Prasanna Mohanty

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

Abstract We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane. 1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ f ) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg 2+ causes an increase in φ f . However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ f . The PMS triggered decrease in φ f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg 2+ and Ca 2+ show that there is a relative increase of System II to System I fluorescence. 2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg 2+ -induced increase of φ f , and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes. 3. In purified pigment System II particles, neither PMS nor Mg 2+ causes any change in φ f . Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg 2+ -induced increase of φ f . These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion ( k h ), whereas the Mg 2+ effect is due to a decrease in the rate of energy transfer ( k t ), between the two photosystems. 4. From the relative ratio of φ f with DCMU and DCMU plus Mg 2+ , we have calculated k t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10 8 s −1 , and φ t (quantum yield of this transfer) to be 0.12.

Action of hydroxylamine in the red alga Porphyridium cruentum

Abstract 1. 1. Oxygen exchange and fluorescence transient studies, made with the intact cells of red alga Porphyridium cruentum, suggest that hydroxylamine (NH2OH) inactivates the oxygen evolving capacity; at high enough concentrations, it also feeds electrons to the reductant side of Photosystem II. This result is in confirmity with the studies made earlier with spinach chloroplasts and green alga Chlorella by other workers. Fluorescence transient data further indicate that the maximal rate of feeding of electrons by this reductant to Photosystem II is not as high as by water. 2. 2. In Porphyridium, the early phase of the fluorescence transient is not markedly changed by NH2OH, but characteristic slow complex decay part of the fluorescence transient is completely abolished. However, the treated cells, like the normal cells, do need a dark exposure before subsequent illumination to fully restore the transient. NH2OH, at substrate concentrations, eliminates the recovery of the fluorescence rise curve (OI) in 3-(3′,4′-dichlorophenyl)-1,1 dimethyl urea (DCMU)-treated cells. Also NH2OH together with DCMU abolishes all delayed light emission measured after a msec of illumination. These observations indicate that NH2OH inhibits the chemical back recombination reaction between Z+ (the primary oxidant produced by light reaction II) and Q− (the primary reductant produced by light reaction II). These results, obtained with Porphyridium were obtained independently of Bennoun (1970) who reached similar conclusions from his studies with green alga Chlorella. 3. 3. Furthermore, the feeding of electrons by NH2OH appears to be very close to the reaction center of Photosystem II, as the photooxidation of NH2OH quenches the F696 band which has been suggested to be linked with the reaction center II.

Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans. I. Relationship of slow fluorescence changes with structural changes

Abstract 1. 1. Both normal and 3-(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned cells of the blue-green alga Anacystis nidulans show an extensive slow rise in the chlorophyll a fluorescence yield upon illumination (Papageorgiou and Govindjee, 1968). It was observed here that uncouplers of phosphorylation notably 5-chloro-3-(p-chlorophenyl)-4′-chlorosalicylanilide and 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide suppress this slow rise in the fluorescence yield both in normal and poisoned samples. In comparison to these salicylanilides, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone seems to be less effective in this respect. 2. 2. Emission spectra measured with the salicylanilides in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells show that the uncouplers prevent a shift in the excitation transfer in favor of pigment system II, i.e. the transformation to the so-called “State 1”. In other words, in the presence of the uncoupler, the pigment systems remain arrested in “State 2”. According to this view, the slow fluorescence rise represents a shift from State 2 to State 1. 3. 3. Furthermore, the uncouplers suppress as well the light-induced structural alterations (as measured by a change in absorbance at 540 nm) in both normal and poisoned Anacystis cells. Fixation of cells with aldehydes, that immobilizes the structural alterations, also abolishes slow fluorescence yield changes. 4. 4. It was observed that the time course of the light-induced macroscopic structural alteration was slower as compared to slow fluorescence yield changes. It, therefore, seems that the microscopic conformational organization, that precedes the macroscopic volume changes, induces the slow changes in fluorescence yield by shifting the mode of excitation energy transfer. 5. 5. In conclusion, we suggest that an energy-dependent specific alteration of the organization between the two photosystems controls and regulates the mode of excitation energy transfer between the two photosystems, the efficiency of such a transfer in Anacystis is approximately 10%.

Fluorescence and delayed light emission in Tris-washed chloroplasts

Yamashita and Butler [I] have shown that extraction of chloroplasts with a high concentration of Tris (0.8 M, pH 8.0) eliminates flow of electrons from water to nicotinamide adenine dinucleotide phosphate (NADP+)* or other Hill oxidants. They suggest that washing with Tris causes a block on the water side of the electron transport chain. Addition of electron donors such as reduced phenylene diamine (PDA) restores the electron flow to NADP’. This donation of electrons by PDA is sensitive to the herbicide diuron (DCMU), a well known inhibitor of photosystem II (PS II) reactions. Measurements of chlorophyll a (Chl) fluorescence in Tris-washed chloroplasts (without any added donor) show a very low level of fluorescence, but the addition of DCMU increases the yield of fluorescence [I]. One could suggest that the low yield of Chl fluorescence in Tris-washed chloroplasts is due to “trickling” of electron flow from some endogenous donor (cf. [ 11). However, if this were so, we should expect the fluorescence yield to slowly rise and reach the maximum yield at longer times. But, this never happens. No satisfactory explanation was available for this peculiar behavior of fluorescence

Inhibition of electron flow and energy transduction in isolated spinach chloroplasts by the herbicide dinoseb.

The mode of action of dinoseb (2-sec-butyl-4-6-dinitrophenol) on chloroplast reactions was studied. Electron flow from water or from an artificial electron donor, diphenylcarbazide (DPC), to dichlorophenol indophenol (DCPIP) was inhibited at low concentrations of the herbicide (5--10 microM) suggesting a site for dinoseb inhibition at the oxidizing side of photosystem II (PS II). Ferricyanide photoreduction was also inhibited by dinoseb. Cyclic and non-cyclic photophosphorylation and Mg2+-ATPase activity were inhibited by dinoseb, which indicates that this herbicide also acts as an energy transfer inhibitor. Among the above mentioned activities, non-cyclic photophosphorylation was the most sensitive to the inhibition by dinoseb. Ca2+-ATPase activity of solubilized heat activated chloroplast coupling factor 1 (CF1) was stimulated by dinoseb. However, the same activity was inhibited in chloroplasts, which perhaps reflect a difference in the mode of interaction of dinoseb with solubilized and membrane bound coupling factor.