Difference between histidine and histamine in the mechanistic pathway of the fluorescence reaction with ortho-phthalaldehyde

Histamine reacts with ortho-phthalaldehyde (OPA) in an alkaline medium to form an unstable fluorescent adduct (Fbase-Hm). Acidification of the solution to pH 2-4 gives a stable and highly fluorescent adduct (Facid-Hm). In contrast, histidine develops a relatively stable fluorescent adduct (Fbase-Hd) with OPA in an alkaline medium. Upon acidification of the solution, however, only trace amounts of the alternative fluorescent adduct (Facid-Hd) are produced although Fbase-Hd disappears similarly to Fbase-Hm. In this paper, the reaction pathway of histidine with OPA was clarified by the kinetic analysis of formation and degradation of Fbase-Hd. In comparing the results of this study with the previous ones for histamine (T. Yoshimura et al., 1987, Anal. Biochem., 164, 132-137), we elucidate the difference between histamine and histidine in the mechanism of fluorescence reaction with OPA. The presence of the carboxyl group in histidine not only stabilizes Fbase-Hd in an alkaline medium but also prevents the formation of a 1:2 adduct of histidine and OPA, the precursor of Facid-Hd.     

Fluorescence stopped-flow study of the o-phthaldialdehyde reaction

The fluorogenic reaction involving three species, namely, a primary amine, o-phthaldialdehyde (OPA), and a thiol compound was studied with the fluorescence stopped-flow technique. The results are consistent with the reaction of the amine with a 1:1 adduct of OPA and the thiol compound. The equilibrium constant for the formation of the adduct, OPAME, from OPA and mercaptoethanol (ME) was determined to be 164 m^?1. A survey of the rates of reaction of OPAME with various amino acids demonstrated that with OPA: ME:amine equal to 1:2.4:1 (total OPA concentration 0.5 to 3.0 × 10^?3m), the reaction followed second-order kinetics, with k = 150 to 450 m^?1s^?1 at pH 9.O. The differences in rates are discussed in relation to structural differences between the amines. The reaction, when conducted under conditions of excess OPAME yielded pseudo-first-order kinetics, with rates consistent with the second-order rate constants. The rate of reaction of OPAME with alanine was maximal at pH 10.5–11, and a great excess of ME resulted in a slower rate. Slower rates were also observed if ME was replaced by dithiothreitol or 1-propanethiol.     

Enhanced chemiluminescence of the fluorescein–KIO4 system by CdTe quantum dots for determination of catechol

In this paper, a novel chemiluminescence (CL) system was introduced based on the use of CdTe quantum dots (QDs) with the mixture solutions of fluorescein and potassium periodate (KIO₄) in alkaline medium. The CL signal of an ultra‐weak system was strongly enhanced in the presence of QDs. The application of CdTe QDs–fluorescein–KIO₄ system is reported for the first time. It was found that catechol had a diminishing effect on the CL reaction. Under optimal experimental conditions, CL intensity decreased linearly in a 1 to 100 μM catechol concentration range, with a 0.18 μM detection limit. A possible reaction mechanism was proposed according to the results of kinetic analyses, CL spectra, ultraviolet–visible and fluorescence spectra. The results pointed to an efficient energy transfer between the CL energy donor CdTe QDs and acceptor fluorescein. Finally, the CL method was successfully applied to the determination of catechol in environmental water samples.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

The effect of thylakoid membrane reorganisation on chlorophyll fluorescence lifetime components: A comparison between state transitions,protein phosphorylation and the absence of Mg2+

Room temperature chlorophyll fluorescence lifetime measurements using single photon counting and low-intensity laser excitation have been carried out on photosynthetic systems which have undergone protein reorganisation by an in vivo state 1-state 2 transition, protein phosphorylation and the absence of Mg²⁺. Analysis of the global changes in average lifetime and total fluorescence yield suggest that each treatment brings about a decrease in Photosystem (PS) II absorption cross-section but that this mechanism of energy redistribution accounts for different proportions of the total fluorescence quenching in the various cases. Further analysis of the overall fluorescence decay into individual kinetic components was carried out using a four-exponential model. The state transition did not alter the lifetimes of the four components but decreased the fluorescence yield of the long-lived decay, at both F₀ and F_M, by 24% and increased the yield of the rapid components. Such changes infer that there is a decrease in PS II absorption cross-section and an increase in PS I excitation on going from state 1 to state 2. Furthermore, these alterations show that the 500 ps component (at F₀) gives rise to the 2 ns decay (at F_M). After in vitro protein phosphorylation at 5 mM Mg²⁺, the changes are very similar to those brought abought by a state transition, except that both long-lived kinetic components exhibit a decrease in yield. When protein phosphorylation was carried out at 2 mM Mg²⁺ a slight decrease in the lifetimes of the two slow components was observed, with a further decrease in the yield of the 2.3 ns decay and a larger increase in the yields of the two rapid decays. Although the fluorescence quenching brought about by the absence of Mg²⁺ (57%) was the largest of all the treatments, only a small part could be explained by a decrease in PS II absorption cross-section (17%). The absence of Mg²⁺ led to a decrease in the lifetimes and yields of the two long-lived decays. A careful comparison of the characteristics of the slowest component in the presence and absence of 5 mM Mg²⁺ on closing the PS II traps suggest that this decay has different origins in the two cases.     

