Sándor Demeter

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

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

Low-pH-induced Ca2+ ion release in the water-splitting system is accompanied by a shift in the midpoint redox potential of the primary quinone acceptor QA

Abstract pH-dependent regulation of Photosystem (PS) II (observed as ‘high-energy quenching’) has been characterized by chlorophyll fluorescence and thermoluminescence measurements in PS II particles, thylakoid membranes, alga cells and leaf tissue. Steady state redox titration of fluorescence yield performed at pH 6.5 revealed that the midpoint redox potential of the primary quinone acceptor, Q A , is shifted towards positive direction from E m = −80 mV to E m = +40 mV (the absolute values for E m were varying by about 40 mV between different preparations) after incubation of PS II particles at pH 4.2 for 15 min in the presence of the Ca 2+ chelator, PP i . The original midpoint potential was restored after the addition of 300 μM CaCl 2 . Low-pH treatment (pH 4.6) of PS II particles also resulted in a decrease of the Q band of thermoluminescence (appearing between 10–14°C after DCMU addition) with a concomitant appearance or intensification of a high temperature band between 42–50°C (C band). In accordance with the results of the redox titration of fluorescence yield the C band is attributed to a low-pH-induced high potential form of Q A . The interconversion of Q band into the C band was more pronounced in the presence of the Ca 2+ chelator, EGTA. Addition of CaCl 2 to the low-pH-treated particles diminished the C band and restored the Q band. Light-induced acidification of the thylakoid lumen (ΔpH formation under illumination conditions of ‘high-energy quenching’) was also accompanied by a transformation of the Q band to the C band in isolated thylakoids, in the green alga, Chlorella vulgaris and in pea leaves. The phenomenon was completely reversed by abolishing the pH gradient with 10 mM NH 4 Cl. Addition of the Ca 2+ -channel inhibitor verapamil to the thylakoid suspension before the formation of a ΔpH suppressed the transformation of Q band into the C band. In contrast, when a ΔpH was first established and then verapamil was added, the ΔpH-induced change in the glow curve was irreversible and conversion of C band back to the Q band was prevented. It is suggested that the appearance of the C band is associated with Ca 2+ -dependent reversible inactivation of the water-splitting system and with a shift in the redox potential of Q A . We propose that pH-dependent Ca 2+ -release is a physiological process which controls the electron transport of PS II in vivo.

Triazine-resistant Nicotiana mutants from photomixotrophic cell cultures

SummaryTriazine-resistant mutants have been isolated in photomixotrophic cell cultures of Nicotiana plumbaginifolia. Triazine herbicides inhibit photosynthesis and cause extensive photodestruction of chloroplasts (bleaching) in sensitive plants. Selection was based on the greening ability of the resistant cells in the presence of 10-4 M terbutryn, under normal culture conditions, but in a medium containing a low sugar concentration. In the mutant plants, as compared to wild type, two to three orders of magnitude higher concentrations of triazines resulted in inhibition of photosynthetic electron transport and greening. The resistance was inherited maternally.

Short- and long-term redox regulation of photosynthetic light energy distribution and photosystem stoichiometry by acetate metabolism in the green alga, Chlamydobotrys stellata

The effect of acetate metabolism on the light energy distribution between the two photosystems, on the PS II/PS I stoichiometry and on the expression of psbA and psbB and psaA genes was investigated in the green alga, Chlamydobotrys stellata during autotrophic (CO2), mixotrophic (CO2 plus acetate) and photoheterotrophic (only acetate) cultivation. It was observed that acetate assimilation in the glyoxylate cycle resulted in a large drop in the ATP content and a concomitant increase in the NADPH content of the cells. The combined effect of high NADPH concentration and linear electron transport brought about an over-reduction of the inter-photosystem electron transport components. The reduced state of the inter-photosystem components initiated a state 1/state 2 transition of LHC II and a decrease in the PS II/PS I ratio. The PS II/ PS I ratio was reduced because the synthesis of PS II reaction centers was repressed and that of the PS I reaction centers was slightly enhanced by acetate cultivation. The amount of PsbA and PsbB proteins of PS II and the abundance of psbA mRNA decreased. The abundance of PS I PsaA protein and psaAmRNA were only slightly increased. All of the acetate-induced effects were reversible when the cells were transferred back to an acetate-free medium. Our observations demonstrate that the expression of the PS II psbA and psbB and PS I psaA genes is regulated by the redox state of the inter-photosystem components at the transcriptional level. Experiments carried out in the presence of DBMIB which facilitates the reduction of plastoquinone pool indicate that the expression of genes encoding the components of PS II and PS I are controlled by the redox state of a component (cytochrome b/f complex) located behind the plastoquinone pool.

