Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Afterglow thermoluminescence band as a possible early indicator of changes in the photosynthetic electron transport in leaves

The afterglow (AG) band of thermoluminescence (TL) has been investigated in leaves of Arabidopsis thaliana. Excitation of dark-adapted leaves with two saturating single turn-over flashes induced the appearance of a complex TL glow curve that could be well simulated by three components: the two components, B₁ and B₂, of the usually called B-band, peaking at 18 and 26 °C, respectively, and a band with t_max at 41 °C, which we attributed to an AG emission. Illumination of dark-adapted leaves with 720 nm monochromatic and FR lights generated the emission of a sharp single band peaking also around at 41 °C, that it is usually assigned to an AG emission band. Dark-incubation of whole plants increased the intensity of AG-band in TL curves induced by two flashes and, in parallel, decreased B-bands. Selective illumination of leaves with light mostly absorbed by PS II (650 nm light) completely abolished the AG-band induced by two flashes, B-band being the only TL band observed. The single AG-band induced by 720 nm light was abolished if leaves were also illuminated with 650 nm light. On the other hand, AG-band could be restored if 650 nm illuminated leaves were afterwards illuminated with 720 nm light. The changes in the intensity of B and AG bands induced by selective illuminations seem to be related to alterations in the redox state of Q_B and plastoquinone pool.     

Interaction of N,N,N′,N′-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the QB niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S₂Q_B⁻ and S₃Q_B⁻ (B-band), S₂Q_A⁻ (Q-band), and Y_D⁺Q_A⁻ (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y_n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q_B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q_B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q_B-site and to reduce the higher S-states of the Mn cluster.     

The role of growth rate, redox-state of the plastoquinone pool and the trans-thylakoid ΔpH in photoacclimation of Chlorella vulgaris to growth irradiance and temperature

The long-term photoacclimation of Chlorella vulgaris Beijer (UTEX 265) to growth irradiance and growth temperature under ambient CO₂ conditions was examined. While cultures grew at a faster rate at 27 than at 5 °C, growth rates appeared to be independent of irradiance. Decreases in light-harvesting polypeptide accumulation, increases in xanthophyll pool size and changes in the epoxidation state of the xanthophyll cycle pigments were correlated linearly with increases in the relative reduction state of Q_A, the primary quinone receptor of photosystem II, when estimated as 1−q_P under steady-state growth conditions. However, we show that there is also a specific temperature-dependent component, in addition to the redox-state of the Q_A, involved in regulating the content and composition of light-harvesting complex II of C. vulgaris. In contrast, modulation of the epoxidation state of the xanthophyll pool in response to increased 1−q_P in cells grown at 5 °C was indistinguishable from that of cells grown at 27 °C, indicating that light and temperature interact in a similar way to regulate xanthophyll cycle activity in C. vulgaris. Because C. vulgaris exhibited a low-light phenotype in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but a high-light phenotype upon addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, we conclude that the plastoquinone pool acts as a sensor regulating the accumulation of light-harvesting polypeptides in C. vulgaris. However, concomitant measurements of non-photochemical fluorescence quenching (q_N) and the epoxidation state of the xanthophyll pool appear to indicate that, in addition to the redox-state of the plastoquinone pool, the trans-thylakoid ΔpH may also contribute to sensing changes in irradiance and temperature that would lead to over-excitation of the photosynthetic apparatus. We suggest that sink capacity as reflected in photosynthate utilization and cell growth ultimately regulate photoacclimation in C. vulgaris. Received: 17 April 2000 / Accepted: 23 May 2000     

A thermoluminescence study of Photosystem II back electron transfer reactions in rice leaves – effects of salt stress

