Illumination does not affect the membrane potential or the plasma membrane H+-ATPase activity in Micrasterias

The membrane potential (MP) of the unicellular green alga Micrasterias torreyi was found to be −46 to −47 mV (when cultured in Waris medium). In contrast to plant cells in general, light-dark changes neither affected the potential or the membrane resistance in Micrasterias . In comparison, the freshwater plant Elodea showed a light-induced hyperpolarization due to the activating effect of light on the plasma membrane adenosine triphosphatases (PM ATPases) through a signal from chloroplasts. In Micrasterias , the PM H+-ATPase inhibitors Na-orthovanadate and diethylstilbestrol depolarized the potential, but it remained at the same level in light and dark. On the other hand, fusicoccin, which activates the PM H+-ATPases, hyperpolarized the potential clearly (to −56 mV). 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, which blocks the electron transport chain from photosystem (PS)II to PSI and thereby prevents the possible signal transmission from chloroplasts to the PM, depolarized the MP slightly, but did not affect the (lacking) light changes either. The results indicate the presence of a continuous (low) activity of PM H+-ATPases in Micrasterias , which is not stimulated by light. The lack of rapid light-induced changes in Micrasterias MP may be due to an unusual functioning of giant chloroplasts in the ion metabolism of the Micrasterias cell.     

5-Aminolevulinic Acid Induced Photodynamic Reactions in Thylakoid Membranes of Cucumber (Cucumis sativus L.) Cotyledons

The cucumber (Cucumis sativus L.) plants were sprayed with 20 mM 5-aminolevulinic acid or distilled water (control) and incubated in dark for 14 hr. The thylakoid membranes prepared from the intact chloroplasts, isolated from the above plants in dark, were illuminated with low light intensity (100 W/m2) for 30 min. Due 10 photodynamic reactions, the photochemical function of photosystem II was damaged by 50% in treated thylakoids whereas it was only slightly (8%) affected in control thylakoids. The photosystem I was, however, not affected. The exogenous electron donors, MnCl2, diphenyl carbazide and NH2OH failed to restore the photosystem II activity suggesting that the photodynamic damage had taken place very close to photosystem II reaction center. Singlet oxygen scavenger, histidine, could protect the photosystem II activity while superoxide radical scavengers, superoxide dismutase and 1, 2-dihydroxybenzene-3, 5-disulphonic acid disodium salt, and hydroxyl radical scavenger, formate, failed to protect the same.     

Dynamic Interaction between the D1 protein,CP43 and OEC33 at the lumenal side of photosystem II in spinach chloroplasts: evidence from light-induced cross-Linking of the proteins in the donor-side photoinhibition

During the donor-side photoinhibition of spinach photosystem II, the reaction center D1 protein cross-linked with the antenna chlorophyll binding protein CP43 of photosystem II lacking the oxygen-evolving complex (OEC) subunit proteins. The cross-linking did not occur upon illumination of photosystem II samples that retained the OEC33, nor when OEC33-depleted photosystem II samples were reconstituted with the OEC33 prior to illumination. These results suggest that the D1 protein, CP43 and the OEC33 are located in close proximity at the lumenal side of photosystem II, and that the OEC33 suppresses the unnecessary contact between the D1 protein and CP43. Previously we presented data showing the D1 protein located adjacent to CP43 on the stromal side of photosystem II [Ishikawa et al. (1999) BIOCHIM: Biophys. Acta 1413: 147]. The present data suggest that the spatial arrangement of the D1 protein and CP43 at the lumenal side of photosystem II in spinach chloroplasts is similar to that at the stromal side of photosystem II and is consistent with the assignment of these proteins recently proposed on the crystal structures of the photosystem II complexes from cyanobacteria [Zouni et al. (2001) Nature 409: 739, Kamiya and Shen 2003 PROC: Natl. Acad. Sci. USA, 100: 98]. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the photosystem II complex from higher plants may be different from those in cyanobacteria.     

