Compared thermoluminescence characteristics of pea thylakoids studied in vitro and in situ (in leaves). The effect of photoinhibitory treatments

Characteristics of thermoluminescence (TL) glow curves were studied in thylakoids (isolated from pea leaves) or in intact pea leaves after an exposure to very high light for 2 min in the TL device. The inhibition of photosynthesis was detected as decreases of oxygen evolution rates and/or of variable fluorescence.In thylakoids exposed to high light, then dark adapted for 5 min, a flash regime induced TL glow curves which can be interpreted as corresponding to special B bands since: 1) they can be fitted by a single B band (leaving a residual band at –5°C) with a lower activation energy and a shift of the peak maximum by –5 to –6°C and, 2) the pattern of oscillation of their amplitudes was normal with a period of 4 and maxima on flashes 2 and 6. During a 1 h dark adaptation, no recovery of PS II activity occurred but the shift of the peak maximum was decreased to –1 to –2°C, while the activation energy of B bands increased. It is supposed that centers which remained active after the photoinhibitory treatment were subjected to reversible and probably conformational changes.Conversely, in intact leaves exposed to high light and kept only some minutes in the dark, TL bands induced by a flash regime were composite and could be deconvoluted into a special B band peaking near 30°C and a complex band with maximum at 2–5°C. In the case of charging bands by one flash, this low temperature band was largely decreased in size after a 10 min dark adaptation period; parallely, an increase of the B band type component appeared. Whatever was the flash number, bands at 2–5°C were suppressed by a short far red illumination given during the dark adaptation period and only remained a main band a 20°C; therefore, the origin of the low temperature band was tentatively ascribed to recombinations in centers blocked in state S₂Q_A ^–Q_B ^2–. In vivo, the recovery of a moderately reduced state in the PQ pool, after an illumination, would be slow and under the dependence of a poising mechanism, probably involving an electron transfer between cytosol and chloroplasts or the so-called chlororespiration process.Abbreviations E_a- activation energy - FR- far-red - MV- methylviologen - pBQ- p-benzoquinone - PQ- plastoquinone - PS II- Photosystem II - Q_A- primary quinone electron acceptor of PS II - Q_B- secondary quinone electron acceptor of PS II - TL- thermoluminescence     

Thermoluminescence Investigation of Low Temperature Stress in Maize

The thermoluminescence (TL) emission of photosynthesising materials originates from the recombination of charge pairs created by a previous excitation. Using a recently described TL set-up the effect of chilling stress on TL bands occurring at positive temperatures (AG, B, and HTL) was investigated in intact leaves. The far-red irradiation of leaves at low, but non-freezing temperatures induced a TL band peaking at around 40–45 °C (AG band), together with a B band peaking between 20 and 35 °C. Low temperature stress first caused a downshift and a temporary increase in the AG band after 4 h at 0 °C in the light, then a decrease in the AG and B TL bands after 1 d at 0 °C in the light. This decrease was less pronounced in cold-tolerant genotypes and in those grown at acclimating temperatures. Furthermore, an additional band appeared above 80 °C after severe cold stress. This band indicates the presence of lipid peroxides. Thus TL is a useful technique for studying the effects of low temperature stress.     

Oxygen-evolving system and secondary quinonic acceptors are highly reduced in dark adapted Euglena cells: A thermoluminescence study