The recombination reactions of Photosystem II have been investigated in vivo in rice leaves by using the thermoluminescence (TL) emission technique. Excitation of dark-adapted leaf segments at 0 °C with different number of single turn-over flashes induced the appearance of complex TL glow curves. The mathematical analysis of these curves showed the existence of four TL components: B1-band (temperature maximum, t_max, at 24 °C, originating from S₃Q_B⁻ recombination), B2-band (t_max at 35 °C, from S₂Q_B⁻), AG-band (t_max at 46 °C) and C-band (t_max at 55 °C, from Tyr_D⁺Q_A⁻). Their contributions to the total TL signal were different depending on the number of flashes given. AG-band seems to reflect a special electron transfer from some unknown stroma donor to PS II. Q-band (t_max at 19 °C), originating from S₂Q_A⁻ recombination, was recorded after flashing samples incubated in the presence of DCMU. The recombination halftimes (t_1/2) at 20 °C of S₂Q_A⁻, S₃Q_B⁻, S₂Q_B⁻ and Tyr_D⁺Q_A⁻ were, respectively, 0.8 s, 48 s, 74 s and about 1 h. A sharp AG-band (t_max at 50 °C and t_1/2 of 210 s) could be also observed after illumination of leaves with far-red light and after a dark incubation period of whole plants. Incubation of leaf segments with 0.5 M NaCl abolished the inductions of AG-band by darkness and far-red illumination, significantly decreased Q-band intensity, whereas induced a strong increase in C-band intensity. The possible inhibition of S₂/S₃ formation and quinone oxidation by saline stress are discussed.     

Compared thermoluminescence characteristics of pea thylakoids studied in vitro and in situ (in leaves). The effect of photoinhibitory treatments

Characteristics of thermoluminescence (TL) glow curves were studied in thylakoids (isolated from pea leaves) or in intact pea leaves after an exposure to very high light for 2 min in the TL device. The inhibition of photosynthesis was detected as decreases of oxygen evolution rates and/or of variable fluorescence.In thylakoids exposed to high light, then dark adapted for 5 min, a flash regime induced TL glow curves which can be interpreted as corresponding to special B bands since: 1) they can be fitted by a single B band (leaving a residual band at –5°C) with a lower activation energy and a shift of the peak maximum by –5 to –6°C and, 2) the pattern of oscillation of their amplitudes was normal with a period of 4 and maxima on flashes 2 and 6. During a 1 h dark adaptation, no recovery of PS II activity occurred but the shift of the peak maximum was decreased to –1 to –2°C, while the activation energy of B bands increased. It is supposed that centers which remained active after the photoinhibitory treatment were subjected to reversible and probably conformational changes.Conversely, in intact leaves exposed to high light and kept only some minutes in the dark, TL bands induced by a flash regime were composite and could be deconvoluted into a special B band peaking near 30°C and a complex band with maximum at 2–5°C. In the case of charging bands by one flash, this low temperature band was largely decreased in size after a 10 min dark adaptation period; parallely, an increase of the B band type component appeared. Whatever was the flash number, bands at 2–5°C were suppressed by a short far red illumination given during the dark adaptation period and only remained a main band a 20°C; therefore, the origin of the low temperature band was tentatively ascribed to recombinations in centers blocked in state S₂Q_A ^–Q_B ^2–. In vivo, the recovery of a moderately reduced state in the PQ pool, after an illumination, would be slow and under the dependence of a poising mechanism, probably involving an electron transfer between cytosol and chloroplasts or the so-called chlororespiration process.Abbreviations E_a- activation energy - FR- far-red - MV- methylviologen - pBQ- p-benzoquinone - PQ- plastoquinone - PS II- Photosystem II - Q_A- primary quinone electron acceptor of PS II - Q_B- secondary quinone electron acceptor of PS II - TL- thermoluminescence     

Voltage changes involving photosystem II quinone–iron complex turnover

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q_A⁻ and the oxidized non-heme iron Fe³⁺ on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Δψ (negative charging of the proteoliposome interior) indicates that the iron–quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (τ ∼ 0.1 μs) of Δψ generation related to electron transfer between the redox-active tyrosine Y_Z and Q_A, an additional phase in the submillisecond time domain (τ ∼ 0.1 ms at 23°C, pH 7.0) and relative amplitude ∼ 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q_A and the secondary quinone Q_B. The rate of the additional electrogenic phase is decreased by about one-half in the presence of D₂O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.     

