Ulrich Schreiber

Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus.

Methods were developed to measure chlorophyll fluorescence yield of intact leaf tissue during heat treatment under varying conditions of light intensity and photosynthetic activity. Fluorescence yield of a dark-adapted leaf increases by 2- to 3-fold with an increase of temperature into the region where heat-damage occurs. The temperatures of the fluorescence transition correlate well with the temperatures where quantum yield of CO2 fixation is irreversibly depressed. Fluorescence-temperature (F-T) curves allow ranking of different species according to their heat sensitivity. Within a single species acclimation to different growth temperatures is reflected by shifts of the transition temperatures in the F-T curves. When F-T curves are recorded in the steady light states at increasing light intensities, substantial shifts (up to 6°C) of transition temperatures to higher values are observed. Quantum yield measurements of CO2 fixation confirm that hight-light conditions protect from heat-damage. It is suggested that chlorophyll acts as an intrinsic fluorescence probe of the thylakoid membrane and responds to the same changes which cause irreversible denaturation of photosynthetic enzymes.

Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heat-damage at the pigment level

The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed.

The ratio of variable to maximum chlorophyll fluorescence from photosystem II, measured in leaves at ambient temperature and at 77K, as an indicator of the photon yield of photosynthesis

The response of a number of species to high light levels was examined to determine whether chlorophyll fluorescence from photosystem (PS) II measured at ambient temperature could be used quantitatively to estimate the photon yield of O2 evolution. In many species, the ratio of the yield of the variable (FV) and the maximum chlorophyll fluorescence (FM) determined from leaves at ambient temperature matched that from leaves frozen to 77K when reductions in FV/FM and the photon yield resulted from exposure of leaves to high light levels under favorable temperatures and water status. Under conditions which were less favorable for photosynthesis, FV/FM at ambient temperature often matched the photon yield more closely than FV/FM measured at 77K. Exposure of leaves to high light levels in combination with water stress or chilling stress resulted in much greater reductions in the photon yield than in FV/FM (at both ambient temperature and 77K) measured in darkness, which would be expected if the site of inhibition was beyond PSII. Following chilling stress, FV/FM determined during measurement of the photon yield in the light was depressed to a degree more similar to that of the depression of photon yield, presumably as a result of regulation of PSII in response to greatly reduced electron flow.

Differences in the capacity for radiationless energy dissipation in the photochemical apparatus of green and blue-green algal lichens associated with differences in carotenoid composition

Green algal lichens, which were able to form zeaxanthin rapidly via the de-epoxidation of violaxanthin, exhibited a high capacity to dissipate excess excitation energy nonradiatively in the antenna chlorophyll as indicated by the development of strong nonphotochemical quenching of chlorophyll fluorescence (FM, the maximum yield of fluorescence induced by pulses of saturating light) and, to a lesser extent, FO (the yield of instantaneous fluorescence). Blue-green algal lichens which did not contain any zeaxanthin were incapable of such radiationless energy dissipation and were unable to maintain the acceptor of photosystem II in a low reduction state upon exposure to excessive photon flux densities (PFD). Furthermore, following treatment of the thalli with an inhibitor of the violaxanthin de-epoxidase, dithiothreitol, the response of green algal lichens to light became very similar to that of the blue-green algal lichens. Conversely, blue-green algal lichens which had accumulated some zeaxanthin following long-term exposure to higher PFDs exhibited a response to light which was intermediate between that of zeaxanthin-free blue-green algal lichens and zeaxanthin-containing green algal lichens. Zeaxanthin can apparently be formed in blue-green algal lichens (which lack the xanthophyll epoxides, i.e. violaxanthin and antheraxanthin) as part of the normal biosynthetic pathway which leads to a variety of oxygenated derivatives of β-carotene during exposure to high light over several days. We conclude that the pronounced difference in the capacity for photoprotective energy dissipation in the antenna chlorophyll between (zeaxanthin-containing0 green algal lichens and (zeaxanthin-free) blue-green algal lichens is related to the presence or absence of zeaxanthin, and that this difference can explain the greater susceptibility to high-light stress in lichens with blue-green phycobionts.

