Inhibition of photosystem I and photosystem II in chloroplasts by UV radiation

The effects of UV radiation on the low temperature fluorescenceand primary photochemistry of PSII and PSI of spinach chloroplastswere studied. Fluorescence induction curves at –196°Cwere measured at 695 nm for PSII fluorescence and at 730 nmfor PSI fluorescence to determine both the initial F_o and finalF_M levels. The primary photochemistry of PSII was measured asthe rate of photoreduction of C-550 at – 196°C, thatof PSI as the rate of photooxidation of P₇₀₀ at –196°C.The results were analyzed in terms of a model for the photosyntheticapparatus which accounts for the yields of fluorescence andprimary photochemistry. According to this analysis UV radiationincreases nonradiative decay processes at the reaction centerchlorophyll of PSII. However, the effect of UV radiation isnot uniform throughout the sample during irradiation so thataccount must be taken of the fraction of PSII reaction centerswhich have been irradiated at any given time. UV radiation alsoinactivates P700 and causes a slight increase in nonradiativedecay in the antenna chlorophyll of PSI. All fluorescence ofvariable yield, F_V = F_M–F_o, at 730 nm is due to energytransfer from PSII to PSI so that the sensitivity of Fv to UVradiation is the same at 730 and 695 nm. ¹Present address: Department of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. ²Present address: Central Research Laboratories, Fuji PhotoFilm Co., Ltd., 105 Mizonuma, Asaka-Shi, Saitama 351, Japan. (Received September 10, 1975; )     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

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.     

Probing the kinetics of photosystem I and photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer.

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.     

Transport of glutamine into isolated pea chloroplasts

Abstract. Uptake of [¹⁴C] glutamine into isolated pea chloroplasts has been examined by using a centrifugal filtration technique. Competition experiments showed that glutamine uptake is mediated by a dicarboxylate carrier with K_m 1.10 mM and V _max. 118 nmol of glutamine min⁻¹ per mg of chlorophyll. Isolated pea chloroplasts accumulated glutamine in the sucrose-impermeable space to concentrations higher than that present in the external solution when the latter was below 0.5 mM. It is suggested that glutamine accumulation is driven by exchange (utilizing the dicarboxylate carrier) with the endogenous pool of dicarboxylates in the chloroplasts. Increasing pH stimulated glutamine uptake but inhibited that of glutamate and 2-oxoglu-tarate. The hypothesis is advanced that when molecules of different charge are exchanged across the chloroplast envelope via the dicarboxylate carrier, electroneutrality is maintained by transport of protons, and that this explains the observed effects of increasing pH. The low rates of glutamine transport coupled with the strong competition of other dicarboxylates for the carrier suggest that export in vivo from the chloroplast of nitrogen in the form of glutamine is not of major importance.     

Ozone increases the permeability of isolated pea chloroplasts

The effect of short-term exposure of chloroplasts isolated from the leaves of Pisum sativum to high concentrations of ozone was examined. The inhibitory effect of O₃ on endogenous photophosphorylation was apparently related to an increased permeability of the chloroplast limiting membranes induced by ozone exposure. A 5 min treatment with 50 ppm O₃ reduced the reflection coefficient of meso-erythritol from 0.84 to 0.58 and that of glycerol from 0.26 to 0.03. Such decreases in reflection coefficients indicate that ozone caused a marked increase in the permeability of the limiting membranes of the chloroplasts, which may result from an oxidation of membrane lipids. The decrease in the reflection coefficient of meso-erythritol was proportional both to ozone concentration (up to 30 ppm for 5 min of bubbling) and to time (up to 5 min at 30 ppm). Extrapolating these results to lower concentrations and longer times, ozone injury should be possible for a 2 hr exposure of plants to 0.3 ppm ozone, as is indeed the case.     

Crystalline arrays of ribosomes in isolated pea chloroplasts

Abstract Transmission electron microscopy of chloroplasts isolated by osmotic lysis of pea leaf protoplasts has revealed crystalline arrays of ribosomal particles associated with the thylakoid membranes. Optical diffraction techniques have established the crystallinity of the arrays and an image-enhancement technique has given an indication of ribosomal macrostructure. A model of crystal-packing is presented. This apparently artefactual induction of ribosome crystals should provide a valuable approach towards the elucidation of the details of the structure of chloroplast ribosomes.     

