Haim Hardt

Kinetic characterization of T-jump thermoluminescence in isolated chloroplasts

Abstract Luminescence triggered by temperature jump in lettuce chloroplasts was measured at different final temperatures in the range of 30–70 °C. The initial temperature was 20 °C. This type of luminescence is suggested to result from a reaction of recombination of precursors formed during the preillumination period. The rate constant for this second order reaction was derived and shown to be proportional to the ratio between the luminescence peak and the total luminescence integral. An Arrhenius plot for these rate constants yields the value of 0.68 ± 0.08 eV for the activation energy. This value is compared with those reported by other investigators and a mechanism in terms of the semi conductor concepts is proposed for luminescence.

Acid—base driven reverse electron flow in isolated chloroplasts

ATP driven reverse electron flow in isolated lettuce chloroplasts has been recently demonstrated in our laboratory [ 1,2] . Upon addition of ATP, the light triggered Mg” dependent ATPase activity of chloroplasts drives both the reduction of Q, the primary acceptor of photosystem II, and the oxidation of cytochrome f. The reduction of Q was measured as the rise in chlorophyll fluorescence yield at low intensity monitoring light . The chemiosmotic theory [3] predicts that ATPase can affect the electron transport only through the formation of a transmembrane proton gradient. Rather large proton gradients were indeed demonstrated in chloroplasts to be induced by either electron transport [4] or light triggered ATPase [5]. ATP driven reverse electron flow [ 1,2] would thus be viewed as effected via the pH gradient formed by the ATPase activity. By this mechanism, it should be possible to drive reverse electron flow directly by an artificially induced pH gradient. Such a phenomenon was indeed observed and is reported on in the present communication.

ATP driven reverse electron transport in chloroplasts

The light triggered thiol dependent ATPase of isolated chloroplasts [I] has been considered to operate through the reversal of the latter stages of ATP formation during photophosphorylation. This view has been supported by the observations that an ATP-Pi exchange reaction accompanied the appearance of light triggered ATPase and could be considered a partial reaction of the ATPase [2, 31. More recently [4] it has been shown that in the dark the hydrolysis of ATP by the light triggered ATPase will cause an uptake by chloroplasts of protons from the medium, leading to the formation of a proton concentration gradient [5]. We wish to report that isolated lettuce chloroplasts, treated so as to show light triggered ATPase activity, exhibit an ATP dependent enhancement of chlorophyll fluorescence which appears to be the consequence of reversed electron flow in the chloroplast driven by ATP hydrolysis. A site for ATP formation in chloroplasts has been located between photosystem II and cytochrome f [6, 71. If reversed electrom flow driven by ATP occurs it would be expected to lead to some reduction of components of the electron transport chain close to Photosystem II. Reduction of Q, the hypothetical

Circular polarization of fluorescence of chlorophyll in solution and in native structures

Chlorophyll dimers in solution, subchlorplast particles and chloroplasts were investigated by their circular dichroism and circular polarization of their fluorescence, which reflect their optical rotatory power in the ground state and electronically excited state, respectively. The chlorophyll dimers in fluid solution lose their optical activity upon electronic excitation, reflecting a marked concomitant change in the structure of the dimers. This change is arrested in a solution of very high viscosity. The pronounced difference between the circular polarization of the dimers in fluid media and that of subchloroplast particles and chloroplasts indicates that the former are not suitable models for associated chlorophyll in native structures in electronically excited states. Impairment of the photochemical activity of chloroplasts by heat treatment is accompanied by a reduction of the circular polarization of the fluorescence, which probably reflects a disorganization in structure. The same extent of circular polarization was observed in the fluorescence of chloroplasts regardless whether the reaction centers are open or closed; thus either the same molecules are emitting in the two cases or, if different molecules emit, they are packed in a similar way.