Studies on thylakoid phosphorylation and noncyclic electron transport

The effect of thylakoid phosphorylation on noncyclic electron transport in spinach chloroplasts was investigated by measuring both the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the steady-state redox level of the primary electron acceptor quinone of photosystem II (Q) during electron flow to NADP. These data are compared with the theoretical predictions for an electron transport model which relates both the redox levels of Q and the photosystem II optical cross section to the overall velocity of noncyclic electron flow. It is demonstrated that transfer of 15-20% of the photosystem II antenna to photosystem I may stimulate electron flow to NADP only if Q is less than 60-70% oxidized (this condition exists with our thylakoids, even at extremely low absorption fluxes, when the illumination is not specifically enriched in photosystem I absorbed wavelengths); in phosphorylated thylakoids the steady-state redox level Q is substantially shifted to a more oxidized one (measurements of this parameter using light of different wavelengths quantitatively support the idea that thylakoid phosphorylation leads to increased photosystem I and decreased photosystem II cross sections); thylakoid phosphorylation leads to stimulated noncyclic electron flow to NADP only when the increased photosystem I antenna is able to bring about large increases in the steady-state level of oxidized Q.     

Carbonic Anhydrase in Subchloroplast Particles of Pea Plants

The partitioning of carbonic anhydrase (CA) activity in chloroplasts isolated from 10–14-day-old pea (Pisum sativum L.) seedlings was investigated. The effect of CA inhibitors on the kinetics of chlorophyll fluorescence in photosystem II (PSII) preparations was also studied. The activity of CA was detected in fractions of soluble proteins and in the polypeptide complexes of the PSI and PSII. Isolated particles of photosystems retained a high photochemical activity similar to that of intact chloroplasts and the high level of polyunsaturated fatty acids. The association of CA with the particles of PSII (PSII-CA) was also tested by Western-blot analysis using antibodies against PSII-CA (Cah3) from Chlamydomonas reinhardtii. The PSII particles isolated with Triton X-100 (T-20) showed a higher activity of the enzyme as calculated on a protein basis than the DT-20 particles isolated with digitonin and Triton X-100. This difference seems to be related to the higher degree of nativity of the chloroplast T-20 fragments as compared to DT-20 particles. The higher level of chlorophyll per reaction center as well as the higher content of chlorophyll b and lipid fatty acids as calculated on protein basis, in particular of E-16:113 acid, which stabilizes the oligomeric structure of the light-harvesting complex of the PSII, also confirms this suggestion. The activity of CA was not detected in the DT-20 preparations treated with Tris–HCl to eliminate manganese ions. This is likely to indicate that one of the extrinsic polypeptides of PSII exhibits CA activity. Specific inhibitors of CA (acetazolamide and imidazole) inhibited the photoinduced yield of chlorophyll fluorescence (F). This might be determined by damaging the water-oxidizing system or its interaction with the PSII reaction centers. The functional role of PSII-CA for ₂-concentrating in carboxylation sites as well as its role in the coupling of light and dark reactions in chloroplasts is discussed.     

Membrane structure and function. II. Alterations in the photo-induced absorption changes after treatment of isolated chloroplasts with large pulses of the ruby laser

Treatment of isolated chloroplasts with high-energy pulses of the ruby laser causes graded structural changes in the chloroplast membranes and is here correlated with the biochemical changes produced. The laser treatment caused decreases in the photoinducible absorption changes of cytochromes b₅₅₉, b₅₆₃, and P₅₂₀ (the carotenoid shift), but smaller decreases in cytochrome f. The decreases correlated with the quantum efficiency alterations produced by the laser treatment. Ferricyanide photoreduction and O₂ evolution was only slightly affected by the laser treatment. The slow phase of the dark recovery kinetics of P₅₂₀ was increased maximally by the lowest laser input energies and NADP⁺ photoreduction induced by carbonylcyanide-P-trifluoromethoxyphenylhydrazone (FCCP) was decreased maximally by the lowest energies, suggesting that uncoupling of the chloroplasts was the most sensitive parameter. This was corroborated by our previous observation (5) that chloroplast membrane bound surface particles (coupling factor) was the ultrastructural change most sensitive to the laser pulses. Electron flow from photosystem II to photosystem I was not altered by the laser treatment. The laser treatments did not cause a detectable decrease in total chlorophyll in the chloroplasts, however, approximately 10% of the total chlorophyll was present in the solution phase after the treatment, whereas no detectable cytochromes were present in the solution phase.