Selective photobleaching of PSI-related chlorophylls in heat-stressed pea chloroplasts

Measurements of electron transport activity point to the occurrence of major changes in the organisation of the photosynthetic apparatus of heat-stressed chloroplasts. One of the consequences of these changes is shown to be a greatly increased susceptibility of chlorophyll to photobleaching. Despite the fact that the threshold temperature for this photobleaching coincides closely with that for the inhibition of PSII activity, the bleached components were found to be specifically associated with PSI. This increased susceptibility of PSI pigments to photobleaching is shown to be a direct consequence of an interruption of the flow of reductants from PSII to PSI that would normally protect PSI from photooxidation.Abbreviations PSI photosystem I - PSII photosystem II - chl a chlorophyll a - chl b chlorophyll b - LHCP chlorophyll a/b light-harvesting protein - CP₁ P700-chlorophyll a protein - DCMU 3-(34 dichlorophenyl)-11-dimethylurea - DCPIP dichlorophenolindophenol - Fecy potassium ferricyanide - MV methyl viologen Biochemistry Department, King's College (KQC), University of London     

Studies on photosystem I. Characteristics of "310 material" isolated from spinach chloroplasts

Exposure of osmotically shocked chloroplasts to dilute pyridine and sonic oscillation results in the extraction of a small molecular-weight factor. Purification of the factor was accomplished using gel filtration chromatography. Due to the spectral nature of the purified species (λ_max at 310 nm) the factor was named “310 material.”Physiologically, the 310 material was found to inhibit a variety of ferredoxin-dependent photoreductions catalyzed by isolated spinach chloroplasts but stimulate both pseudocyclic photophosporylation and the ferredoxin-independent photoreduction of mammalian cytochrome c. The latter reaction was found to involve, at least partially, the formation of a Superoxide radical. Dark-reduction studies have further established that the 310 material is an autooxidizable electron carrier.Chemically, the 310 material is a water-soluble, low molecular-weight phenolic-type compound; possibly a derivative of coumaric acid. No proteinaceous material is observed in physiologically active preparations of 310 material.Based on these findings, it is concluded that the isolated 310 material acts on the reducing side of Photosystem I at or near the site of reduction of ferredoxin and competes with ferredoxin for the reducing power generated by the Photosystem I reaction center. The exact physiological role of the 310 material in the intact photosynthetic system, however, remains unknown.The similarities between the 310 material and a variety of other factors previously isolated from chloroplasts are discussed.     

Polarographic study of oxaloacetate reduction by isolated pea chloroplasts

Suspensions of pea chloroplasts, prepared by differential centrifugation, catalyzed oxaloacetate-dependent O(2) evolution (mean rate of 29 determinations 10.9 micromoles per milligram of chlorophyll per hour, sd 3.2) with the concomitant production of malate. At optimum concentrations of oxaloacetate, both reactions were light-dependent, inhibited by 3-(3,4- dichlorophenyl)-1, 1-dimethylurea and oxalate, and enhanced 2.5- to 4-fold by 10 millimolar NH(4)Cl. At concentrations of oxaloacetate (<50 micromolar), 10 millimolar NH(4)Cl was inhibitory. The ratio of O(2) evolved to malate produced was 0.39 to 0.58. The ratio of O(2) evolved to oxaloacetate supplied was commensurate with the theoretical value of 0.5.Chloroplast suspensions contained both NAD- and NADP-malate dehydrogenase activities. It was concluded from oxalate inhibition studies and the promotion of oxaloacetate-dependent O(2) evolution by shocked chloroplasts by NADPH (but not NADH) that the reaction was mediated via the NADP enzyme.     

Transport of purines and pyrimidines into isolated pea chloroplasts

Abstract. Uptake of [¹⁴C] adenine and [¹⁴C] cytosine into isolated pea chloroplasts has been examined by using a centrifugal-filtration technique. These chloroplasts accumulate both compounds in the sucrose-impermeable space to concentrations higher than that present in the external solution when the latter was below 1.0 mM. Competition experiments suggested that uptake is mediated by a carrier mechanism.     