Thermal sensitivity of mitochondrial function in the Antarctic Notothenioid Lepidonotothen nudifrons

The thermal sensitivity of mitochondrial function was investigated in the stenothermal Antarctic fish Lepidonotothen nudifrons. State 3 respiration increases with increasing temperature between 0 °C and 18 °C with a Q ₁₀ of 2.43–2.63. State 4 respiration in the presence of oligomycin, an inhibitor of mitochondrial ATP synthase, quantifies the leakage of protons through the inner mitochondrial membrane, which causes oxygen consumption without concomitant ATP production. This parameter shows an unusually high Q ₁₀ of 4.21 ± 0.42 (0–18 °C), which indicates that proton leakage does not depend merely on ion diffusion but is an enzyme-catalysed process. The differential thermal sensitivity of oxidative phosphorylation (=state 3) and proton leakage (=state 4 in the presence of oligomycin) leads to progressive uncoupling of the mitochondria and decreased efficiency of oxidative phosphorylation under in vivo conditions if the body temperature of L. nudifrons increases. Accepted: 2 September 1999     

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S₂Q⁻_A state while that at 25°C is attributed to recombination of S₂Q_AQ⁻_B (and S₃Q_AQ⁻_B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S₂Q_AQ⁻_B and S₃Q_AQ⁻_B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q_A and thence to Q_B or Q⁻_B) results in predictable changes in the extent of flash-induced thermoluminescence.     

Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone Q_B⁻ with the S₂ state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S₂Q_A⁻ recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a Tyr_D⁺-Q_A⁻ recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII Q_B-nonreducing centers.     

The function of water channels in Chara: The temperature dependence of water and solute flows provides evidence for composite membrane transport and for a slippage of small organic solutes across water channels

Using the cell pressure probe, the effects of temperature on hydraulic conductivity (Lp; osmotic water permeability), solute permeability (permeability coefficient, P_s), and reflection coefficients (σ_s) were measured on internodes of Chara corallina, Klein ex Willd., em R.D.W.. For the first time, complete sets of transport coefficients were obtained in the range between 10 and 35 °C which provided evidence about pathways of water and solutes as they move across the plasma membrane (water channel and bilayer arrays). Test solutes used to check for the selectivity of water channels were monohydric alcohols of different molecular size and shape (ethanol, n-propanol, iso-propanol, and tert-butanol) and heavy water (HDO). Within the limits of accuracy, Q₁₀ values for Lp and for the diffusive water permeability (P_d) were identical (Q₁₀ for Lp = 1.29 ± 0.17 (± SD; n = 15 cells) and Q₁₀ for P_d = 1.25 ± 0.16 (n = 5 cells)). The Q₁₀ values were equivalent to activation energies of E_a = 16.8 ± 6.4 and 16.6 ± 10.0 kJ · mol⁻¹, respectively, which is similar to that of self-diffusion or of viscous flow of water. The Q₁₀ values and activation energies for P_s of the alcohols were significantly larger (ethanol: Q₁₀ = 1.68 ± 0.16, E_a = 37.1 ± 5.9 kJ · mol⁻¹; n-propanol: Q₁₀ =  1.75 ± 0.40, E_a = 43.1 ± 15.3 kJ · mol⁻¹; iso-propanol: Q₁₀ = 2.12 ± 0.42, E_a =  52.2 ± 14.6 kJ · mol⁻¹; tert-butanol: Q₁₀ = 2.13 ± 0.56, E_a = 51.6 ± 17.1 kJ · mol⁻¹; ±SD; n = 5 to 6 cells). Effects of temperature on reflection coefficients were most pronounced. With increasing temperature, σ_s values of the alcohols decreased and those of HDO increased. The data indicate that water and solutes use different pathways when crossing the membrane. Ordinary and isotopic water use water channels and the other test solutes use the bilayer array (composite transport model of membrane). Changes in σ_s values with temperature were found to be a sensitive measure for the open/closed state of water channels. The decrease of σ_s with temperature was theoretically predicted from the temperature dependence of P_s and Lp. Differences between predicted and measured values of σ_s allowed estimation of the bypass flow (slippage) of solutes through water channels which did not completely exclude test solutes. The permeability of channels depended on the structure and size of test solutes. It is concluded that water channels are much less selective than is usually thought. Since water channels represent single-file or no-pass pores, solutes drag along considerable amounts of water as they diffuse across channels. This results in low overall values of σ_s. The σ_s of HDO was extremely low. Its response to temperature was opposite to that for the σ_s of the alcohols. This suggested a stronger effect of temperature on the hydraulic (osmotic) than on the diffusive water flow across individual water channels, i.e. a differential sensitivity of different mechanisms to temperature. Received: 10 October 1996 / Accepted: 2 December 1996     