Effects of in vivo CO2-depletion on electron transport and photoinhibition in the green algae, Chlamydobotrys stellata and Chlamydomonas reinhardtii

Abstract Short-term illumination of the green algae, Chlamydobotrys stellata and Chlamydomonas reinhardtii in CO2-depleted cultivation medium under low photon flux density (50 and 150 μmol m−2 s−1, respectively) resulted in an inhibition of Photosystem II electron transport from water to diaminodurene, but only slightly affected the electron flow from water to 2,6-dichlorobenzoquinone. The intermediary fluorescence level, Fi was raised to the maximum level of fluorescence, Fm. The initial level of fluorescence, Fo was considerably enhanced. The development of the Fo rise was facilitated by low pH, but inhibited in the presence of an acceptor, dichlorobenzoquinone, or by chemical cross-linking of proteins with glutaraldehyde. The uninhibited electron transport and the original Fo level were restored by readdition of CO2 or by dark adaptation of algae. The observations suggest that in green alga cells CO2-depletion in the light results in a reversible inhibition of steady-state electron flow between the primary (QA) and secondary quinone electron acceptor (QB). Following the inhibition of electron transport a long-lived but reversible state of singly-reduced and probably protonated QA is formed which manifests itself as an apparent Fo rise. Prolonged photoinhibitory illumination of the CO2-depleted green alga cells resulted in an irreversible loss of variable fluorescence and electron transport. The photoinactivation developed more slowly in the CO2-depleted than in the CO2-containing cells. It is concluded that in the bicarbonate-depleted redox state, which is accompanied with an enhanced Fo level of fluorescence, the Photosystem II reaction center is less susceptible to photoinhibition than in the bicarbonate-containing state.

Thermoluminescence characteristics of granal and agranal chloroplasts of maize

Materials illuminated at temperatures below 0°C emit light upon heating. This phenomenon is called thermoluminescence and was first observed with chloroplasts by Arnold and Sherwood [ 11. The emission bands of thermoluminescence are a result of charge recombination between the various positively charged electron donors and negatively charged electron acceptors of the photosystems [2-41. According to most authors thermoluminescence originates only from photosystem II [5-71 but Sane et al. [2] as well as Shuvalov and Litvin [S] attributed some of the glow peaks to photosystem I. Inoue and Shibata [3,9] demonstrated that the emission of the bands at +25”C and +4O”C is linked to the positively charged S states of the water-splitting enzyme. Few data, however, are available on the location of negative charges participating in the charge recombination [2-41. The aim of the present study was to obtain further information about the involvement of photosystem I in thermoluminescence and to correlate the main bands of the glow curve with the different components of the electron transport chain.

Classification of Photosystem II inhibitors by thermodynamic characterization of the thermoluminescence of inhibitor-treated chloroplasts

Computer-assisted curve resolution was used to analyse the thermoluminescence of chloroplasts treated by different inhibitors in order to determine the free energy of activation, ΔF, activation energy, ΔE, and activation entropy, ΔS, of the radiative charge-recombination reaction. It was found that the activation energies and entropies related to the different inhibitor treatments of chloroplasts exhibit a compensation relationship. On the basis of the values of the activation parameters ΔE and ΔS, the Photosystem II inhibitors, which block electron transport between the primary acceptor Q and plastoquinone, could be classified into two main groups.

Macromolecular organization of chlorophyll a in aggregated chlorophyll ab protein complex as shown by circular dichroism at room and cryogenic temperatures

This report concerns the circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll a/b complex at 25 degrees C and -250 degrees C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.

EFFECT OF INACTIVATION OF THE OXYGEN‐EVOLVING SYSTEM ON THE THERMOLUMINESCENCE OF ISOLATED CHLOROPLASTS

Abstract— The effect of inactivation of water‐splitting enzyme on the thermoluminescence of isolated chloroplasts was investigated. The inhibitory treatments used included Tris‐washing, alkaline pH in the presence of the uncoupler gramicidin, incubation with a high concentration of magnesium ions and different chaotropic agents. It was found that inhibition of oxygen evolution resulted in the disappearance of the main thermoluminescence band at +20°C. The A band which appears at — 10°C and has been related to the S4 state of the water‐splitting enzyme (Inoue, 1981) was not considerably affected by the inhibition of oxygen evolution. The results presented here indicate that the participation of the S4 state of water‐splitting enzyme in the generation of the A band should be reinvestigated.

Photoinhibition of Electron Transport Activity of Photosystem II in Isolated Thylakoids Studied by Thermoluminescence and Delayed Luminescence

für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution 4.0 International License. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung 4.0 Lizenz. Photoinhibition of Electron Transport Activity of Photosystem II in Isolated Thylakoids Studied by Thermoluminescence and Delayed Luminescence