Inhibition of Energy-Transfer to Photosystem II in Lichens by Dehydration: Different Properties of Reversibility with Green and Blue-green Phycobionts

Summary Dry lichens with green algal phycobionts are able to recover net photosynthesis through rehydration with water vapor, whereas lichens with blue-green algae lack this ability (Lange, Kilian and Ziegler, Oecologia 71, 104–110, 1986). By measurements of 77 K fluorescence emission and excitation spectra, it is investigated whether this basic difference in rehydration properties of green and blue-green lichens is due to the different organization of the antenna pigments. Emission spectra obtained from a lichen with a blue-green alga ( Peltigera rufescens ) and from a free-living blue-green alga ( Nostoc cf. commune ) were essentially identical after normalization at 710 nm and revealed the following major features: Equilibration of a dry organism with air of 99 % r.h. resulted in a pronounced increase of fluorescence emission at 650 nm when excited at 580 nm (i.e. at maximal absorbance for the phycobilin pigments). Additional spraying with liquid water led to a strong fluorescence increase around 690 nm. With excitation of chlorophyll at 430 nm no such changes could be observed with both procedures. Excitation spectra for emission at 695 nm revealed that the efficiency for fluorescence excitation at 565 nm compared to that at 435 nm was minimal after equilibration at 99 % r.h., whereas spraying with liquid water resulted in a strong enhancement. It is concluded that desiccation induces a functional detachment of the phycobilisomes (PBS) from photosystem II (PS II). Energy transfer from PBS to PS II is restored only when rehydration occurs with liquid water. Similar experiments with the lichen Ramalina maciformis , which contains a green phycobiont ( Trebouxia spec.) yielded distinctly different responses. The desiccation induced lowering of fluorescence emission was almost totaly reversed by equilibration with air of 93 % r.h. Excitation spectra of 695 nm emission showed a large reduction of the emission ratio with 480 nm excitation over that with 435 nm induced by desiccation. Rehydration by increasing air humidity resulted in a gradual increase of this ratio until it was almost maximal at about 99 % r.h. It is concluded that with green algal symbionts desiccation induces a functional interruption of energy transfer between the light harvesting chl a/b pigment complex and PS II and that this can be largely restored by rehydration with humidified air, in contrast to the situation with blue-green algal symbionts.

Properties of ATP-driven reverse electron flow in chloroplasts☆

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.

Proton gradients as possible intermediary energy transducers during ATP-driven reverse electron flow in chloroplasts

After light activation of the latent adenosine triphosphatase (ATPase), addition of ATP in the dark drives reverse electron flow in isolated chloroplasts as evidenced by the oxidation of cytochrome f and the reduction of Q [ 1,2]. According to the chemiosmotic hypothesis [3] proton gradients should be obligatory energy transducing intermediates in this process. Indeed, transmembrane proton gradients were shown to be created during the action of the ATPase [4,5] and, when artificially Induced, drive reverse electron flow by themselves [6,7]. As obligatory intermediates, the buildup of proton gradients would be expected to be kinetically at least as fast as the inductlon of reverse electron flow under all experimental conditions. Thus, treatments which slow down the development of the transmembrane proton gradient would be expected to induce a corresponding slow-down in the development of the ATP-driven reverse electron flow. In this report we shall describe the construction of an apparatus which enabled us to simultaneously monitor the ATP-driven development of ApH and reduction of Q under a variety of conditions, and thus test the above prediction.

Properties of ATP-induced chlorophyll luminescence in chloroplasts.

1. The recently described reaction of ATP-induced luminescence is analyzed for its relation to other ATP-induced reactions such as ATP-driven transmembrane proton gradient formation and ATP-driven reverse electron flow. 2. In the absence of phenazine methosulfate ATP-induced luminescence is optimal while the main phase of ATP-driven reverse electron flow is eliminated. 3. DCMU which by itself causes a much smaller luminescence, inhibits the ATP-induced luminescence. 4. Nigericin plus valinomycin, but not each by itself, fully inhibit the ATP-induced luminescence. 5. The observations are interpreted as indicating that ATP stimulates luminescence by a 2-fold mechanism: (a) increasing the amount of the reducing primary electron acceptor of Photosystem II, Q, and (b) creating a transmembrane electrochemical potential which serves to decrease the activation energy required for the charge recombination reaction which leads to luminescence.

ATP-induced chlorophyll luminescence in isolated spinach chloroplasts.

Upon a light-dark transition chloroplasts emit post-illumination luminescence [l] , which is commonly considered to result from the recombination of positive charges (Z’) and negative charges (Q-) at the photosystem II reaction centers [ 1,2] . Stimulation of post-illumination luminescence by a variety of treatments has been reported (for a recent review, see [3]) such as transthylakoidal ApH [4], ApH-reduced, reverse electron flow [S] , salt addition [6], a rapid temperature increase {7], addition of DCMU [8] or dithionite [9]. These treatments have in common, that they either increase the supply of electrons at Q or provide an additional source of activation energy for the recombination reaction (see [31). After light-activation of the latent adenosine triphosphatase (ATPase), addition of ATP in the dark to isolated chloroplasts leads to formation of a transthylakoidal ApH (see [lo]) and reduction of Q by reverse electron flow [ 11,121. In this report we wish to describe a procedure by which ATP-induced luminescence can be readily