Thermoluminescence study of charge recombination in Photosystem II at low temperatures. II. Oscillatory properties of the Zv and A thermoluminescence bands in chloroplasts dark-adapted for various time periods

The oscillations of the Z_V and A thermoluminescence bands were investigated in spinach chloroplasts which had been dark-adapted for various time periods and subjected to a series of flashes at +2°C before continuous illumination at various low temperatures. When excited with continuous light below −65°C, the Z_V band exhibited period-4 oscillation, with maxima on preflashes 0, 4 and 8. Above −65°C, the oscillation pattern depended greatly on the dark-adaptation period of the chloroplasts. In preilluminated samples (15 s light followed by 3 min dark), when the Q_B pool is half oxidized, the oscillation of the thermoluminescence intensity measured at −50°C was similar to that observed below −65°C. However, after the thorough dark-adaptation of the chloroplasts (6 h), when the major fraction of the Q_B pool is assumed to be oxidized, a binary oscillation appeared in the oscillation pattern, with maxima at odd flash numbers. Below −65°C, period-2 oscillation of the Z_V band could not be induced by the dark-adaptation of the chloroplasts, suggesting an inhibition of electron exchange between Q_A and Q_B. Upon excitation of the chloroplasts with continuous light at −30°C, the A band oscillated with a periodicity of 4 with maxima at preflash numbers 2 and 6. At pH 7.5, the period-4 oscillation was converted into a period-2 oscillation by thorough dark-adaptation of the chloroplasts (24 h). Model calculations of the oscillatory patterns suggest that the period-4 oscillations of the Z_V and A bands are determined by the concentrations [S₀] + [S₁] and [S₂] + [S₃], respectively, which are present after the preflashes prior to the low-temperature continuous illumination. The period-2 oscillations in the amplitudes of the Z_V and A bands reflect the changes occurring in the redox state of the Q_B pool in a sequence of flashes. The possible relationship between the characteristics of the Z_V and A bands and the temperature-dependence of the S state transitions was investigated. Comparison of the amplitudal changes of the B (S₂Q⁻_B and S₃Q⁻_B recombination) and Q (S₂Q⁻_A recombination) thermoluminescence bands as a function of the excitation temperature suggests that the S₂ → S₃ and S₃ → S₄ transitions are blocked at about −65 and −40°C, respectively. It is also concluded that the thermoluminescence intensity emitted by the reaction center is about twice as high in the S₃ state as in the S₂ state.     

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C

A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C₁₄ to C₁₇) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry. Received: 5 October 1998 / Received revision: 4 December 1998 / Accepted: 5 December 1998     

Determination of shelf life of <Emphasis Type="Italic">Spirulina platensis</Emphasis> (MI<Subscript>2</Subscript>) grown in the Philippines

Gamma linolenic acid (GLA) degradation in Spirulina followed first-order reaction kinetics. At an accelerated temperature range of 45 to 55°C, the degradation rate constants (k _r) of GLA obtained were 4.0 × 10⁻² to 8.8 × 10⁻² day⁻¹. The energy of activation (E _a) was 16.53 kcal mol⁻¹, and the Q₁₀ was 2.22. Based on 20% GLA degradation, the shelf life of sun-dried Spirulina at 30°C is 263 days or 8.6 months using the Arrhenius plot, and 258 days or 8.5 months using the Q ₁₀ approach. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Effects of artificially-induced anaemia on sudomotor and cutaneous blood flow responses to heat stress