A Microscope for Two-Dimensional Measurements of In Vivo Chlorophyll Fluorescence Kinetics Using Pulsed Measuring Radiation,Continuous Actinic Radiation,and Saturating Flashes

Transients of chlorophyll fluorescence in photosynthetic objects are often measured using short pulses of exciting radiation, which has recently been employed to capture kinetic images of fluorescence at the macroscopic level. Here we describe an instrument introducing this principle to recording of two dimensional fluorescence transients in microscopic objects. A modified fluorescence microscope is equipped with a CCD camera intensified by a micro-channel plate image amplifier. The microscopic field is irradiated simultaneously by three types of radiation: actinic radiation, saturating flashes, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 µs. The detection of fluorescence images (300×400 pixels, 8 bit) has a maximum time resolution of 40 ms and is gated in synchrony with the exciting pulses. This allows measuring on a background of a continuous actinic radiation up to irradiance that can elicit the maximal fluorescence yield (F_M). On the other hand, the integral irradiance of the objects by the measuring radiation is very low, e.g., 0.08 µmol m^–2 s^–1 at 0.05 µm spatial resolution and 0.006 µmol m^–2 s^–1 at 4 µm spatial resolution. This allows a reliable recording of F₀ even in very short time intervals (e.g., 5×80 ms). The software yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetics parameters (F_M, F_O, F_V, F_V/F_M, etc.) showing a two-dimensional distribution of their values. Several examples demonstrate that records of fluorescence kinetics can be obtained with a reasonable signal-to-noise ratio with all standard microscope objectives and with object sizes reaching from segments of leaf tissue to individual algal cells or chloroplasts.     

Evaluation of chlorophyll fluorescence as a tool for salt stress detection in roses

The induction kinetic of the chlorophyll (Chl) fluorescence and the F_v/F_m ratio have been tested in order to find out the suitability of this technique to evaluate damage caused by salinity in plants of Rosa hybrida cv. Ilseta grafted on R. manetti growing in a greenhouse under non-saturating irradiance. Under these conditions salinity induced changes in plants morphology, nutrient and Chl contents and in the gas exchange parameters, but not in the F_v/F_m ratio. The Rfd index did not reveal more information. The F_v/F_m ratio as well as the fluorescence induction curves were more affected by salinity when an irradiation stress was added, therefore as an indicator of salt stress in roses, Chl fluorescence is of limited use when the plants are grown without additional stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photoinhibition in outdoor Spirulina platensis cultures assessed by polyphasic chlorophyll fluorescence transients

Photoinhibition in outdoor cultures of Spirulina platensis was studied by measuring the polyphasic rise of chlorophyll fluorescence transients, which provide information on the primary photochemistry of PSII. The maximum efficiency of PSII photochemustry (F_v/F_m) declined in response to daily increasing irradiance and recovered as daily irradiance decreased. The greatest inhibition (15%) in F_v/F_m was observed at 12:00 hr which responded to the highest irradiance. The absorption flux, the trapping flux, and the electron transport flux per PSII reaction center increased in response to daily increasing irradiance and decreased as irradiance decreased. The daily change in the concentration of PSII reaction centers followed the same pattern as F_v/F_m. However, no significant changes in the probability of electron transport beyond Q_A (Ψ_o) were observed during the day. The results suggest that the decrease in F_v/F_m induced by photoinhibition in outdoor Spirulina cultures was a result of the inactivation of PSII reaction centers. The results also suggest that the measurement of polyphasic fluorescence transients is a powerful tool to study the mechanism of photoinhibition in outdoor Spirulina cultures and to screen strains for photoinhibition tolerance. This revised version was published online in June 2006 with corrections to the Cover Date.     

