Assessment of photosystem II thermoluminescence as a tool to investigate the effects of dehydration and rehydration on the cyclic/chlororespiratory electron pathways in wheat and barley leaves.

Thermoluminescence emission from wheat leaves was recorded under various controlled drought stress conditions: (i) fast dehydration (few hours) of excised leaves in the dark (ii) slow dehydration (several days) obtained by withholding watering of plants under a day/night cycle (iii) overnight rehydration of the slowly dehydrated plants at a stage of severe dessication. In fast dehydrated leaves, the AG band intensity was unchanged but its position was shifted to lower temperatures, indicating an activation of cyclic and chlororespiratory pathways in darkness, without any increase of their overall electron transfer capacity. By contrast, after a slow dehydration the AG intensity was strongly increased whereas its position was almost unchanged, indicating respectively that the capacity of cyclic pathways was enhanced but that they remained inactivated in darkness. Under more severe dehydration, the AG band almost disappeared. Rewatering caused its rapid bounce significantly above the control level. No significant differences in AG emission could be found between the two drought-sensitive and drought-tolerant wheat cultivars. The afterglow thermoluminescence emission in leaves provides an additional tool to follow the increased capacity and activation of cyclic electron flow around PSI in leaves during mild, severe dehydration and after rehydration.

Pitfalls, artefacts and open questions in chlorophyll thermoluminescence of leaves or algal cells.

Thermoluminescence of intact photosynthetic organisms, leaves or algal cells, raises specific problems. The constitutive S2/3Q B (-) B bands constitute major probes of the state of photosystem II in vivo. The presence of a dark-stable acidic lumen causes a temperature downshift of B bands, specially the S3 B band, providing a lumen pH indicator. This is accompanied by a broadening of the S3 B band that becomes an envelope of elementary B bands. The occasional AT, Q and C bands are briefly examined in an in vivo context. It is emphasized that freezing below the nucleation temperature is not necessary for physiological studies, but a source of artefacts, hence should be avoided. In intact photosynthetic structures, a dark-electron transfer from stroma reductants to the quinonic acceptors of photosystem II via the cyclic/chlororespiratory pathways, strongly stimulated by moderate warming, gives rise to the afterglow (AG) luminescence emission that reflects chloroplast energy status. The decomposition of complex TL signals into elementary bands is necessary to determine the maximum temperature T m and the area of each of them. A comparison of TL signals after 1 flash and 2 flashes prevents from confusing the three main bands observed in vivo, i.e. the S2 and S3 B bands and the AG band. Finally, the thermoluminescence bands arising sometimes above 50 °C are mentioned. The basic principles of (thermo)luminescence established on isolated thylakoids should not be applied directly without a careful examination of in vivo conditions.

Thermoluminescence and P700 redox kinetics as complementary tools to investigate the cyclic/chlororespiratory electron pathways in stress conditions in barley leaves.

Cyclic electron flow around photosystem I drives additional proton pumping into the thylakoid lumen, which enhances the protective non-photochemical quenching and increases ATP synthesis. It involves several pathways activated independently. In whole barley leaves, P700 oxidation under far-red illumination and subsequent P700(+) dark reduction kinetics provide a major probe of the activation of cyclic pathways. Two 'intermediate' and 'slow' exponential reduction phases are always observed and they become faster after high light illumination, but dark inactivation of the Benson-Calvin cycle causes the emergence of both a transient in the P700 oxidation and a 'fast' phase in the P700(+) reduction. We investigate here the afterglow (AG) thermoluminescence emission as another tool to detect the activation of cyclic electron pathways from stroma reductants to the acceptor side of photosystem II. This transfer is activated by warming, yielding an AG band at about 45°C. However, treatments that accelerate the 'intermediate' and 'slow' P700(+) reduction phases (brief anoxia, hexose infiltration, fast dehydration of excised leaves) also produced a downshift of this AG band. This pathway ascribable to NADPH dehydrogenase (NDH) would be triggered by a deficit in ATP, while the 'fast' reduction phase corresponding to the ferredoxin plastoquinone reductase pathway is triggered by an overreduction of the photosystem I acceptor pool and is undetected in thermoluminescence. Contrastingly, slow dehydration of unwatered plants did not cause faster reduction of P700(+) nor temperature downshift of the AG band, that is no induction of the NDH pathway, whereas an increased intensity of the AG band indicated a strong NADPH + ATP assimilatory potential.

Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii.

The microalgae Chlamydomonas reinhardtii and Chlorella sp. CCAP 211/84 were grown autotrophically and mixotrophically and their thermoluminescence emissions were recorded above 0 °C after excitation by 1, 2 or 3 xenon flashes or by continuous far-red light. An oscillation of the B band intensity according to the number of flashes was always observed, with a maximum after 2 flashes, accompanied by a downshift of the B band temperature maximum in mixotrophic compared to autotrophic grown cells, indicative of a dark stable pH gradient. Moreover, new flash-induced bands emerged in mixotrophic Chlamydomonas grown cells, at temperatures higher than that of the B band. In contrast to the afterglow band observed in higher plants, in Chlamydomonas these bands were not inducible by far-red light, were fully suppressed by 2 µM antimycin A, and peaked at different temperatures depending on the flash number and growth stage, with higher temperature maxima in cells at a stationary compared to an exponential growth stage. These differences are discussed according to the particular properties of cyclic electron transfer pathways in C. reinhardtii.

Thermoluminescence: experimental.

Thermoluminesence measurements are useful for the study of Photosystem II electron transport in intact leaves, in algal and cyanobacterial cells, as well as in isolated membrane complexes. Here an overview of the experimental approaches is provided. In the present review, instruments and the experimental procedures for measuring thermoluminescence emission from photosynthetic systems of various origins are summarized and discussed. Major pitfalls frequently encountered in measurements with isolated membranes, suspensions of intact organisms or solid leaf samples are highlighted. Analytical and numeric methods for the analysis of measured thermoluminescence curves are also discussed.

Photosynthesis limitations during water stress acclimation and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandierixV. rupestris).

The hybrid Richter-110 (Vitis berlandierixVitis rupestris) has the reputation of being a genotype strongly adapted to drought. A study was performed with plants of R-110 subjected to sustained water-withholding to induce acclimation to two different levels of water stress, followed by rewatering to induce recovery. The goal was to analyse how photosynthesis is regulated during acclimation to water stress and recovery. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence and thermoluminescence), and biochemistry (V(c,max)) were assessed. During water stress, g(s) declined to 0.1 and less than 0.05 mol CO(2) m(-2) s(-1) in moderately and severely water-stressed plants, respectively, and was kept quite constant during an acclimation period of 1-week. Leaf photochemistry proved to be very resistant to the applied water-stress conditions. By contrast, g(m) and V(c,max) were affected by water stress, but they were not kept constant during the acclimation period. g(m) was initially unaffected by water stress, and V(c,max) even increased above control values. However, after several days of acclimation to water stress, both parameters declined below (g(m)) or at (V(c,max)) control values. For the latter two parameters there seemed to be an interaction between water stress and cumulative irradiance, since both recovered to control values after several cloudy days despite water stress. A photosynthesis limitation analysis revealed that diffusional limitations and not biochemical limitations accounted for the observed decline in photosynthesis during water stress and slow recovery after rewatering, both in moderately and severely stressed plants. However, the relative contribution of stomatal (SL) and mesophyll conductance (MCL) limitations changes during acclimation to water stress, from predominant SL early during water stress to similar SL and MCL after acclimation. Finally, photosynthesis recovery after rewatering was mostly limited by SL, since stomatal closure recovered much more slowly than g(m).

Changes in photosynthetic metabolism induced by tobamovirus infection in Nicotiana benthamiana studied in vivo by thermoluminescence.