Characteristics of thermoluminescence glow curves were compared in three types of Euglena cells: (i) strictly autotrophic, Cramer and Myers cells; (ii) photoheterotrophic cells sampled from an exponentially growing culture containing lactate as substrate repressing the photosynthetic activity; (iii) semiautotrophic cells, sampled when the lactate being totally exhausted, the photosynthesis was enhanced.In autotrophic and semiautotrophic cells, composite curves were observed after series of two or more actinic flashes fired at –10°C, which can be deconvoluted into a large band peaking in the range 12–22°C and a smaller one near 40°C, This second band presents the characteristics of a typical B band (due to S_2/3Q_B ^- recombination), whereas the first one resembled the band, shifted by -15–20°C, which is observed in herbicide resistant plants. The amplitude of this major band, which was in all cases very low after one flash, exhibited oscillations of period four but rapidly damping, with maxima after two and six flashes. In contrast, photoheterotrophic Euglena displayed single, non-oscillating curves with maxima in the range 5–10°C.In autotrophic and semiautotrophic cells, oxidizing pretreatments by either a preillumination with one or more (up to twenty-five) flashes, or a far-red preillumination in the presence of methylviologen, followed by a short dark period, induced thermoluminescence bands almost single and shifted by +3–5°C, or +12°C, respectively. In autotrophic cells, far-red light plus methyl viologen treatment induced a band peaking at 31°C, as in isolated thylakoids from Euglena or higher plants, while it had barely any effect in photoheterotrophic cells.Due to metabolic activities in dark-adapted cells, a reduction of redox groups at the donor and acceptor sides of PS II dark-adapted cells is supposed to occur. Two different explanations can be proposed to explain such a shift in the position of the main band in dark-adapted autotrophic control. The first explanation would be that in these reducing conditions a decreasing value of the equilibrium constant for the reaction: S_nQ_A ^-Q_BS_nQ_AQ_B ^-, would determine the shift of the main TL band towards low temperatures, as observed in herbicide resistant material. The second explanation would be that the main band would correspond to peak III already observed in vivo and assigned to S_2/3Q_B ^2- recombinations.Abbreviations CM Cramer and Myers - D₁ a 32 kDa protein component of the PS II reaction center, psbA.gene product - D₂ a 34 kDa protein component of the PS II reaction center, psbD gene product - FR lar-red illumination - Lexpo and Lstat cells from lactate culture samples at exponential and stationary phase of growth - MV methylviologen - pBQ parabenzoquinone - PQ plastoquinone - PS II photosystem II - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - TL thermoluminescence     

Effects of dark- and light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands

Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S₂ and S₃ states of the oxygen evolving complex with Q_A ^- and Q_B ^-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S₂Q_A ^- (Q band), S₂Q_B ^- and S₃Q_B ^- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S₃Q_B ^- than for S₂Q_B ^- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S₃S₂ and S₂S₁ reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA 9-aminoacridine - E_A activation energy - F₀ constant fluorescence level - F_M maximum fluorescence, when all PS-II centers are closed - F_V variable fluorescence (F_M–F₀) - PS I, PS II Photosystem I, photosystem II - PQ plastoquinone - TL thermoluminescence     

Thermoluminescence study of the in vivo effects of bicarbonate depletion and acetate/formate presence in the two algae Chlamydobotrys stellata and Chlamydomonas reinhardtii

We investigated the influence of CO₂/HCO₃ ^–-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae Chlamydobotrys stellata and Chlamydomonas reinhardtii by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO₂ from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO₂-deficient Chl. stellata with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of Chl. reinhardtii DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO₂-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO₂-depletion and of the presence of anions were fully reversible.Abbreviations DCMU 3-(3,4)-dichlorophenyl-1,1-dimethylurea - HT band high temperature TL band - P₆₈₀ reaction center chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively - PS II Photosystem II - S_2/3 redox states of the oxygen evolving complex of PS II - TL thermoluminescence     

Afterglow thermoluminescence band as a possible early indicator of changes in the photosynthetic electron transport in leaves

The afterglow (AG) band of thermoluminescence (TL) has been investigated in leaves of Arabidopsis thaliana. Excitation of dark-adapted leaves with two saturating single turn-over flashes induced the appearance of a complex TL glow curve that could be well simulated by three components: the two components, B₁ and B₂, of the usually called B-band, peaking at 18 and 26 °C, respectively, and a band with t_max at 41 °C, which we attributed to an AG emission. Illumination of dark-adapted leaves with 720 nm monochromatic and FR lights generated the emission of a sharp single band peaking also around at 41 °C, that it is usually assigned to an AG emission band. Dark-incubation of whole plants increased the intensity of AG-band in TL curves induced by two flashes and, in parallel, decreased B-bands. Selective illumination of leaves with light mostly absorbed by PS II (650 nm light) completely abolished the AG-band induced by two flashes, B-band being the only TL band observed. The single AG-band induced by 720 nm light was abolished if leaves were also illuminated with 650 nm light. On the other hand, AG-band could be restored if 650 nm illuminated leaves were afterwards illuminated with 720 nm light. The changes in the intensity of B and AG bands induced by selective illuminations seem to be related to alterations in the redox state of Q_B and plastoquinone pool.     

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 S_2/3Q _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 A_T, 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 characteristics of sodium chloride salt‐stressed Indian mustard seedlings

The thermoluminescence (TL) parameters in the intact leaves and the thylakoids isolated from leaves of NaCl treated seedlings showed different patterns of change. NaCl treatment brings about a destabilization of Q_A and Q_B, leading to a decrease in Q and B bands in the leaves. However, the Q and B band intensity of isolated thylakoids increased in NaCl‐treated seedlings. The differences in the TL intensities are described as the action of NaCl on the density of quinones per unit leaf area and on chlorophyll units in isolated thylakoids. Copyright © 2002 John Wiley & Sons, Ltd.     

