in leaves.

Lettuce (Lactuca sativa) and benth (Nicotiana benthamiana) leaves were illuminated with 720 nm background light to mix S-states and oxidize electron carriers. Green-filtered xenon flashes of different photon dose were applied and O2 evolution induced by a flash was measured. After light intensity gradient across the leaf was mathematically considered, the flash-induced PSII electron transport (= 4·O2 evolution) exponentially increased with the flash photon dose in any differential layer of the leaf optical density. This proved the absence of excitonic connectivity between PSII units. Time courses of flash light intensity and 680 nm chlorophyll fluorescence emission were recorded. While with connected PSII the sigmoidal fluorescence rise has been explained by quenching of excitation in closed PSII by its open neighbors, in the absence of connectivity the sigmoidicity indicates gradual rise of the fluorescence yield of an individual closed PSII during the induction. Two phases were discerned: the specific fluorescence yield immediately increased from Fo to 1.8Fo in a PSII, whose reaction center became closed; fluorescence yield of the closed PSII was keeping time-dependent rise from 1.8Fo to about 3Fo, approaching the flash fluorescence yield Ff = 0.6Fm during 40 µs. The time-dependent fluorescence rise was resolved from the quenching by 3Car triplets and related to protein conformational change. We suggest that QA reduction induces a conformational change, which by energetic or structural means closes the gate for excitation entrance into the central radical pair trap-efficiently when QB cannot accept the electron, but less efficiently when it can.

Variable fluorescence of closed photochemical reaction centers.

Chlorophyll fluorescence induction during 0.4 to 200 ms multiple-turnover pulses (MTP) was measured in parallel with O2 evolution induced by the MTP light. Additionally, a saturating single-turnover flash (STF) was applied at the end of each MTP and the total MTP +STF O2 evolution was measured. Quantum yield of O2 evolution during the MTP transients was calculated and related to the number of open PSII centers, found from the STF O2 evolution. Proportionality between the number of open PSII and their running photochemical activity showed the quantum yield of open PSII remained constant independent of the closure of adjacent centers. During the induction, total fluorescence was partitioned between Fo of all the open centers and Fc of all the closed centers. The fluorescence yield of a closed center was 0.55 of the final Fm while less than a half of the centers were closed, but later increased, approaching Fm to the end of the induction. In the framework of the antenna/radical pair equilibrium model, the collective rise of the fluorescence of centers closed earlier during the induction is explained by an electric field, facilitating return of excitation energy from the Pheo- P680+ radical pair to the antenna.

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction.

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and QA as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F m post-flash fluorescence yield F f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from QA and requires that electron transfer from QA- to QB be slower than that from QA- to QB-. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O2 evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced QA and reduced donor side.

Kinetics of plastoquinol oxidation by the Q-cycle in leaves.

Electrochromic shift measurements confirmed that the Q-cycle operated in sunflower leaves. The slow temporarily increasing post-pulse phase was recorded, when ATP synthase was inactivated in the dark and plastoquinol (PQH(2)) oxidation was initiated by a short pulse of far-red light (FRL). During illumination by red light, the Q-cycle-supported proton arrival at the lumen and departure via ATP synthase were simultaneous, precluding extreme build-up of the membrane potential. To investigate the kinetics of the Q-cycle, less than one PQH(2) per cytochrome b(6)f (Cyt b(6)f) were reduced by illuminating the leaf with strong light pulses or single-turnover Xe flashes. The post-pulse rate of oxidation of these PQH2 molecules was recorded via the rate of reduction of plastocyanin (PC(+)) and P700(+), monitored at 810 and 950 nm. The PSII-reduced PQH(2) molecules were oxidized with multi-phase overall kinetics, τ(d)=1, τ(p)=5.6 and τ(s)=16 ms (22 °C). We conclude that τ(d) characterizes PSII processes and diffusion, τ(p) is the bifurcated oxidation of the primary quinol and τ(s) is the Q-cycle-involving reduction of the secondary quinol at the n-site, its transport to the p-site, and bifurcated oxidation there. The extraordinary slow kinetics of the Q-cycle may be related to the still unsolved mechanism of the "photosynthetic control."

Oxidation of plastohydroquinone by photosystem II and by dioxygen in leaves.

