The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Non-photochemical fluorescence quenching in photosystem II antenna complexes by the reaction center cation radical

In direct experiments, rate constants of photochemical (k_P) and non-photochemical (k_P⁺) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the “dark” reaction center (RC) state, antenna fluorescence decay kinetics were measured under lowintensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a “closed” [P680⁺Q_A^–] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680⁺Q_A^–] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680⁺) state was found to be ～1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ₁ obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680⁺Pheo^– charge recombination. Thus, for the first time the ratio of k_P⁺/k_P = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.     

Crystal structure of plant light‐harvesting complex shows the active,energy‐transmitting state

Plants dissipate excess excitation energy as heat by non‐photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC‐II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC‐II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC‐II emit strong, orientation‐dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC‐II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC‐II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy‐transmitting state of LHC‐II. We conclude that quenching of excitation energy in the light‐harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment–protein complexes such as PsbS in vivo, and does not require a conformational change within the complex.     

Photophysical processes of energy conversion in thylakoid membranes of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> mutants D1-R323H,D1-R323D,and D1-R323L

Chlamydomonas reinhardtii mutants D1-R323H, D1-R323D, and D1-R323L showed elevated chlorophyll fluorescence yields, which increased with decline of oxygen evolving capacity. The extra step K ascribed to the disturbance of electron transport at the donor side of PS II was observed in OJIP kinetics measured in mutants with a PEA fluorometer. Fluorescence decay kinetics were recorded and analyzed in a pseudo-wild type (pWt) and in mutants of C. reinhardtii with a Becker and Hickl single photon counting system in pico- to nanosecond time range. The kinetics curves were fitted by three exponentials. The first one (rapid, with lifetime about 300 ps) reflects energy migration from antenna complex to the reaction center (RC) of photosystem II (PS II); the second component (600–700 ps) has been assigned to an electron transfer from P680 to Q_A, while the third one (slow, 3 ns) assumingly originates from charge recombination in the radical pair [P680^+• Pheo^−•] and/or from antenna complexes energetically disconnected from RC II. Mutants showed reduced contribution of the first component, whereas the yield of the second component increased due to slowing down of the electron transport to Q_A. The mutant D1-R323L with completely inactive oxygen evolving complex did not reveal rapid component at all, while its kinetics was approximated by two slow components with lifetimes of about 2 and 3 ns. These may be due to two reasons: a) disconnection between antennae complexes and RC II, and b) recombination in a radical pair [P680^+• Pheo^−•] under restricted electron transport to Q_A. The data obtained suggest that disturbance of oxygen evolving function in mutants may induce an upshift of the midpoint redox potential of Q_A/Q_A⁻ couple causing limitation of electron transport at the acceptor side of PS II.     

Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 Of photosystem II

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P+ are fluorescence quenchers, and Q- is a weak quencher, and that the reduction time for P+ is 20-35 ns. 2. The P+ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P+ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.     

Lipid‐polymorphism of plant thylakoid membranes. Enhanced non‐bilayer lipid phases associated with increased membrane permeability

Earlier experiments, using ³¹P‐NMR and time‐resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non‐bilayer (or non‐lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in ³¹P‐NMR signatures. However, changes in the lipid‐phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording ³¹P‐NMR spectra on isolated thylakoid membranes that were suspended in sorbitol‐ or NaCl‐based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol‐based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash‐induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a‐fluorescence transients; all variations were much less pronounced in the NaCl‐based medium. These observations suggest that non‐bilayer lipids and non‐lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

Time-resolved monitoring of flash-induced changes of fluorescence quantum yield and decay of delayed light emission in oxygen-evolving photosynthetic organisms

The present contribution describes a new experimental setup that permits time-resolved monitoring of the rise kinetics of the relative fluorescence yield, Phi(rel)(t), and simultaneously of the decay of delayed light emission, L(t), induced by strong actinic laser flashes. The results obtained by excitation of dark-adapted samples with a train of eight flashes reveal (a) in suspensions of spinach thylakoids, Phi(rel)(t) exhibits a typical period four oscillation that is characteristic for a dependence on the redox states S(i)() of the water oxidizing complex (WOC), (b) the relative extent of the unresolved "instantaneous" rise to the level (100 ns) at 100 ns and the maximum values of Phi(rel)(t) attained at about 45 s after each actinic flash, (45 s) synchronously oscillate and exhibit the largest values at flash nos. 1 and 5 and minima after flash nos. 2 and 3, (c) opposite effects are observed for the normalized extent of the rise kinetics in the 100 ns to 5 s time domain of relative fluorescence yield, Phi(rel)(5 s) - Phi(rel)(100 ns), i.e., both parameters attain minimum and maximum values after the first/fifth and second/third flash, respectively, and (d) analogous features for the "fast" and "slow" ns-kinetics of the fluorescence rise were observed in suspensions of Chlamydomas reinhardtii cells. A slight phase shift by one flash is ascribed to physiological differences. The applicability of this noninvasive technique to study reactions of photosystem II, especially the reduction kinetics of P680(*)(+) and their dependence on the redox state S(i)() of the WOC, is discussed.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q_A of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F _o towards a maximum F _m^STF when Q_A becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20–30% increase when the second partner is applied within 20–100 μs after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2–0.4 and 2–5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1–4 kHz) shows (i) in the first 3–4 flashes a transient overshoot 20–30% above the F _m^STF = 3*F _o level reached in the 1st flash with a partial decline towards a dip D in the next 2–3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F _m = 5*F _o in the 3–60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S₀ fraction with Q_B-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q_B-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.     