A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem I was stimulated approx. 20 % by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea were frozen to ?196 °C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at ?196 °C with the preilluminated sample was approx. 25 % faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.     

Evidence for the ultraviolet-B (280–320 nm) radiation induced structural reorganization and damage of photosystem II polypeptides in isolated chloroplasts

Direct evidence for the possible loss of photosystem II (PS II) activity in chloroplasts of Vigna sinensis L. cv. Walp after ultraviolet-B (UV-B, 280–320 nm) radiation treatment was provided by polyacrylamide gel electrophoretic analysis of PS II polypeptides. A 30 min UV-B treatment of chloroplasts caused a 50% loss of PS II activity. The artificial electron donor. Mn²⁺ failed to restore UV-B radiation induced loss of PS II activity, while diphenyl carbazide (DPC) and NH₂OH only partially restored activity. Such a loss in PS II activity was found to be primarily due to a loss of 23 and 33 kDa extrinsic polypeptides. UV-B treatment induced the synthesis of a few polypeptides and a 29 kDa light-harvesting chlorophyll protein.     

Volume contraction of isolated pea chloroplasts promoted by bivalent cations

1. Chloroplasts isolated from pea seedlings were incubated in sucrose–tris medium reinforced with salts of calcium, magnesium, manganese or iron, at concentrations up to 10mm. 2. Measurements of chloroplast-pellet volume and water content showed that the bivalent cations brought about a contraction in chloroplast volume and a loss of chloroplast water. This was further substantiated by density-gradient centrifugations. 3. Measurements of the light-scattering and apparent fluorescence of chloroplast suspensions confirmed this conclusion and eliminated the possibility of contraction being caused by centrifugal forces. 4. The uptake of ⁴⁵Ca²⁺ was measured and shown to be competitive with diluent Ca²⁺, Mg²⁺ or Mn²⁺ ions, indicating a mechanism of low specificity. 5. The chloroplast contraction was insensitive to light but could be made sensitive by the addition of ferric EDTA. This light-sensitivity was inhibited by added 3-(p-chlorophenyl)-1,1-dimethylurea and so probably involves the Hill reaction. 6. On the basis of these observations it is suggested that the process of contraction does not consume much energy, but that in light-activated contraction a previous step occurs that is conducive to contraction and that is energy-transducing. It is postulated that this step results in a local increase in concentration of bivalent ions, which promotes contraction.     

Selective thiol inhibition of ferricyanide reduction in photosystem II of spinach chloroplasts

Selective inhibition of ferricyanide reduction in photosystem II by lipophilic thiols indicates a unique pathway of electron transport, which is not involved in reduction of class III acceptors or transfer of electrons to photosystem I. Both aromatic and aliphatic thiols induce the inhibition, but thiol binding reagents such as p-hydroxymercuribenzoate or N-ethylmaleimide do not inhibit. The inhibition can be observed using either dibromothymoquinone or bathophenanthroline to direct electrons away from photosystem I. No pretreatment of chloroplasts with thiols in the light was necessary to inhibit ferricyanide reduction by photosystem II or the O₂ evolution associated with ferricyanide reduction.     

Photosynthetic control and photophosphorylation in photosystem II of isolated spinach chloroplasts

Two cycles of photosynthetic control have been observed in isolated spinach chloroplasts in the presence of lipophilic class III electron acceptors, which may accept electrons at PS II. $\text{ADP}\text{O}$ ratios of 0.8 to 0.9 were recorded;rates of oxygen evolution were stimulated by phosphorylating reagents and uncouplers. Addition of the plastoquinone antagonist DBMIB decreased photosynthetic control, oxygen evolution and photophosphorylation. We believe that there is a coupling site associated with PSII which can be rate limiting. Comparison of the $\text{P}\text{2e}$ ratios observed with class I and class III electron acceptors leads us to propose that more than 0.6 and possibly approaching one molecule of ATP can be formed for every pair of electrons transported from water to PSII acceptors.     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.