Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde

Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O₂ flash yields, pH exchange, and fluorescence induction show that the O₂ evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

On the site of electron donation to the photosynthetic electron transport chain by 1,5-diphenylcarbazide

A solubilized base-exchange enzyme activity was dependent upon the addition of phospholipids, added Ca⁺⁺ ion, and had optimum pH of 7.2. Phosphatidyl-ethanolamine was found to be the best stimulator of both[¹⁴C]-ethanolamine and [¹⁴C]-serine incorporation. Preliminary evidence suggests the presence of phospholipase D type activity in this solubilized preparation.     

Influence of ultraviolet-B (UV-B) radiation on photosynthetic and growth characteristics in field-grown cassava (Manihot esculentum Crantz)

The effects of ultraviolet-B (UV-B between 290 and 320 nm) on photosynthesis and growth characteristics were investigated in field grown cassava (Manihot esculentum Crantz). Plants were grown at ambient and ambient plus a 5.5kJ m^?2 d^?1 supplementation of UV-B radiation for 95 d. The supplemental UV-B fluence used in this experiment simulated a 15% depletion in stratospheric ozone at the equator (0°N). Carbon dioxide exchange, oxygen evolution, and the ratio of variable to maximum fluorescence (F_v/F_m) were determined for fully expanded leaves after 64–76 d of UV-B exposure. AH plants were harvested after 95 d of UV-B exposure, assayed for chlorophyll and UV-B absorbing compounds, and separated into leaves, petioles, stems and roots. Exposure to UV-B radiation had no effect on in situ rates of photosynthesis or dark respiration. No difference in the concentration of UV-B absorbing compounds was observed between treatments. A 2-d daytime diurnal comparison of F_v to F_m ratios indicated a significant decline in F_v/F_m ratios and a subsequent increase in photoinhibition under enhanced UV-B radiation if temperature or PPF exceeded 35°C or 1800μmol m^?2 s^?1, respectively. However, UV-B effects on fluorescence kinetics appeared to be temporal since maximal photosynthetic rates as determined by oxygen evolution at saturated CO₂ and PPF remained unchanged. Although total biomass was unaltered with UV-B exposure, alterations in the growth characteristics of cassava grown with supplemental UV-B radiation are consistent with auxin destruction and reduced apical dominance. Changes in growth included an alteration of biomass partitioning with a significant increase in shoot/root ratio noted for plants receiving supplemental UV-B radiation. The increase in shoot/root ratio was due primarily to a significant decrease in root weight (–32%) with UV-B exposure. Because root production determines the harvest-able portion of cassava, UV-B radiation may still influence the yield of an important tropical agronomic species, even though photosynthesis and total dry biomass may not be directly affected.     

Light-dependent inhibition of photosynthetic electron transport by zinc

The effects of zinc concentrations up to 400 μ M were examined on three photosynthetic electron transport reactions of thylakoids isolated from Pisum sativum L. cv. Meteor. Zinc (400 μ M ) had no effect on photosystem I mediated electron transport from reduced N,N,N',N'-tetramethyl- p -phenylenediamine to methyl viologen, but inhibited uncoupled electron flow from water to methyl viologen by ca 50% and to 2,6-dichlorophenol-indophenol (DCPIP) by ca 30% at saturating light levels. Zinc inhibition of DCPIP photoreduction was independent of the light intensity to which thylakoids were exposed. Decreasing the photon flux density below 400 μmol m⁻² s⁻¹ produced a logarithmic reduction in the zinc-induced inhibition of methyl viologen photoceduction; a stimulation of this reaction was observed below 80 μmol photons m⁻² s⁻¹. Increasing light intensity decreased the amount of zinc tightly bound to the thylakoid membranes, but increased the weakly associated zinc which could be removed by washing the membranes with buffer containing Mg². The results suggest that zinc acts on the photosynthetic electron transport system at two sites. Site 1 is on the oxidizing side of photosystem 2 and the inhibition by zinc is independent of the light intensity. Site 2 is between photosystems 1 and 2 and the electron flow can be positively or negatively affected by zinc depending on the light intensity.     

On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

The inhibition of photosynthetic electron transport by methyl parathion

The effect of methyl parathion (metacid-50), an organophosphorous insecticide, on the Hill reactions of isolated mesophyll chloroplasts ofSorghum vulgare was studied. The pesticide was found to inhibit the Hill reaction with all the Hill oxidants tested, namely potassium ferricyanide,2,6-dichlorophenol indophenol and para-benzoquinone. The concentration of the pesticide required to inhibit 50% of the control Hill activity (I₅₀value) was found to vary with the different Hill oxidants.     

Damages of photosynthetic apparatus inAnacystis nidulans by ultraviolet-B radiation

Changes in photosystem (PS) 2 activity were measured inAnacystis nidulans cells exposed to UV-B irradiation. The decrease in PS 2 activity was biphasic in cells exposed at the surface (0 cm) and at 2 cm depth in the water column, while gradual in those exposed at 10 and 30 cm depth. Addition of supplemental “white light” reduced the extent of UV-B damage. Decrease in photosynthetic activity was primarily due to the loss of energy transfer from phycobilisome to chlorophyll as the former in cyanobacteria acts as the primary light-harvesting complex. This was supported by the absorption and fluorescence excitation and emission spectral studies. All these changes were proportionally reduced by the thickness of the water column that reduced UV-B irradiance. This work was supported in part by a grant from the Ministerio de Education y Ciencia (ref SB 94-AM086772) to N. N.     