The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K₂IrCl₆) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K₂IrCl₆ to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (F_v). At concentrations lower than 20 M, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (F_M). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F₀) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 M, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at F_M. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.Abbreviations DNB m-dinitrobenzene - F_M maximum Chl fluorescence - F₀ initial fluorescence - F_v variable fluorescence - I pheophytin a primary electron acceptor of PS2 - P₆₈₀ chlorophyll a of photochemical centre - PS2 photosystem 2 - Q_A primary stable electron acceptor of PS2 - Chl chlorophyll - LHC2 light harvesting Chl a/b complex of PS2 - MES 2(N-morpholino) ethanesulfonic acid - DCMU 3-(3-4-dichlorophenyl) 1-1 dimethylurea - PPBQ phenyl-p-benzo-quinone - BBY PS2-enriched membranes prepared as in Berthold et al. (1981) - Q₄₀₀ PS2 electron acceptor with a midpoint potential of 400 mV     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Simulation of Pulse-Amplitude-Modulated (PAM) fluorescence: Limitations of some PAM-parameters in studying environmental stress effects

Fluorescence parameters obtained during steady-state electron transport are frequently used to evaluate photosynthetic efficiency of plants. We studied the behaviour of those parameters as a function of irradiance-adapted fluorescence yields F_S and F'_M. Applied simulations showed that photochemical quenching evaluated by q_P is greatly influenced by the steady-state fluorescence level (F_S), and that its evolution is not complementary to non-photochemical quenching (q_N). On the other hand, the relative photochemical and non-photochemical quenching coefficients (q_P(rel) and q_N(rel)) proposed by Buschmann (1995) represent better the balance between the energy dissipation pathways. However, these relative parameters are also non-linearly related when the F_S level is varied. We investigated the application of a new parameter, the relative unquenched fluorescence (UQF_(rel)) which takes into account the fraction of non-quenched fluorescence yield (F_S), which is related to closed photosystem 2 reaction centres not participating in electron transport. By using computer simulations and real in vivo measurements, we found that this new parameter is complementary to q_P(rel) and q_N(rel), which may facilitate the use of PAM fluorescence as diagnostic tool in environmental studies.     

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" _m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H⁺extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO₂in acid and alkaline regions. The acid zones are enriched with CO₂that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO^– ₃is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.     

Mechanistic aspects of the Fenton reaction under conditions approximated to the extracellular fluid

The Fenton reaction was investigated, in a medium approximating to that of the extracellular fluid (ECF), by rapid-mixing stopped flow experiments and HPLC analysis using sodium terephthalate (TA²⁻). The reactive intermediate of the Fenton reaction hydroxylates the essentially nonfluorescent, TA²⁻ to the brilliant fluorophor 2-hydroxy-terephthalate (OH-TA), which allows the Fenton reaction to be monitored in stopped-flow experiments. There was no artefactual quenching of the fluorescence by substances present in the Fenton-reaction mixture or in the artificial cerebrospinal fluid (aCSF) that might have influenced OH-TA quantification. A mathematical model based on kinetic considerations was developed. This explains the observed independence of the OH-TA concentration on the amount of TA²⁻ present in aCSF as well as its dependence on TA²⁻ concentration in potassium acetate buffer. A mechanism based on this model, involving complex formation between Fe(II), TA²⁻ and H₂O₂, followed by an intra-molecular hydroxylation accompanied by an intra-molecular electron transfer was developed. The results are consistent with a reactive intermediate, which causes oxidative stress in vivo, not being a free hydroxyl radical, but a ferryl species or a “crypto” radical. The biological implications of these results are discussed.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ～1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.     

Photoprotective mechanisms in photosystem II of Ephedra monosperma during development of frost tolerance

Dissipation of light energy absorbed by photosystem II (PSII) in assimilating shoots of an evergreen shrub Ephedra monosperma was investigated during its transition from the vegetative to frost-tolerant state under natural conditions of Central Yakutia. The dynamics of modulated chlorophyll fluorescence and carotenoid content was analyzed during seasonal decrease in ambient temperature. The seasonal cooling was accompanied by a stepwise decrease in photochemical activity of PSII (F _v/F _m = (F _m ? F ₀)/F _m). The decrease in F _v/F _m occurred from the beginning of September to the end of October, when the temperature was lowered from 10 to ?8°C. During winter period the residual activity of PSII was retained at about 30% of the summer values. The seasonal decrease in temperature was accompanied by a significant stimulation of pH-independent dissipative processes in reaction centers and antenna of PSII. The increase in energy losses was paralleled by a proportional increase in zeaxanthin content on the background of decreasing content of violaxanthin and β-carotene as possible zeaxanthin precursors. At the same time, inhibition of light-induced non-photochemical quenching in the PSII antenna was observed. The results suggest that principal photoprotective mechanisms during seasonal lowering of temperature are: (1) inactivation of PSII and dissipation of excitation energy in PSII reaction centers and (2) zeaxanthin-mediated energy dissipation in the antenna complexes. The first mechanism seems to prevail at early stages of seasonal cooling, whereas both mechanisms are recruited from the onset of sustained freezing temperatures.     

