Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Expression of 5-amino levulinic acid induced photodynamic damage to the thylakoid membranes in dark sensitized by brief pre-illumination

The 5-amino levulinic acid treated cucumber (Cucumis sativus L., CV. Pointsette) plants upon exposure to light (≃30,000 lux) wilted within 6 h and died after 36 h due to photodynamic reactions. Thylakoid membranes, the site of accumulation of porphyrins, were damaged due to photodynamic reactions leading to the inhibition of membrane linked functions of photosystem II, photosystem I and the whole chain electron transport. Photosystem II was more susceptible to photodynamic damage than photosystem I. The exogenous electron donors Mn²⁺, diphenyl carbazide and NH₂OH failed to donate electrons to photosystem II suggesting that the damage has taken place close to P680. The 5-amino levulinic acid treated plants exposed to 30 min of light did not show any damage to the thylakoid membranes. However, when the above plants were transferred to dark for 12 h there was substantial damage to the thylakoid membrane system.     

5-Aminolevulinic Acid Induced Photodynamic Damage of the Photosynthetic Electron Transport Chain of Cucumber (Cucumis sativus L.) Cotyledons

Cucumber (Cucumis sativus L. cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid (ALA) and then incubated in dark for 14 hours. Upon transfer to sunlight (800 watts per square meter) the plants died after 5 hours of exposure due to photosensitization reaction of metalloporphyrins. Due to the photodynamic damage, photosystem II (PSII), photosystem I (PSI), and whole chain reactions were impaired. PSII activity was more susceptible to photodynamic damage than PSI. The variable fluorescence was significantly reduced in ALA-treated plants within 1 hour of exposure to sunlight. At low temperature (77°K), the PSI fluorescence peak height (F₇₃₄) was drastically reduced and blue shifted by 6 nanometers. The photodynamic damage was irreversible; rather, it continued upon dark incubation of ALA-treated cucumber plants exposed to sunlight for 15 minutes. In the latter experiment, continued production of malondialdehyde during dark treatment suggests the degradation of unsaturated membrane lipids.     

Effects of High Temperature Exposure of Spinach Intact Plants and Isolated Thylakoids on Light-Harvesting Complex 2 Protein Phosphorylation

After a 6 min exposure of isolated thylakoids to 43 °C, the extent of phosphorylation of light-harvesting complex of photosystem 2 (LHC2) was higher than in control thylakoids kept at 25 °C. Similarly, the exposure of intact spinach plants to 43 °C in dark for 11 h induced higher extent of thylakoid LHC2 phosphorylation than in control plants kept at 25 °C. The induced ability of LHC2 for enhanced phosphorylation may enable better energy distribution in favour of photosystem 1.     

Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5°C or 20°C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol → methylviologen) after dark preincubation at temperatures between 0 and 60°C. Thylakoids isolated in the presence or absence of Na⁺/Mg²⁺ from 20°C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60°C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20°C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20°C grown rye, thylakoids isolated from 5°C grown rye in the presence of Na⁺/Mg²⁺ exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60°C. This was not due to damage to PSI electron transport in thylakoids isolated from 5°C grown plants since light saturated PSI activity was 60% higher in 5°C thylakoids than 20°C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5°C thylakoids could be observed after dark preincubation only when 5°C thylakoids were initially isolated in the absence of Na⁺/Mg²⁺. We suggest that 5°C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20°C thylakoids and hence cannot be increased further by dark preincubation.     

Thylakoids from pea seedlings grown under intermittent light: biochemical and flash-spectrophotometric properties.

Thylakoid membranes were isolated from pea seedlings grown under intermittent light (2-min light/118-min dark cycles). These preparations differed from controls (thylakoids from plants grown under 16-h light/8-h dark cycles) in the following respects: 15 times smaller chlorophyll/protein ratio, 10 times greater chlorophyll a/b ratio, absence of light-harvesting chlorophyll a/b binding proteins, and 2-3-fold greater ratio of photosystem II over photosystem I. In addition we found the following: (1) Electrogenic electron transfer around cytochrome b6/f under flashing light was greatly enhanced, probably as a consequence of the greater photosystem II/photosystem I ratio. (2) The rate of proton uptake from the medium at the acceptor side of photosystem II was enhanced, probably by unshielding of the quinone binding domain. (3) The N,N'-dicyclohexylcarbodiimide sensitivity of the proton-pumping activity of photosystem II was absent, which was consistent with the attribution of a N,N'-dicyclohexylcarbodiimide-induced protonic short circuit to chlorophyll a/b binding proteins. (4) The sensitivity of oxygen evolution under continuous light to variations of pH or the concentration of Ca2+ was altered. Chlorophyll a/b binding proteins serve as light-harvesting antennas. We found in addition that they modulated the activity of water oxidation and, in particular, the proteolytic reactions around photosystem II.     