Functional size of photosynthetic electron transport chain determined by radiation inactivation

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H₂O to methylviologen) was 623 ± 37 kilodaltons; for photosystem II (H₂O to dimethylquinone/ferricyanide), 174 ± 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 ± 11 kilodaltons. The difference between 364 ± 22 (the sum of 174 ± 11 and 190 ± 11) kilodaltons and 623 ± 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b₆/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.     

The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

Reversal of dibromothymoquinone inhibition of photosynthetic electron transport by bovine serum albumin

Addition of bovine serum albumin to cholorplasts inhibited by prior addition of 1 μm dibromothymoquinone results in a time- and light-dependent restoration of electron transport activity. The kinetics of this reversal reaction are complex, and indicate that it is controlled by the degree to which the thylakoid membranes are energized. The presence of ADP and inorganic phosphate, or of uncouplers, serves to retard the rate of reversal, whereas an acceleration of reversal is observed if the thylakoid membranes have been intentionally unstacked by exposure to low-salt medium. The reversal reaction reported here is unique to bovine serum albumin, and does not require the function of the free sulfhydryl group on the protein. It is concluded that the site of DBMIB inhibition associated with chloroplast membranes is situated in a position whose access to contact by bovine serum albumin is regulated by the structural changes induced by illumination and energization.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport

The binding of radioactively labelled atrazin, metribuzin and phenmedipham by broken chloroplasts was studied. From the double-reciprocal plots (bound vs. free inhibitors) a high affinity binding reaction is graphically isolated which is related to the inhibition of photosynthetic electron transport. It is concluded that the specific binding sites correspond to the electron carrier molecules which are attacked by the inhibitors. The relative concentration of specific binding sites is 1 per 300–500 chlorophyll molecules.The binding of the labelled substances is competitively inhibited by each of the indicated unlabelled substances, by DCMU and by several pyridazinone derivatives. These results suggest that triazines, triazinones, pyridazinones, biscarbamates and phenylureas interfere with the same electron carrier of the photosynthetic electron transport chain, according to the same molecular mechanism.     

Impact of elevated UV-B radiation on photosynthetic electron transport, primary productivity and carbon allocation in estuarine epipelic diatoms

Epipelic diatoms are important components of microphytobenthic biofilms. Cultures of four diatom species (Amphora coffeaeformis, Cylindrotheca closterium, Navicula perminuta and Nitzschia epithemioides) and assemblages of mixed diatom species collected from an estuary were exposed to elevated levels of ultraviolet-B (UV-B) radiation. Short exposures to UV-B resulted in decreases in photosystem II (PSII) photochemistry, photosynthetic electron transport, photosynthetic carbon assimilation and changes in the pattern of allocation of assimilated carbon into soluble colloidal, extracellular polysaccharides (EPS) and glucan pools. The magnitude of the effects of the UV-B treatments varied between species and was also dependent upon the photosynthetically active photon flux density (PPFD) to which the cells were also exposed, with effects being greater at lower light levels. Both increases in nonphotochemical quenching of excitation energy in the pigment antennae and photodamage to the D1 reaction centres contributed to decreases in PSII photochemistry. All species demonstrated a rapid ability to recover from perturbations of PSII photochemistry, with some species recovering during the UV-B exposure period. Some of the perturbations induced in carbon metabolism were independent of effects on PSII photochemistry and photosynthetic electron transport. Elevated UV-B can significantly inhibit photosynthetic performance, and modify carbon metabolism in epipelic diatoms. However, the ecological effects of UV-B at the community level are difficult to predict as large variations occur between species.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Inhibition of photosynthetic activity by UV-B radiation in radish seedlings

Inhibition of primary photosynthetic reactions by UV-B radiation (280 nm-320 nm) was demonstrated in radish leaves ( Raphanus sativus cv. Saxa Treib). Detached radish cotyledons from 10-day-old seedlings were irradiated with continuous white light and increasing UV-B irradiances using cut-off filters with increasing transmission for shorter wavelengths (WG 360, WG 345, WG 320, WG 305, WG 295, WG 280). Photosynthetic activity measured in terms of chlorophyll fluorescence induction (Kautsky effect) after 2, 4, 6, 8 and 24 h irradiation decreased in a wavelength dependent way with increasing UV-B irradiance and irradiation time.
Radish seedlings grown for 10 days from the time of germination under the same UV-B irradiation conditions exhibited similar reductions of the variable fluorescence as detached cotyledons irradiated for short time periods. They additionally had lower initial fluorescence at high UV-B radiation levels, although the chlorophyll content per leaf area increased. In contrast to short term experiments, the plastoquinone and flavonoid content increased with increasing UV-B irradiance when based on leaf area.