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn₄ cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S₂-, S₃-, and S₀-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Primary radical ion pairs in photosystem II core complexes

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700–710 nm at 278 K. It was found that the initial charge separation between P680* and Chl_D1 (Chl-670) takes place with a time constant of ～1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680^*(1?δ) (P680^δ+1Chl _D1 ^δ? ), where δ ～ 0.5. The subsequent electron transfer from P680^δ+Chl _D1 ^δ? to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (Chl _D1 ^δ? ) and bleaching of the Pheo_D1 bands at 420, 545, and 680 nm with development of the Pheoband at 460 nm. Further electron transfer to Q_A occurs within 250 ps in accordance with earlier data. The spectra of P680⁺ and Pheo^? formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ～1 and ～13 ps due to the formation of P680^δ+Chl _D1 ^δ? and P680⁺Pheo _D1 ^? , respectively.     

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.     

Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton,and the relevance for pulse‐amplitude‐modulated and fast‐repetition‐rate fluorometry1

We studied the variability of in vivo absorption coefficients and PSII‐scaled fluorescence excitation (fl‐ex) spectra of high light (HL) and low light (LL) acclimated cultures of 33 phytoplankton species that belonged to 13 different pigment groups (PGs) and 10 different phytoplankton classes. By scaling fl‐ex spectra to the corresponding absorption spectra by matching them in the 540–650 nm range, we obtained estimates for the fraction of total chl a that resided in PSII, the absorption of light by PSII, PSI, and photoprotective carotenoids. The in vivo red peak absorption maxima ranged from 673 to 679 nm, reflecting bonding of chl a to different pigment proteins. A simple approach is presented for quantifying intracellular self‐shading and evaluating the impact of photoacclimation on biooptical characteristics of the different PGs examined. In view of these results, parameters used in the calculation of oxygenic photosynthesis based on pulse‐amplitude‐modulated (PAM) and fast‐repetition‐rate (FRR) fluorometers are discussed, showing that the ratio between light available to PSII and total absorption, essential for the calculation of the oxygen release rate (using the PSII‐scaled fluorescence spectrum as a proxy) was dependent on species and photoacclimation state. Three subgroups of chromophytes exhibited 70%–80%, 60%–80%, and 50%–60% chl a in PSII‐LHCII; the two subgroups of chlorophytes, 70% or 80%; and cyanobacteria, only 12%. In contrast, the mean fraction for chromo‐ and chlorophytes of quanta absorbed by PSII was 73% in LL‐ and 55% in HL‐acclimated cells; thus, the corresponding ratios 0.55 and 0.73 might be used as correction factors adjusting for quanta absorbed by PSII for PAM and FRR measurements.     

Simultaneously measuring pulse-amplitude-modulated (PAM) chlorophyll fluorescence of leaves at wavelengths shorter and longer than 700 nm

PAM fluorescence of leaves of cherry laurel (Prunus laurocerasus L.) was measured simultaneously in the spectral range below 700 nm (sw) and above 700 nm (lw). A high-sensitivity photodiode was employed to measure the low intensities of sw fluorescence. Photosystem II (PSII) performance was analyzed by the saturation pulse method during a light response curve with subsequent dark phase. The sw fluorescence was more variable, resulting in higher PSII photochemical yields compared to lw fluorescence. The variations between sw and lw data were explained by different levels of photosystem I (PSI) fluorescence: the contribution of PSI fluorescence to minimum fluorescence (F₀) was calculated to be 14% at sw wavelengths and 45% at lw wavelengths. With the results obtained, the validity of an earlier method for the quantification of PSI fluorescence (Genty et al. in Photosynth Res 26:133–139, 1990, https://doi.org/10.1007/BF00047085) was reconsidered. After subtracting PSI fluorescence from all fluorescence levels, the maximum PSII photochemical yield (F_V/F_M) in the sw range was 0.862 and it was 0.883 in the lw range. The lower F_V/F_M at sw wavelengths was suggested to arise from inactive PSII reaction centers in the outermost leaf layers. Polyphasic fluorescence transients (OJIP or OI₁I₂P kinetics) were recorded simultaneously at sw and lw wavelengths: the slowest phase of the kinetics (IP or I₂P) corresponded to 11% and 13% of total variable sw and lw fluorescence, respectively. The idea that this difference is due to variable PSI fluorescence is critically discussed. Potential future applications of simultaneously recording fluorescence in two spectral windows include studies of PSI non-photochemical quenching and state I–state II transitions, as well as measuring the fluorescence from pH-sensitive dyes simultaneously with chlorophyll fluorescence.