In sunflower leaves linear electron flow LEF=4O2 evolution rate was measured at 20 ppm O2 in N2. PSII charge separation rate CSRII=aII∙PAD∙(Fm-F)/Fm, where aII is excitation partitioning to PSII, PAD is photon absorption density, Fm and F are maximum and actual fluorescence yields. Under 630 nm LED+720 nm far-red light (FRL), LEF was equal to CSRII with aII=0.51 to 0.58. After FRL was turned off, plastoquinol (PQH2) accumulated, but LEF decreased more than accountable by F increase, indicating PQH2-oxidizing cyclic electron flow in PSII (CEFII). CEFII was faster under conditions requiring more ATP, consistent with CEFII being coupled with proton translocation. We propose that PQH2 bound to the QC site is oxidized, one e- moving to P680+, the other e- to Cyt b559. From Cyt b559 the e- reduces QB- at the QB site, forming PQH2. About 10-15% electrons may cycle, causing misses in the period-4 flash O2 evolution and lower quantum yield of photosynthesis under stress. We also measured concentration dependence of PQH2 oxidation by dioxygen, as indicated by post-illumination decrease of Chl fluorescence yield. After light was turned off, F rapidly decreased from Fm to 0.2 Fv, but further decrease to F0 was slow and O2 concentration dependent. The rate constant of PQH2 oxidation, determined from this slow phase, was 0.054 s(-1) at 270 µM (21%) O2, decreasing with Km(O2) of 60 µM (4.6%) O2. This eliminates the interference of O2 in the measurements of CEFII.

Fluorescence F 0 of photosystems II and I in developing C3 and C 4 leaves, and implications on regulation of excitation balance.

This work addresses the question of occurrence and function of photosystem II (PSII) in bundle sheath (BS) cells of leaves possessing NADP-malic enzyme-type C4 photosynthesis (Zea mays). Although no requirement for PSII activity in the BS has been established, several component proteins of PSII have been detected in BS cells of developing maize leaves exhibiting O2-insensitive photosynthesis. We used the basal fluorescence emissions of PSI (F 0I) and PSII (F 0II) as quantitative indicators of the respective relative photosystem densities. Chl fluorescence induction was measured simultaneously at 680 and 750 nm. In mature leaves, the F m(680)/F 0(680) ratio was 10.5 but less in immature leaves. We propose that the lower ratio was caused by the presence of a distinct non-variable component, F c, emitting at 680 and 750 nm. After F c was subtracted, the fluorescence of PSI (F 0I) was detected as a non-variable component at 750 nm and was undetectably low at 680 nm. Contents of Chls a and b were measured in addition to Chl fluorescence. The Chl b/(a + b) was relatively stable in developing sunflower leaves (0.25-0.26), but in maize it increased from 0.09 to 0.21 with leaf tissue age. In sunflower, the F 0I/(F 0I + F 0II) was 0.39 ± 0.01 independent of leaf age, but in maize, this parameter was 0.65 in young tissue of very low Chl content (20-50 mg m(-2)) falling to a stable level of 0.53 ± 0.01 at Chl contents >100 mg m(-2). The values of F 0I/(F 0I + F 0II) showed that in sunflower, excitation was partitioned between PSII and PSI in a ratio of 2:1, but the same ratio was 1:1 in the C4 plant. The latter is consistent with a PSII:PSI ratio of 2:1 in maize mesophyll cells and PSI only in BS cells (2:1:1 distribution). We suggest, moreover, that redox mediation of Chl synthesis, rather than protein accumulation, regulates photosystem assembly to ensure optimum excitation balance between functional PSII and PSI. Indeed, the apparent necessity for two Chls (a and b) may reside in their targeted functions in influencing accumulation of PSI and PSII, respectively, as opposed to their spectral differences.

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.

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.

Photosystem II antennae are not energetically connected: evidence based on flash-induced O2 evolution and chlorophyll fluorescence in sunflower leaves.