Photoadaptation in the Green Alga Spongiochloris Sp. A Three-Fluorometer Study

The dark-adapted cells of the green alga Spongiochloris sp. were exposed to "white light" of 1000 μmol(photon) m⁻² s⁻¹ for 2 h and then dark adapted for 1.5 h. Changes of photochemical activities during photoadaptation were followed by measurement of chlorophyll (Chl) fluorescence kinetics, 77 K emission spectra, photosynthetic oxygen evolution, and pigment composition. We observed a build-up of slowly-relaxing non-photochemical quenching which led to a decrease of the F_v/F_m parameter and the connectivity. In contrast to the depression of F_v/F_m (35 %) and the rise of non-photochemical quenching (∼ 1.6), we observed an increase in effective absorption cross-section (20 %), Hill reaction (30 %), photosynthetic oxygen evolution (80 %), and electron transport rate estimated from the Chl fluorescence analysis (80 %). We showed an inconsistency in the presently used interpretation schemes, and ascribe the discrepancy between the increase of effective absorption cross-section and the photosynthetic activities on one side and the effective non-photochemical quenching on the other side to the build-up of a quenching mechanism which dissipates energy in closed reaction centres. Such a type of quenching changes the ratio between thermal dissipation and fluorescence without any effect on photochemical yield. In this case the F_v/F_m ratio cannot be used as a measure of the maximum photochemical yield of PS2. This revised version was published online in August 2006 with corrections to the Cover Date.     

Functioning of photosystems I and II in pea leaves exposed to heat stress in the presence or absence of light

Fluorimetric, photoacoustic, polarographic and absorbance techniques were used to measure in situ various functional aspects of the photochemical apparatus of photosynthesis in intact pea leaves (Pisum sativum L.) after short exposures to a high temperature of 40 ° C. The results indicated (i) that the in-vivo responses of the two photosystems to high-temperature pretreatments were markedly different and in some respects opposite, with photosystem (PS) II activity being inhibited (or down-regulated) and PSI function being stimulated; and (ii) that light strongly interacts with the response of the photosystems, acting as an efficient protector of the photochemical activity against its inactivation by heat. When imposed in the dark, heat provoked a drastic inhibition of photosynthetic oxygen evolution and photochemical energy storage, correlated with a marked loss of variable PSII-chlorophyll fluorescence emission. None of the above changes were observed in leaves which were illuminated during heating. This photoprotection was saturated at rather low light fluence rates (around 10 W · m^–2). Heat stress in darkness appeared to increase the capacity for cyclic electron flow around PSI, as indicated by the enhanced photochemical energy storage in far-red light and the faster decay of P ₇₀₀ ⁺ (oxidized reaction center of PSI) monitored upon sudded interruption of the far-red light. The presence of light during heat stress reduced somewhat this PSI-driven cyclic electron transport. It was also observed that heat stress in darkness resulted in the progressive closure of the PSI reaction centers in leaves under steady illumination whereas PSII traps remained largely open, possibly reflecting the adjustment of the photochemical efficiency of undamaged PSI to the reduced rate of photochemistry in PSII.Abbreviations B₁ and B₂ fraction of closed PSI and PSII reaction centers, respectively - ES photoacoustically measured energy storage - F_o, F_m and F_s initial, maximal and steady-state levels of chlorophyll fluorescence - P₇₀₀ reaction center of PSI - PS (I, II) photosystem (I, II) - V = (F_s – F_o)/(F_m – F_o) relative variable chlorophyll fluorescence We wish to thank Professor R. Lannoye (ULB, Brussels) for the use of this photoacoustic spectrometer and Mrs. M. Eyletters for her help.     

Black leaf-clips of a commercial fluorometer increased leaf temperature during dark adaptation under high solar radiation

The use of black leaf-clips for dark adaptation under high solar radiation conditions is reported to underestimate the maximum quantum yield of PSII photochemistry (F_v/F_m) measured by the continuous-excitation fluorometer Pocket PEA. The decrease in F_v/F_m was due to a rise in minimum fluorescence emission (F_o), probably resulting from increased leaf temperature (T_l). In field-grown tomato and pepper, fluorescence parameters and T_l in the region covered by the black leaf clip were measured in clipped leaves exposed to solar radiation during dark adaptation (clipped-only leaves) and in clipped leaves protected from solar radiation by aluminium foil (shrouded clipped leaves). Results confirmed significant F_v/F_m underestimates in clipped-only leaves primarily due to increased F_o. In one tomato experiment, T_l increased from 30 to 44.5°C in clipped-only leaves, with a negligible rise in shrouded clipped leaves. In two respective pepper experiments, T_l in clipped-only leaves increased from 27 to 36.2°C and 33 to 40.9°C. Based on the results of this study, a clip-effect parameter (P_CE) on fluorescence emission is proposed as the difference for F_v/F_m (or ?F_o/F_m) between shrouded clipped leaves and clipped-only leaves, which resulted to be 0.706 for tomato, and 0.241 and 0.358 for the two pepper experiments.