     

Detecting <Emphasis Type="Italic">Suaeda salsa</Emphasis> L<Emphasis Type="Italic">.</Emphasis> chlorophyll fluorescence response to salinity stress by using hyperspectral reflectance

Measurements of chlorophyll fluorescence and hyperspectral reflectance were used to detect salinity stress in Suaeda salsa L., beach of Dongtai, Jiangsu Province, China. Three experimental sites were used in our study, which belong to low salinity, middle salinity and high salinity. The results showed that leaf chlorophyll fluorescence changed along salinity gradient. To select the sensitive hyperspectral ranges of leaf chlorophyll fluorescence, the correlationship between leaf chlorophyll fluorescence and hyperspectral reflectance was regressed and analyzed. Statistical results indicated that the 680 and 935 nm were the most sensitive hyperspectral bands for estimating leaf chlorophyll fluorescence. Then, 11 relative hyperspectral indices were selected based on the sensitive bands and previous literature. (R ₆₈₀ − R ₉₃₅)/(R ₆₈₀ + R ₉₃₅) and R ₆₈₀/R ₉₃₅ have higher correlationship coefficient (R) and lower root mean square error, may be used for detecting chlorophyll fluorescence, such as F _o, F _m, F _v/F _m, qP, and ΦPSII, while NPQ may be detected by (R ₇₈₀ − R ₇₁₀)/(R ₇₈₀ − R ₆₈₀). These results suggest that chlorophyll fluorescence of halophyte response to salinity stress could be identified by remote sensing.     

DIFFERENTIAL RESPONSES OF ANABAENA SP. PCC 7120 (CYANOPHYCEAE) CULTURED IN NITROGEN‐DEFICIENT AND NITROGEN‐ENRICHED MEDIA TO ULTRAVIOLET‐B RADIATION1

Stratospheric ozone depletion increases the amount of ultraviolet‐B radiation (UVBR) (280–320 nm) reaching the surface of the earth, potentially affecting phytoplankton. In this work, Anabaena sp. PCC 7120, a typically nitrogen (N)‐fixing filamentous bloom‐forming cyanobacterium in freshwater, was individually cultured in N‐deficient and N‐enriched media for long‐term acclimation before being subjected to ultraviolet‐B (UVB) exposure experiments. Results suggested that the extent of breakage in the filaments induced by UVBR increases with increasing intensity of UVB stress. In general, except for the 0.1 W · m^?2 treatment, which showed a mild increase, UVB exposure inhibits photosynthesis as evidenced by the decrease in the chl fluorescence parameters maximum photochemical efficiency of PSII (F_v/F_m) and maximum relative electron transport rate. Complementary chromatic acclimation was also observed in Anabaena under different intensities of UVB stress. Increased total carbohydrate and soluble protein may provide some protection for the culture against damaging UVB exposure. In addition, N‐deficient cultures with higher recovery capacity showed overcompensatory growth under low UVB (0.1 W · m^?2) exposure during the recovery period. Significantly increased (～830%) ATPase activity may provide enough energy to repair the damage caused by exposure to UVB.     

PSII photochemistry, thermal energy dissipation, and the xanthophyll cycle in Kalanchoë daigremontiana exposed to a combination of water stress and high light

Kalanchoë daigremontiana, a CAM plant grown in a greenhouse, was subjected to severe water stress. The changes in photosystem II (PSII) photochemistry were investigated in water‐stressed leaves. To separate water stress effects from photoinhibition, water stress was imposed at low irradiance (daily peak PFD 150 μmol m^?2 s^?1). There were no significant changes in the maximal efficiency of PSII photochemistry (F_v/F_m), the traditional fluorescence induction kinetics (OIP) and the polyphasic fluorescence induction kinetics (OJIP), suggesting that water stress had no direct effects on the primary PSII photochemistry in dark‐adapted leaves. However, PSII photochemistry in light‐adapted leaves was modified in water‐stressed plants. This was shown by the decrease in the actual PSII efficiency (Φ_PSII), the efficiency of excitation energy capture by open PSII centres (F_v′/F_m′), and photochemical quenching (q_P), as well as a significant increase in non‐photochemical quenching (NPQ) in particular at high PFDs. In addition, photoinhibition and the xanthophyll cycle were investigated in water‐stressed leaves when exposed to 50% full sunlight and full sunlight. At midday, water stress induced a substantial decrease in F_v/F_m which was reversible. Such a decrease was greater at higher irradiance. Similar results were observed in Φ_PSII, q_P, and F_v′/F_m′. On the other hand, water stress induced a significant increase in NPQ and the level of zeaxanthin via the de‐epoxidation of violaxanthin and their increases were greater at higher irradiance. The results suggest that water stress led to increased susceptibility to photoinhibition which was attributed to a photoprotective process but not to a photodamage process. Such a photoprotection was associated with the enhanced formation of zeaxanthin via de‐epoxidation of violaxanthin. The results also suggest that thermal dissipation of excess energy associated with the xanthophyll cycle may be an important adaptive mechanism to help protect the photosynthetic apparatus from photoinhibitory damage for CAM plants normally growing in arid and semi‐arid areas where they are subjected to a combination of water stress and high light.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.