Oxygen evolution was measured in sunflower leaves in steady-state and during multiple-turnover pulses (MTP) of different light (630 nm LED plus far-red light) intensity and duration. In parallel, Chl fluorescence yields F(0) (minimum), F(s) (steady-state), and F(m) (pulse-saturated), as well as fluorescence induction during MTPs were recorded. Extra O(2) evolution was measured in response to a saturating single-turnover Xe flash (STF) applied immediately subsequently to the actinic light in the steady-state and to each MTP. Under the used anaerobic conditions and randomized S-states electron transport per STF was calculated as 4O(2) evolution. The STF-induced electron transport (=the number of open PSII) was maximal at the low background light, but decreased with progressing light saturation in steady-state and with the increasing duration of MTP. The quantum yield (effective antenna size) of open PSII centers remained constant when adjacent centers became closed. The photochemical quenching of fluorescence q(P) = (F(m) - F(s))/(F(m) - F(0)) was proportional with the portion of open PSII centers in the steady-state (variable non-photochemical quenching, NPQ) and with increasing MTP duration (NPQ absent). Comparison of experimental responses to a model based on PSII dimers with well-connected antennae showed no energetic connectivity between PSII antennae in intact leaves, suggesting that in vivo PSII exist as monomers, or dimers with energetically disconnected antennae.

Oxygen evolution and chlorophyll fluorescence from multiple turnover light pulses: charge recombination in photosystem II in sunflower leaves.

Oxygen evolution and Chl fluorescence induction were measured during multiple turnover light pulses (MTP) of 630-nm wavelength, intensities from 250 to 8,000 µmol quanta m(-2) s(-1) and duration from 0.3 to 200 ms in sunflower leaves at 22 °C. The ambient O(2) concentration was 10-30 ppm and MTP were applied after pre-illumination under far-red light (FRL), which oxidized plastoquinone (PQ) and randomized S-states because of the partial excitation of PSII. Electron (e ( - )) flow was calculated as 4·O(2) evolution. Illumination with MTP of increasing length resulted in increasing O(2) evolution per pulse, which was differentiated against pulse length to find the time course of O(2) evolution rate with sub-millisecond resolution. Comparison of the quantum yields, Y (IIO) = e ( - )/hν from O(2) evolution and Y (IIF) = (F (m) - F)/F (m) from Chl fluorescence, detected significant losses not accompanied by fluorescence emission. These quantum losses are discussed to be caused by charge recombination between Q (A) (-) and oxidized TyrZ at a rate of about 1,000 s(-1), either directly or via the donor side equilibrium complex Q(A) â†’ P (D1) (+)  â†” TyrZ(ox), or because of cycling facilitated by Cyt b (559). Predicted from the suggested mechanism, charge recombination is enhanced by damage to the water-oxidizing complex and by restricted PSII acceptor side oxidation. The rate of PSII charge recombination/cycling is fast enough for being important in photoprotection.

Oxygen evolution from single- and multiple-turnover light pulses: temporal kinetics of electron transport through PSII in sunflower leaves.

Oxygen evolution per single-turnover flash (STF) or multiple-turnover pulse (MTP) was measured with a zirconium O(2) analyzer from sunflower leaves at 22 °C. STF were generated by Xe arc lamp, MTP by red LED light of up to 18000 µmol quanta m(-2) s(-1). Ambient O(2) concentration was 10-30 ppm, STF and MTP were superimposed on far-red background light in order to oxidize plastoquinone (PQ) and randomize S-states. Electron (e(-)) flow was calculated as 4 times O(2) evolution. Q (A) → Q (B) electron transport was investigated firing double STF with a delay of 0 to 2 ms between the two. Total O(2) evolution per two flashes equaled to that from a single flash when the delay was zero and doubled when the delay exceeded 2 ms. This trend was fitted with two exponentials with time constants of 0.25 and 0.95 ms, equal amplitudes. Illumination with MTP of increasing length resulted in increasing O(2) evolution per pulse, which was differentiated with an aim to find the time course of O(2) evolution with sub-millisecond resolution. At the highest pulse intensity of 2.9 photons ms(-1) per PSII, 3 e(-) initially accumulated inside PSII and the catalytic rate of PQ reduction was determined from the throughput rate of the fourth and fifth e(-). A light response curve for the reduction of completely oxidized PQ was a rectangular hyperbola with the initial slope of 1.2 PSII quanta per e(-) and V (m) of 0.6 e(-) ms(-1) per PSII. When PQ was gradually reduced during longer MTP, V (m) decreased proportionally with the fraction of oxidized PQ. It is suggested that the linear kinetics with respect to PQ are apparent, caused by strong product inhibition due to about equal binding constants of PQ and PQH(2) to the Q (B) site. The strong product inhibition is an appropriate mechanism for down-regulation of PSII electron transport in accordance with rate of PQH(2) oxidation by cytochrome b(6)f.