Light‐independent thermoluminescence from thylakoids of greening barley leaves. Evidence for involvement of oxygen radicals and free chlorophyll

A study was conducted to identify biophysical markers which change in response to changing chlorophyll organization during plant development. When heated to around 70°C in the dark, barley thylakoids emit red thermoluminescence (TL). This is a pure chemiluminescence signal and distinct from the lower-temperature TL bands of thylakoids that are seen only with preillumination. The development of the light-independent, 70°C TL band was investigated following transfer of dark-grown barley leaves to the light. Because of the rapidly increasing chlorophyll content of the plastid membrane, the TL signal was normalized against either chlorophyll or tissue mass of the starting material. In either case, the extent of the TL signal reached a maximum in the early hours of greening and then declined. The drop in signal over 20 h was approximately 50% for TL per unit tissue mass, and well over 90% for TL per unit chlorophyll. Exposure of plastid membrane samples to hydrogen peroxide for several minutes caused a large increase in light-independent TL, while addition of ascorbate caused substantial quenching. The fluorescence profiles of dark-grown barley leaves were recorded following transfer to the light. Basal fluorescence (F₀) reaches a substantial level after just seconds of illumination. Over the next few hours, F₀ increases only slightly and then starts to decline. The decline in F₀ is correlated with an increase in variable fluorescence (F_v) which indicates the appearance of active photosystem II. It is concluded that the early peak in F₀ reflects a state in which the leaves contain a maximum amount of disorganized chlorophyll. Considering the TL and fluorescence data together, we propose the following: When chlorophyll first appears in the system, it is not properly assembled into the complexes that offer photochemical or non-photochemical quenching of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen resulting in lipid peroxidation and/or other radical-generating processes. When the membranes are heated, increased interaction of the radicals with chlorophyll generates chemiluminescence. We thus conclude that light-independent thermoluminescence is a marker for actual damage arising from poor chlorophyll organization and propose that this parameter might be usefully applied for assessing stress effects.     

Thermoluminescence properties of the isolated photosystem two reaction centre

Formation of thermoluminescence signals is characteristics of energy- and charge storage in Photosystem II. In isolated D1/D2/cytochrome b-559 Photosystem II reaction centre preparation four thermoluminescence components were found. These appear at -180 (Z band), between -80 and -50 (Z_v band), at -30 and at +35°C. The Z band arises from pigment molecules but not correlated with photosynthetic activity. The Z_v and -30°C bands arise from the recombination of charge pairs stabilized in the Photosystem II reaction centre complex. The +35°C band probably corresponds to the artefact glow peak resulting from a pigment-protein-detergent interaction in subchloroplast preparations (Rózsa Zs, Droppa M and Horváth G (1989) Biochim Biophys Acta 973, 350–353).Abbreviations Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - D1 psbA gene product - D2 psbD gene product - P680 primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - RC reaction centre of PS II - TL thermoluminescence     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermoluminescence as a probe of Photosystem II in intact leaves: Non-photochemical fluorescence quenching in peas grown in an intermittent light regime

We have measured thermoluminescence (TL) and chlorophyll fluorescence from leaves of peas grown under an intermittent light regime (IML) and followed changes in those leaves during greening. IML peas show low variable fluorescence and a certain capacity for reversible non-photochemical quenching. It has been suggested that reversible quenching may be caused by pH-dependent release of Ca²⁺ from Photosystem II (PS II) (Krieger and Weis (1992) Photosynthetica 27: 89–98). Under conditions in which reversible non-photochemical quenching occurs, a TL band at around 50 °C is observed, in the presence of DCMU, in IML leaves. A band in this temperature range has previously been observed in PS II depleted of Ca²⁺ (Ono and Inoue (1989) Biochimica et Biophysica Acta 973: 443–449). The 50 °C band disappears upon dark adaptation. In mature leaves, no significant band is seen at 50 °C. It is concluded that, in IML leaves, reversible quenching may be related to the release of Ca²⁺ from Photosystem II. However, it seems that in the mature system, under most conditions, such release does not contribute significantly to quenching     

The mechanism of the ozone-induced changes in thermoluminescence glow curves of barley leaves