* In thylakoids from Nicotiana benthamiana infected with the pepper mild mottle virus (PMMoV), a decreased amount of the PsbP and PsbQ proteins of photosystem II and different proteins of the Calvin cycle have been previously observed. We used thermoluminescence to study the consequences in vivo. * Measurements on unfrozen discs from symptomatic and asymptomatic leaves of plants infected by two tobamovirus PMMoV-S and PMMoV-I strains were compared with homologous samples in control plants. * Thermoluminescence emission did not reveal noticeable alteration of PSII electron transfer activity in infected symptomatic leaves. In these leaves, the relative intensity of the 'afterglow' emission indicated an increase of the NADPH + ATP assimilatory potential, contrasting with its decrease in asymptomatic leaves. High-temperature thermoluminescence, as a result of peroxides, increased in symptomatic and asymptomatic leaves. * In young infected leaves, PSII activity is preserved, producing a high assimilatory potential. Older asymptomatic leaves export more nutrients towards young infected leaves. This depresses their assimilatory potential and weakens their defence mechanisms against reactive oxygen species, resulting in higher peroxide content.

Chlorophyll thermofluorescence and thermoluminescence as complementary tools for the study of temperature stress in plants.

The photosynthetic apparatus, especially the electron transport chain imbedded in the thylakoid membrane, is one of the main targets of cold and heat stress in plants. Prompt and delayed fluorescence emission originating from photosystem II have been used, most often separately, to monitor the changes induced in the photosynthetic membranes during progressive warming or cooling of a leaf sample. Thermofluorescence of F (0) and F (M) informs on the effects of heat on the chlorophyll antennae and the photochemical centers, thermoluminescence on the stabilization and movements of charges and Delayed Light Emission on the permeability of the thylakoid membranes to protons and ions. Considered together and operated simultaneously, these techniques constitute a powerful tool to characterize the effect of thermal stress on intact photosynthetic systems and to understand the mechanisms of constitutive or induced tolerance to temperature stresses.

Probing the FQR and NDH activities involved in cyclic electron transport around Photosystem I by the 'afterglow' luminescence.

Far-red illumination of plant leaves for a few seconds induces a delayed luminescence rise, or afterglow, that can be measured with the thermoluminescence technique as a sharp band peaking at around 40-45 degrees C. The afterglow band is attributable to a heat-induced electron flow from the stroma to the plastoquinone pool and the PSII centers. Using various Arabidopsis and tobacco mutants, we show here that the electron fluxes reflected by the afterglow luminescence follow the pathways of cyclic electron transport around PSI. In tobacco, the afterglow signal relied mainly on the ferredoxin-quinone oxidoreductase (FQR) activity while the predominant pathway responsible for the afterglow in Arabidopsis involved the NAD(P)H dehydrogenase (NDH) complex. The peak temperature T(m) of the afterglow band varied markedly with the light conditions prevailing before the TL measurements, from around 30 degrees C to 45 degrees C in Arabidopsis. These photoinduced changes in Tm followed the same kinetics and responded to the same light stimuli as the state 1-state 2 transitions. PSII-exciting light (leading to state 2) induced a downward shift while preillumination with far-red light (inducing state 1) caused an upward shift. However, the light-induced downshift was strongly inhibited in NDH-deficient Arabidopsis mutants and the upward shift was cancelled in plants durably acclimated to high light, which can perform normal state transitions. Taken together, our results suggest that the peak temperature of the afterglow band is indicative of regulatory processes affecting electron donation to the PQ pool which could involve phosphorylation of NDH. The afterglow thermoluminescence band provides a new and simple tool to investigate the cyclic electron transfer pathways and to study their regulation in vivo.

Role of the oxidized secondary acceptor QB of photosystem II in the delayed 'afterglow' chlorophyll luminescence.

Leaf discs of dark-adapted tobacco plants were excited by 2 flashes and kept in darkness at 20 degrees C for various time periods, then thermoluminescence emission was recorded without freezing the sample. The B band at 30 degrees C decreased with a half-time t1/2 approximately 1 min and the AG band at 45 degrees C with a t1/2 approximately 5 min. This corresponds to the decay kinetics of S2/3 in PS II centres in the state S2/3 QB - (B band) or S2/3 QB. Assuming that the 45 degrees C band is an 'afterglow' emission originating from those centres with an oxidized QB on which an electron is back-transferred from stroma reductants through a pathway induced by warming, the theoretical ratio of the B and AG band was compared to that measured experimentally. After 2 or 3 flashes producing mainly S3, the intensity of AG band encompassed several fold that of the B band, because recombining S3 recreated S2 QB AG-emitting centres. In order to confirm that the AG band is governed by the heat-induced activation of a dark QB-reducing pathway rather than by PS II charge recombination, the AG emission was characterized in triazine-resistant Chenopodium album weed biotypes. In these mutants where the QB pocket is altered, the B band is strongly downshifted to 18 degrees C, compared to 32 degrees C in the wild type, whereas the AG band is only downshifted by 3 or 4 degrees C, demonstrating that S2/3 QB - is not the limiting step of the AG emission.