Chromium increases photosystem 2 activity in <Emphasis Type="Italic">Brassica juncea</Emphasis>

In 7-d-old seedlings of Brassica juncea chromium (VI) promoted photosystem 2 (PS 2) mediated photoreactions. The increase in PS 2 activity in the thylakoids from Cr-treated seedlings, in the presence of uncoupler (5 mM NH₄Cl), was similar to that recorded with the control thylakoids. Thus Cr enhanced PS 2 activity was not due to uncoupling of electron transport from photophosphorylation. Photon saturation kinetics revealed that the PS 2 activity of thylakoids from Cr-treated seedlings was significantly higher at almost all irradiances in comparison to that of controls. PS 2 activity of thylakoids from Cr-treated plants at 25 % of the saturating irradiance was in par with the PS 2 activity of the thylakoids from control plants at saturating irradiance. Thylakoids from both control and Cr-treated seedlings exhibited maximum PS 2 activity at pH 7.5. The PS 2 activity of thylakoids from Cr-treated plants remained high even at pH 8.0 and 8.5, demonstrating Cr enhances tolerance of PS 2 to alkaline pH.     

Active oxygen produced during selective excitation of photosystem I is damaging not only to photosystem I, but also to photosystem II

With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4 degrees C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.     

Oxidative damage to thylakoid proteins in water-stressed leaves of wheat (Triticum aestivum)

The production of reactive oxygen species in the chloroplast may increase under water deficit. To determine if this causes oxidative damage to the photosynthetic apparatus, we analyzed the accumulation of oxidatively damaged proteins in thylakoids of water-stressed wheat ( Triticum aestivum L.) leaves. Water stress was imposed on 4-week-old plants by withholding watering for 10 days to reach a soil water potential of about −2.0 MPa. In thylakoids of water-stressed leaves there was an increase in oxidative damage, particularly in polypeptides of 68, 54, 41 and 24 kDa. High molecular mass oxidized (probably cross-linked) proteins accumulated in chloroplasts of droughted leaves. Oxidative damage was associated with a substantial decrease in photosynthetic electron transport activity and photosystem II (PSII) efficiency (F_v/F_m). Treatment of stressed leaves with l -galactono-1,4-lactone (GL) increased their ascorbic acid content and enhanced photochemical and non-photochemical quenching of chlorophyll fluorescence. GL reduced oxidative damage to photosynthetic proteins of droughted plants, but it reverted the decrease in electron transport activity and PSII efficiency only partially, suggesting that other factors also contributed to loss of photosystem activity in droughted plants. Increasing the ascorbic acid content of leaves might be an effective strategy to protect thylakoid membranes from oxidative damage in water-stressed leaves.     

Cause for dark, chilling-induced inactivation of photosynthetic oxygen-evolving system in cucumber leaves

Effects on oxygen evolution of the storage of detached cucumber (Cucumis sativus) leaves at 0°C in the dark were investigated with thylakoids and oxygen-evolving photosystem II membranes isolated from stored leaves. The cold and dark treatment of leaves selectively inactivated electron transport on the oxidizing side of photosystem II. Photosystem II membranes isolated from treated leaves were largely depleted of two proteins of 20 and 14 kilodaltons, which correspond to the extrinsic 23- and 17- kilodalton proteins of spinach functioning in oxygen evolution. The manganese content of photosystem II membranes was also markedly reduced by the treatment. Thus, the inactivation of oxygen evolution induced by the dark, chilling treatment is ascribed to solubilization of the 20- and 14-kilodalton proteins and extraction of manganese.     

Over-expression of PaSOD in transgenic potato enhances photosynthetic performance under drought

Drought stress enhances the production of superoxide radical (O₂ ^._) and superoxide dismutase catalyses dismutation of it to H₂O₂ and O₂, and hence provides a first line of defense against oxidative stress. Over-expression of a cytosolic copper-zinc superoxide dismutase, cloned from Potentilla atrosanguinea (PaSOD), in potato (Solanum tuberosum ssp. tuberosum L. cv. Kufri Sutlej) resulted in enhanced net photosynthetic rates (P_N) and stomatal conductance (g_s) compared to that in the wild type (WT) plants under control (irrigated) as well as drought stress conditions. Drought stress declined leaf water potential, P_N, g_s, photosystem II activity, and chlorophyll content, but increased proline and O₂ ^._ content more in WT than transgenic potato plants (SS5). The significantly higher SOD activity in SS5 coincided well with lower O₂ ^._ content suggesting its role in maintaining higher g_s and P_N in transgenic potato plants.     