The influence of artificially induced anaemia on thermal strain was evaluated in trained males. Heat stress trials (38.6°C, water vapour pressure 2.74 kPa) performed at the same absolute work rates [20 min of seated rest, 20 min of cycling at 30% peak aerobic power (V˙O_2peak), and 20 min cycling at 45% V˙O_2peak] were completed before (HST1) and 3–5 days after 3 units of whole blood were withdrawn (HST2). Mild anaemia did not elevate thermal strain between trials, with auditory canal temperatures terminating at 38.5°C [(0.16), HST1] and 38.6°C [(0.13), HST2; P > 0.05]. Given that blood withdrawal reduced aerobic power by 16%, this observation deviates from the close association often observed between core temperature and relative exercise intensity. During HST2, the absolute and integrated forearm sweat rate (m˙ _sw) exceeded control levels during exercise (P < 0.05), while a suppression of forehead m˙ _sw occurred (P < 0.05). These observations are consistent with a possible peripheral redistribution of sweat secretion. It was concluded that this level of artificially induced anaemia did not impact upon heat strain during a 60-min heat stress test. Accepted: 17 April 1997     

Interaction between photon flux density and elevated temperatures on photoinhibition in Alocasia macrorrhiza

The effects of light and elevated temperatures on the efficiency of energy conversion in PSII [?_PSII = (Fm′−Fs)/Fm′], pigment composition and heat tolerance of shade-acclimated Alocasia macrorrhiza were investigated. Leaf discs were exposed for 3 h to high light (HL; 1600 μmol photons · m⁻² · s⁻¹) or low light (LL; 20 μmol photons · m⁻² · s⁻¹) and a series of constant temperatures ranging from 30 to 49 °C. All HL treatments led to rapid and severe decreases in ?_PSII. During the 2-h recovery period (LL, 25 °C) following the HL treatments, fast and slow recovery phases could be distinguished. Leaf discs that had experienced HL and 30 °C recovered completely while no recovery of ?_PSII was seen after a 3-h exposure to HL and 45 °C. A 3-h exposure to 45 °C at LL led to a less severe decrease in ?_PSII and complete recovery was accomplished after less than 1 h. Under LL conditions a temperature of 49 °C was necessary to cause an irreversible decrease in ?_PSII, followed by necrosis the next day. Streptomycin had no effect on the degree of reduction and recovery in ?_PSII discs exposed to HL and 35–45 °C, but partially inhibited recovery in discs exposed to HL and 30 °C. Streptomycin led to a more severe decrease in ?_PSII at LL and 49 °C and completely inhibited recovery. Streptomycin had no effect on the conversion of the xanthophyll-cycle pigments during the treatment or the recovery. The epoxidation state was roughly the same in all leaf discs after a 3-h HL treatment (0.270–0.346) irrespective of the exposure temperature. The back-conversion of zeaxanthin into violaxanthin after a 2-h recovery period was only seen in leaf discs that had been exposed to HL and 30 °C. The thermotolerance of shade A. macrorrhiza leaves of 49.0 ± 0.7 °C (determined by fluorescence) coincided with the temperature at which damage occurred in leaf discs exposed to LL. However, under HL the critical temperature under which necrosis occurred was much lower (42 °C). The thermotolerance of A. macrorrhiza shade leaves could be increased by a short exposure (<20 min) to slightly elevated temperatures. Received: 11 June 1997 / Accepted: 9 September 1997     

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m⁻²) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA₃₂₅(t), as an indicator of redox changes in Q_A, and of the relative fluorescence quantum yield, F(t)/F_o, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA₃₂₅, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA₃₂₅ dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl₂, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680^+·Q_A ^−● is almost invariant to this UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 O₂ 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 O₂ 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 O₂ evolution. Q _A → Q _B electron transport was investigated firing double STF with a delay of 0 to 2 ms between the two. Total O₂ 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 O₂ evolution per pulse, which was differentiated with an aim to find the time course of O₂ 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 PQH₂ 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 PQH₂ oxidation by cytochrome b₆f.