Jean-Marc Ducruet

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

Membrane lipid peroxidation, cell viability and Photosystem II activity in the green alga Chlorella pyrenoidosa subjected to various stress conditions

Abstract The unicellular green alga Chlorella pyrenoidosa was subjected to a variety of stress conditions (strong illumination, incubation with Cu 1+ or Zn 2+ , exposure to high temperatures). The amplitude of thermoluminescence (TL) peak at 125°C, accumulation of thiobarbituric acid reactive substances (TBARS), which indicate an accumulation of lipid peroxidation products, efficiency of Photosystem II reactions ( F v / F M ratio) and the percentage of viable cells were measured in stressed culture. Exposure of algae to strong (5000 μmol photons m 2 s 1 ) or to low (60 μmol photons m −2 s −1 ) light combined with the addition of 1.6 μM Cu 2+ or 30 μM Zn 2+ inactivated Photosystem II, decreased the viability of Chlorella cells, and, finally, significantly enhanced TL and the accumulation of TBARS, which was accompanied by chlorophyll bleaching. TL emission started to rise after a lag-period of about 30 min in algae subjected to strong illumination, 2–3 h in copper-treated algae, and 10 h in zinc-treated algae. A vast majority of cells were nonviable to the end of the lag-period. The addition of Cu 2+ or ZN 2+ in darkness caused a slight decrase in the F v / F M ratio without significant changes in TL emission. Incubation of algae at 50°C for 10 min did not affect the F v / F M ratio nd cell viability, whereas no viable cells and Photosystem II activity were detected in the culture incubated at 55°C. Heat stress at temperatures above 55°C significantly enhanced the amplitude of the 125°C TL peak and the accumulation of TBARS when the algae were further incubated at low light at room temperature. We conclude that, under the stress conditions used in this study, (i) lipid peroxides and products of their degradation are not responsible for the cytolethal effect in Chlorella and (ii) lipid peroxidation arises mainly upon illumination of dead cells.

Graphical and numerical analysis of thermoluminescence and fluorescence F0 emission in photosynthetic material

A set-up for recording thermoluminescence emission together with the constant F0 fluorescence yield is described briefly. It is driven by a microcomputer through plugged-in cards.Practical aspects of the simulation of TL bands and of decomposition of complex TL signals are examined. A reproducible and linear temperature gradient and the use of photon counting for luminescence detection are important features for further analyzing the recorded signal. The simulation procedure used is a step-by-step calculation of the number of charge recombinations, which is then substracted from the number of remaining charge pairs able to produce luminescence. This procedure consists first of a graphical fitting, followed by a numerical minimization, with a maximum of five simulated components. The quality of the simulation is evaluated by the sum of squares of differences (signal-simulation), related to the signal area. Equivalent decomposition patterns may be found for the same recording and additional information is needed for interpretation of TL data. Averaging signals is feasible, provided that maximum temperatures Tm of averaged bands are sufficiently similar (±3°C). Simultaneous measurement of the antenna fluorescence yield F0, using an ultra-weak pulsed blue LED, gives an estimate of the luminescence yield. This has to be taken into account in the analysis of the Q band and of high temperature (>40°C) bands.The simulation parameters appear to be dependent on plant growth conditions. Quantitative analysis of thermoluminescence emission could be useful in the study of the effects of climatic factors on the photosynthetic apparatus in plants.

The Origin of115–130°C Thermoluminescence Bands in Chlorophyll‐Containing Material

High‐temperature thermoluminescence (TL) emitted in the temperature region from +50 to +150°C has been studied in a variety of chlorophyll‐containing samples that were allowed to dry during the TL measurement. Analysis of the recorded traces by a multicomponent‐fit‐ting procedure revealed the existence of up to three bands of nonphotosynthetic origin with peak positions at62–75,114–128 and151–157°C and apparent activation energies of 27.0‐28.8, 14.1‐15.4 and 22.1‐23.3 kcal/mol (the bands are denoted as HT1 HT2 and HT3, respectively). Low‐temperature treatment of leaves, incubation of algae in the presence of paraquat, exposure of algae or isolated thylakoids to a strong light, all conditions known to stimulate oxidative damage to membrane lipids, caused appearance of a small HT1, band and significant rise in the intensity of the HT2 band. The increase in the HT2 component correlated positively with accumulation of conjugated dienes and malondialdehyde in thylakoids illuminated with a strong light. Different quenchers of active oxygen species and scavengers of free radicals added to preilluminated thylakoids or thylakoid lipid extracts before the TL measurements, as well as injection of argon into the TL measuring chamber, caused no changes in the intensity of the HT2 emission. The HT2 band in the thylakoids increased strongly upon addition of linoleate peroxidized by hydroxyradicals generated in the Fenton reaction but remained unchanged if the linoleate was oxidized with the use of lipoxygenase. We suggest that the HT2 band arises due to thermal decomposition of lipid cyclic peroxides present in the samples. In turn, the decomposition reaction leads to formation of carbonyls in triplet state with following migration of excitation energy toward chlorophyll. Contrary to the HT1, and HT2 bands, the HT3 band of TL cannot be associated with the thermolysis of lipid peroxidation products already present in the samples before starting the TL gradient.