The changes in thermoluminescence (TL) signals induced by short-term ozone exposure of leaves are characterized by a down-shift of the peak-temperature of the TLB-band and an increase of a TL band at 55°C. We investigated the relationship of these changes to photosystem 2 (PS2) photochemistry. The changes were not only detectable in the presence of ozone, but also after irradiation of dark-adapted leaves and after aging of irradiated detached leaf segments. The opposite effect on TL, an up-shift of the peak-temperature of the B-band and the decrease of the intensity of the band at 55°C were found after infiltration of leaves with nigericin, antimycin A, and diphenyleneiodonium chloride (DPI). Propyl gallate down-shifted the peak-temperature of the B-band. 2,5-dimethyl-1,4-benzoquinone up-shifted the peak-temperature of the B-band and decreased the intensity of the 55°C band. The intensity of the 55°C band did not change significantly in the presence of oxygen in comparison to that in nitrogen atmosphere. It decreased with time of dark adaptation (50% intensity was observed after 3 h of dark adaptation at room temperature), however, it was reactivated to its initial value (at 5 min of dark adaptation) after 1 single-turnover flash. The 55°C band was not significantly changed in the presence of DCMU. Thus the ozone-induced band at 55°C is assigned to charge recombination in PS2. Changes in the electron transport chain at the acceptor side of PS2, probably related to the cyclic electron transport around photosystem 1 and/or chlororespiration, could play an important role in the increase of the 55°C band and the down-shift of the B-band. The changes at the acceptor side indicated by TL can be an ex pression of a physiological regulatory mechanism functional under stress conditions.     

Role of the oxidized secondary acceptor Q B 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 °C for various time periods, then thermoluminescence emission was recorded without freezing the sample. The B band at 30 °C decreased with a half-time t_1/2~1 min and the AG band at 45 °C with a t_1/2~5 min. This corresponds to the decay kinetics of S_2/3 in PS II centres in the state S_2/3 Q_B^- (B band) or S_2/3 Q_B. Assuming that the 45 °C band is an ‘afterglow’ emission originating from those centres with an oxidized Q_B 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 Q_B AG-emitting centres. In order to confirm that the AG band is governed by the heat-induced activation of a dark Q_B-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 Q_B pocket is altered, the B band is strongly downshifted to 18 °C, compared to 32 °C in the wild type, whereas the AG band is only downshifted by 3 or 4 °C, demonstrating that S_2/3 Q_B^- is not the limiting step of the AG emission.     

Effects of High Temperature Exposure of Spinach Intact Plants and Isolated Thylakoids on Light-Harvesting Complex 2 Protein Phosphorylation

After a 6 min exposure of isolated thylakoids to 43 °C, the extent of phosphorylation of light-harvesting complex of photosystem 2 (LHC2) was higher than in control thylakoids kept at 25 °C. Similarly, the exposure of intact spinach plants to 43 °C in dark for 11 h induced higher extent of thylakoid LHC2 phosphorylation than in control plants kept at 25 °C. The induced ability of LHC2 for enhanced phosphorylation may enable better energy distribution in favour of photosystem 1.     

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S₂Q_B ^- (B band) and S₂Q_A ^– (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of Q_A ^- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block Q_A to Q_B electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced Q_B. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the Q_B/DCMU binding site. The preferential loss of dark stable Q_B ^- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.Abbreviations Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A first quinone electron acceptor of PS II - Q_B second quinone electron acceptor of PS II - Tyr-D accessory electron donor of PS II - S₀-S₄ charge storage states of the water-oxidizing complex     

Isolation of chestnut chloroplasts: membrane potentials of chestnut and spinach thylakoids

Typical chestnut thylakoid extracts isolated by mechanical disruption of leaf tissues had an equivalent of 0.28 kg m⁻³ chlorophyll (Chl) which is six times less than in thylakoids obtained from spinach, although Chl content in leaves was only half as small. According to optical microscopy, the vesicles showed a good integrity, exhibiting at 21 °C a high capacity of photon-induced potential membrane generation, which was demonstrated by the almost full 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in a hyper-saline medium containing 150 mM KCl and having osmotic potential of −1.5 MPa. The half-time of the thylakoid potential generation was 11.7 s with the time of dissipation around 8.9 s. In such conditions, spinach thylakoids showed an increased swelling and also differences in the half-time generation which was almost four times faster than was observed in chestnut. However, when spinach thylakoids were incubated in a typical hypo-saline medium without KCl with osmotic potential −0.8 MPa, no additional swelling was observed. Consequently the half-time of potential dissipation was 35 s. Studies with nigericin suggested a chestnut thylakoid ΔpH significantly smaller than that observed in spinach, which was confirmed by the measurements of the ATP driven pumping activity.     

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.