The size of the lumenal proton pool in leaves during induction and steady-state photosynthesis.

This report describes a new method to measure the chloroplastic lumenal proton pool in leaves (tobacco and sunflower). The method is based on measurement of CO(2) outbursts from leaves caused by the shift in the CO(2) + H(2)O ↔ HCO(3)(-) + H(+) equilibrium in the chloroplast stroma as protons return from the lumen after darkening. Protons did not accumulate in the lumen to a significant extent when photosynthesis was light-limited, but a large pool of >100 µmol H(+) m(-2) accumulated in the lumen as photosynthesis became light-saturated. During thylakoid energization in the light, large amounts of protons are moved from binding sites in the stroma to binding sites in the lumen. The transthylakoidal difference in the chemical potential of free protons (ΔpH) is largely based on the difference in the chemical potential of bound protons in the lumenal and stromal compartments (pK). Over the course of the dark-light induction of photosynthesis protons accumulate in the lumen during reduction of 3-phosphoglycerate. The accumulation of electrons in reduced compounds of the stroma and cytosol is the natural cause for accumulation of a stoichiometric pool of lumenal protons during this transient event.

Equilibrium or disequilibrium? A dual-wavelength investigation of photosystem I donors.

Oxidation of photosystem I (PSI) donors under far-red light (FRL), slow re-reduction by stromal reductants and fast re-reduction in the dark subsequent to illumination by white light (WL) were recorded in leaves of several C(3) plants at 810 and 950 nm. During the re-reduction from stromal reductants the mutual interdependence of the two signals followed the theoretical relationship calculated assuming redox equilibrium between plastocyanin (PC) and P700, with the equilibrium constant of 40 +/- 10 (Delta E (m) = 86-99 mV) in most of the measured 24 leaves of nine plant species. The presence of non-oxidizable PC of up to 13% of the whole pool, indicating partial control of electron transport by PC diffusion, was transiently detected during a saturation pulse of white light superimposed on FRL or on low WL. Nevertheless, non-oxidizable PC was absent in the steady state during fast light-saturated photosynthesis. It is concluded that in leaves during steady state photosynthesis the electron transport rate is not critically limited by PC diffusion, but the high-potential electron carriers PC and P700 remain close to the redox equilibrium.

Fast cyclic electron transport around photosystem I in leaves under far-red light: a proton-uncoupled pathway?

Fast cyclic electron transport (CET) around photosystem I (PS I) was observed in sunflower (Helianthus annuus L.) leaves under intense far-red light (FRL) of up to 200 mumol quanta m(-2) s(-1). The electron transport rate (ETR) through PS I was found from the FRL-dark transmittance change at 810 and 950 nm, which was deconvoluted into redox states and pool sizes of P700, plastocyanin (PC) and cytochrome f (Cyt f). PC and P700 were in redox equilibrium with K(e) = 35 (ΔE(m) = 90 mV). PS II ETR was based on O(2) evolution. CET [(PS I ETR) - (PS II ETR)] increased to 50-70 mumol e(-) m(-2) s(-1) when linear electron transport (LET) under FRL was limited to 5 mumol e(-) m(-2) s(-1) in a gas phase containing 20-40 mumol CO(2) mol(-1) and 20 mumol O(2) mol(-1). Under these conditions, pulse-saturated fluorescence yield F(m) was non-photochemically quenched; however, F(m) was similarly quenched when LET was driven by low green or white light, which energetically precluded the possibility for active CET. We suggest that under FRL, CET is rather not coupled to transmembrane proton translocation than the CET-coupled protons are short-circuited via proton channels regulated to open at high ΔpH. A kinetic analysis of CET electron donors and acceptors suggests the CET pathway is that of the reversed Q-cycle: Fd -> (FNR) -> Cyt c(n) -> Cyt b(h) -> Cyt b(l) -> Rieske FeS -> Cyt f -> PC -> P700 ->-> Fd. CET is activated when PQH(2) oxidation is opposed by high ΔpH, and ferredoxin (Fd) is reduced due to low availability of e(-) acceptors. The physiological significance of CET may be photoprotective, as CET may be regarded as a mechanism of energy dissipation under stress conditions.