Comparative studies on electron transfer in Photosystem II of herbicide-resistant mutants from different organisms

Abstract We have studied the electron transfer properties of Photosystem II using several techniques (fluorescence, oxygen emission and thermoluminescence measurements) in a series of herbicide-resistant mutants from widely different organisms. Five mutants of Synechocystis 6714, of which we have determined the D1 sequence, one mutant of Synechococcus 7942, one mutant of Chlamydomonas reinhardtii , a triazine-resistant biotype of Chenopodium album and their herbicide-susceptible controls were analyzed. Two mutants have an almost unimpaired Photosystem II electron transfer. For five mutants of the different organisms, the initial phase of the electron transfer Q − A to Q B is unaltered but the electron transfer equilibrium between these two acceptors is displaced. In the Chlamydomonas -resistant mutant, the electron transfer from Q − A to Q B is slowed down.

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower QA− reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.

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 F0 and FM 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.

Lipid peroxidation in tobacco leaves treated with the elicitor cryptogein: evaluation by high-temperature thermoluminescence emission and chlorophyll fluorescence

Abstract Treatment of excised tobacco leaves with the fungal elicitor cryptogein progressively induced lipid peroxidation. In a first step, evidence was provided by the accumulation of thiobarbituric acid reactive substances (TBARS) and in a second step, the process was monitored for a 26 h period by high-temperature thermoluminescence (TL) emission, showing a close correlationship with the TBARS data. Differences in the temperature-associated F 0 rise (constant fluorescence) and in fluorescence emission spectra point to a progressive destabilization of the thylakoid membrane, especially affecting Photosystem II (PS II). In parallel, the PS II quantum efficiency ΔF / F m and the F v / F m ratio of chlorophyll fluorescence induction decreased significantly over the 24 h period.

Primary events occurring in photoinhibition in Synechocystis 6714 wild-type and an atrazine-resistant mutant

Abstract Exposure of photosynthetic organisms to a light intensity higher than that needed to saturate photosynthesis causes the inhibition of Photosystem II activity (photoinhibition). We induced photoinhibition in cells of Synechocystis 6714 strains by exposure to light intensities between 1000 and 4000 μE.m−2.s−1. Fluorescence, thermoluminescence and oxygen measurements were used to follow the inhibition of electron transfer in Photosystem II centers. We demonstrated that, in oxygen evolving Photosystem II centers, the electron transfer from QA to QB was not slowed down. Photoinhibited samples presented a normal oscillation pattern of oxygen yield. The rate of deactivation of S2 state was similar in control and photoinhibited cells. This indicates that the Q A Q B equilibrium is not modified in the centers which evolve oxygen. By studying the kinetics of the decrease of the amplitudes of the thermoluminescence Q and B bands in Synechocystis 6714 we confirmed our previous conclusions drawn from electron transfer measurements: the inhibition of electron transfer to QB is faster than the inhibition of electron transfer to QA. We conclude that a succession of two different states of Photosystem II centers is produced during the process of photoinhibition: (1) A state in which the electron transfer is inhibited between QA and QB. (2) an inactivated low fluorescent state where Q−A formation is also inhibited. Restoration of the photoactive fluorescent Photosystem II requires de novo synthesis of D1. Our main conclusion is that the first damage during photoinhibition in cyanobacteria cells is at the level of the QA to QB electron transfer step.

Cytochrome c550 in the Cyanobacterium Thermosynechococcus elongatus STUDY OF REDOX MUTANTS

Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 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 c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{S}_{2}Q_{B}^{-}\) \end{document} state, rather than a stabilized S2 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 c550.