Cyclic electron flow around PSI monitored by afterglow luminescence in leaves of maize inbred lines (Zea mays L.): correlation with chilling tolerance.

Maize (Zea mays L.) inbred lines of contrasting chilling sensitivity (three tolerant, three sensitive lines) were acclimated to 280 mumol photons m(-2) s(-1) white light at a 17 degrees C sub-optimal temperature. They showed no symptoms of photoinhibition, despite slight changes in photosystem II (PSII) fluorescence and thermoluminescence properties in two tolerant lines. A luminescence "afterglow" emission [Bertsch and Azzi (1965) Biochim Biophys Acta 94:15-26], inducible by a far-red (FR) illumination of unfrozen leaf discs, was detected either as a bounce in decay kinetics at constant temperatures or as a sharp thermoluminescence afterglow band at about 45 degrees C, in dark-adapted leaves. This band reflects the induction by warming of an electron pathway from stromal reductants to plastoquinones and to the Q(B) secondary acceptor of PSII, resulting in a luminescence-emitting charge recombination in the fraction of centres that were initially in the S(2/3)Q(B) non-luminescent state. A 5-h exposure of plants to growth chamber light shifted this luminescence emission towards shorter times and lower temperatures for several hours in the three chilling-tolerant lines. This downshift was not observed, or only transiently, in the three sensitive lines. In darkness, the downshifted afterglow band relaxed within hours to resume its dark-adapted location, similar for all maize lines. A faster dark re-reduction of P700(+) oxidized by FR light (monitored by 820-nm absorbance) and an increase of photochemical energy storage under FR excitation (determined by photoacoustic spectroscopy) confirmed that a cyclic pathway induced by white actinic light remained activated for several hours in the tolerant maize lines.

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: study of redox mutants.

Cytochrome c(550) is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c(550) we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and -30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c(550) is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c(550) was also constructed. It showed a lowered affinity for Cl(-) and Ca(2+) as reported earlier for the cytochrome c(550)-less Synechocystis 6803 mutant, but it showed a shorter lived S(2)Q(B)(-) state, rather than a stabilized S(2) state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c(550).

Chlorophyll thermoluminescence of leaf discs: simple instruments and progress in signal interpretation open the way to new ecophysiological indicators.

Luminescence from photosynthetic material observed in darkness following illumination is a delayed fluorescence produced by a recombination of charge pairs stored in photosystem II, i.e. the back-reaction of photosynthetic charge separation. Thermoluminescence (TL) is a technique consisting of a rapid cooling followed by the progressive warming of a preilluminated sample to reveal the different types of charge pairs as successive emission bands, which are resolved better than the corresponding decay phases recorded at constant temperatures. Progress in thermoelectric Peltier elements and in compact light detectors made the development of simple, affordable and transportable instruments possible. These instruments take advantage of multifurcated light guides for combined TL, fluorescence and absorbance/reflectance measurements. Meanwhile, experiments on unfrozen leaf discs, with excitation by single turn-over flashes or far red light and infiltration by specific inhibitors/uncouplers, have led to a better understanding of in vivo TL signals. Much like chlorophyll fluorescence and in a complementary way, TL in the 0-60 degrees C temperature range not only informs on the state of photosystem II in leaf tissues and its possible alterations, but also gives a broader insight into the energetic state inside the chloroplast by probing (1) the light-induced or dark-stable thylakoid proton gradient through the protonation of the Mn oxygen-evolving complex, (2) the induction of cyclic/chlororespiratory electron flow towards the plastoquinone pool, (3) the [NADPH+ATP] assimilatory potential. By a different mechanism, warming above 60 degrees C without preillumination reveals chemiluminescence high temperature thermoluminescence (HTL) bands due to the radiative thermolysis of peroxides, which are indicators of oxidative stress in leaves.