Rates and roles of cyclic and alternative electron flow in potato leaves.

Measurements of 810 nm transmittance changes in leaves, simultaneously with Chl fluorescence, CO(2) uptake and O(2) evolution, were carried out on potato (Solanum tuberosum L.) leaves with altered expression of plastidic NADP-dependent malate dehydrogenase. Electron transport rates were calculated: J(C) from the CO(2) uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, J(O) from the O(2) evolution rate, J(F) from Chl fluorescence parameters and J(I) from the post-illumination re-reduction speed of PSI donors. In the absence of external O(2), J(O) equaled (1.005 +/- 0.003) J(C), independent of the transgenic treatment, light intensity and CO(2) concentration. This showed that nitrite and oxaloacetate reduction rates were very slow. The Mehler-type O(2) reduction was evaluated from the rate of electron accumulation at PSI after the O(2) concentration was decreased from 210 to 20 mmol mol(-1), and resulted in <1% of the linear flow. J(F) and J(I) did not differ from J(C) while photosynthesis was light-limited, but considerably exceeded J(C) at saturating light. Then, typically, J(F) = 1.2 J(C) and J(I) = 1.3 J(C), and J(F) -J(C) and J(I) -J(C) depended little on CO(2) and O(2) concentrations. The results showed that the alternative and cyclic electron flow necessary to compensate variations in the ATP/NADPH ratio were only a few percent of the linear flow. The data do not support the requirement of 14H(+)/3ATP by the chloroplast ATP synthase. We suggest that the fast PSI cyclic electron flow J(I) - J(C), as well as the fast J(F) - J(C) are energy-dissipating cycles around PSI and PSII at light saturation.

Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves.

The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC), and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal were measured immediately before and after FRL was turned on or off. The same derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied to fit the model to the measurements. Typical best-fit pool sizes were 1.0-1.5 micromol m(-2) of P700, 3 PC/P700 and 1 Cyt f/P700, apparent equilibrium constants were 15 between P700 and PC and 3 between PC and Cyt f. Cyclic electron flow (CET) was calculated from the slope of the signal after FRL was turned off. CET activated as soon as electrons accumulated on the PSI acceptor side. The quantum yield of CET was close to unity. Consequently, all PSI in the leaf were able to perform in cycle, questioning the model of compartmentation of photosynthetic functions between the stroma and grana thylakoids. The induction of CET was very fast, showing that it was directly redox-controlled. After longer dark exposures CET dominated, because linear e- transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase.

Calibration of simultaneous measurements of photosynthetic carbon dioxide uptake and oxygen evolution in leaves.

The stoichiometric ratio of O2 evolution to CO2 uptake during photosynthesis reveals information about reductive metabolism, including the reduction of alternative electron acceptors, such as nitrite and oxaloacetate. Recently we reported that in simultaneous measurements of CO2 uptake and O2 evolution in a sunflower leaf, O2 evolution changed by 7% more than CO2 uptake when light intensity was varied. Since the O2/CO2 exchange ratio is approximately 1, small differences are important. Thus, these gas exchange measurements need precise calibration. In this work, we describe a new calibration procedure for such simultaneous measurements, based on the changes of O2 concentration caused by the addition of pure CO2 or O2 into a flow of dry air (20.95% O2) through one and the same capillary. The relative decrease in O2 concentration during the addition of CO2 and the relative increase in O2 concentration during the addition of O2 allowed us to calibrate the CO2 and O2 scales of the measurement system with an error (relative standard deviation, RSD) of <1%. Measurements on a sunflower leaf resulted in an O2/CO2 ratio between 1.0 and 1.03 under different CO2 concentrations and light intensities, in the presence of an ambient O2 concentration of 20-50 micromol mol(-1). This shows that the percentage use of reductive power from photochemistry in synthesis of inorganic or organic matter other than CO2 assimilation in the C3 cycle is very low in mature leaves and, correspondingly, the reduction of alternative acceptors is a weak source of coupled ATP synthesis.