OSCILLATIONS OF THE TRIGGERED LUMINESCENCES OF ISOLATED CHLOROPLASTS PREILLUMINATED BY SHORT FLASHES

Abstract— Four types of triggered luminescence of isolated lettuce chloroplast (HCl‐induced, methanol‐induced, sodium benzoate‐induced and T‐jump‐induced) were examined after preillumination by a series (from 1 to 10) of short flashes. Oscillations were observed in the luminescence peaks, with a period of four flashes. These oscillations had maxima after the second and the sixth flash, similar to those of delayed light emission. The maxima were shifted forward two flashes by 50 μM hydroxylamine, as in oxygen evolution, and were abolished by 5 μM DCMU, as for delayed light. These results may show that the mechanism of triggered luminescence is influenced directly by the oxidation states (S1) on the donor side of photo‐system II.

KINETIC STUDIES ON CHEMILUMINESCENCE OF CHLOROPLASTS INDUCED BY CHANGES IN ION CONCENTRATION

Abstract— Acid‐base, acid‐, and salt‐induced chemiluminescences of chloroplasts were investigated in relation to their dependences upon several time parameters:

Luminescence of isolated chloroplasts induced by organic solvents

Abstract Preilluminated lettuce chloroplasts emit light when injected with pure organic solvents. A positive linear correlation was found between the luminescence intensity and the dielectric constant of the solvent. It is suggested that the dissolution of the solvent in the chloroplast membrane increases its dielectric constant. This modifies the energy levels of charged trapped photoproducts and enables their recombination leading to luminescence.

Enhancement of luminiscence in chloroplasts by electron transport inhibitors

Summary DCMU, NQNO and BDHB caused pronounced enhancement of the triggered luminiscences and of the delayed light in chloroplasts. The enhancement induced by DCMU was higher at lower levels and at shorter periods of the preillumination and decayed during the few seconds of the dark period after the preillumination. These findings support the possibility that the enhancement is caused by an increase of the emission yield from the excited state, as shown by the parallel enhancement of the variable fluorescence. However, a reservation to this explanation is the absence of emission enhancement at short exposures of high preillumination intensity, in difference from the variable fluorescence that shows considerable enhancement.

Effect of uncouplers and ADRY reagents on delayed and triggered emission from isolated chloroplasts

is related to the abolishment by uncouplers of transmembrane potential and pH gradients which enhance delayed luminescence considerably [3,6,7]. The situation with respect to delayed luminescence in longer time region (> 100 msec) and triggered luminescence is much less certain. Mayne and Clayton reported that acid-base triggered luminescence was inhibited by uncouplers [S] . In our lab we noticed previously [9] that in the case of solvent triggered luminescence the only uncoupler that inhibited this luminescence was FCCP (or CCCP). Several authors noticed the inhibitory effect of some uncouplers on delayed and triggered light. It is known that some uncouplers affect also the precursors of oxygen evolution, besides their effect as uncouplers of phosphorylation. These reagents were termed Accelerating of the Deactivation of the Reaction of the water splitting enzyme Y (ADRY) reagents. Their effect was shown by the decrease of the yield of oxygen evolution excited by flashes at longer time intervals between the flashes [lo] ; more specifically they accelerate the deactivation of higher S-states [ 1 l] .

Emission spectra of the triggered luminescences in isolated chloroplasts obtained in a flow apparatus

Since the discovery of the ability of chloroplasts to emit a pulse of luminescence as a result of an acid -base transition (Mayne and Clayton [ 1 ] ) extensive studies were carried out in several laboratories, including ours, on the subject of triggered luminescence. These led to the discoveries of a series of different treatments which can induce preilluminated chloroplasts to emit a pulse of luminescence in a similar manner. For reviews on these studies, cf. that by Mayne and Fleischman [2] and that by Lavorel [3]. The emission spectra of some luminescences were either not measured at all or measured only roughly. In the two cases where a spectrum was reported (acid -base type as shown by Mayne and Clayton [1] ; acid and salt induced types as described by Miles and Jagendorf, [4] ) these spectra were obtained point by point by measuring separately the emission peak at each wavelength. The wavelength setting was chan, ged either by use of interference filters or by a monochromator. From our experience, the scattering of results obtained in this way is quite considerable. The purpose of the present work was to construct a flow apparatus capable of measuring continuously the spectra of the triggered emissions from chloroplasts suspensions. We present below the spectra of the luminescences induced by HC1, by sodium benzoate, by methanol, by dimethyl-sulfoxide, and by T-jump.