Reactive oxygen intermediates produced by photosynthetic electron transport are enhanced in short-day grown plants

Leaves of tobacco plants grown in short days (8h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16h light). A two fold higher level of superoxide production was observed even in isolated thylakoids from short day plants. By using specific inhibitors of photosystem II and of the cytochrome b(6)f complex, the site of O(2) reduction could be assigned to photosystem I. The higher rate of O(2) reduction led to the formation of a higher proton gradient in thylakoids from short day plants. In the presence of an uncoupler, the differences in O(2) reduction between thylakoids from short day and long day plants were abolished. The pigment content and the protein content of the major protein complexes of the photosynthetic electron transport chain were unaffected by the growth condition. Addition of NADPH, but not of NADH, to coupled thylakoids from long day plants raised the level of superoxide production to the same level as observed in thylakoids from short day plants. The hypothesis is put forward that the binding of an unknown protein permits the higher rate of pseudocyclic electron flow in thylakoids from short-day grown plants and that this putative protein plays an important role in changing the proportions of linear, cyclic and pseudocyclic electron transport in favour of pseudocyclic electron transport. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Articifical.     

Changes in the Cationic Regulation of Structure and Function in Thylakoids Isolated from Wheat Seedlings Treated with BASF 13.338

When wheat seedlings (Triticum vulgare cf HD 2189) were grown in the presence of BASF 13.338 (4-chloro-5-[dimethylamino]-2-phenyl-3[2H]-pyridazinone), there was a decrease in the ratio of linolenic acid to linoleic acid in the thylakoid membrane lipids (JB St John 1976 Plant Physiol 57: 38) and an increase in the ratio of photosystem II to photosystem I (RM Mannan, S Bose 1984 Photochem Photobiol 41: 63). Accompanying these gross structural changes were alterations in the cationic regulation of structure and functioning of the thylakoid membranes: (a) Mg²⁺-induced increase in the room temperature fluorescence was totally absent; (b) Mg²⁺-induced increase in absorbance at 560 nm, indicative of granal stacking, was slightly higher in thylakoids isolated from the BASF 13.338 treated plants suggesting an increased degree of stacking; and (c) absorption changes in the red and Soret regions of the absorption spectrum, normally resulting from the addition of divalent cation or alkyl anion, or from osmotic shrinkage were almost totally absent in thylakoid membranes isolated from BASF 13.338 treated plants. These observations have been interpreted in terms of: (a) significant alterations in the lipid matrix of the thylakoids from treated plants, (b) absence of cation-induced reorganization of the pigment-protein complexes in the horizontal plane of the treated thylakoid membranes suspended in low salt medium, and (c) absence of dynamic changes even within the individual pigment-protein complexes of treated thylakoids.     

Oxidative stress in cucumber (Cucumis sativus L) seedlings treated with acifluorfen

Treatment of diphenyl ether herbicide acifluorfen-Na (AF-Na) to intact cucumber (Cucumis sativus L cv Poinsette) seedlings induced overaccumulation of protoporphyrin IX in light (75 mumole m-2 s-1). The extra-plastidic protoporphyrin IX accumulated during the light exposure disappeared within two hours of transfer of acifluorofen-treated seedlings to darkness. The dark disappearance was due to re-entry of migrated protoporphyrin IX into the plastid and its subsequent conversion to protochlorophyllide. In light, protoporphyrin IX acted as a photosensitizer and caused generation of active oxygen species. The latter caused damage to the cellular membranes by peroxidation of membrane lipids that resulted in production of malondialdehyde. Damage to the plastidic membranes resulted in damage to photosystem I and photosystem II reactions. Dark-incubation of herbicide-sprayed plants before their exposure to light enhanced photodynamic damage due to diffusion of the herbicide to the site of action. Compared to control, in treated samples the cation-induced increases in variable fluorescence/maximum fluorescence ratio and increase in photosystem II activity was lower due to reduced grana stacking in herbicide-treated and light-exposed plants.     

Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl-3(2H)Pyridazinone)

When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H₂O → methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30°C. Below 30°C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6-dichlorophenol indophenol-reduced → methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30°C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide → methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H₂O → methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1°C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55°C; this decrease was biphasic in the control and monophasic in the treated plants.     

Photosystems activities and polypeptide composition of Cyamopsis tetragonoloba and Vigna mungo thylakoids as affected by exclusion of solar UV radiation

The impacts of solar UV (280–400 nm) radiation on photosynthetic activities and polypeptide composition of thylakoids of cluster bean (Cyamopsis tetragonoloba L, UV-B sensitive) and black gram (Vigna mungo L., UV-B resistant) plants were compared. The activity of photosystem 1 and especially photosystem 2 increased in cluster bean while decreased in black gram, when either UV-B or UV-B + UV-A radiation was removed as compared to control plants. Exclusion of UV-B radiation caused changes in the protein composition of the thylakoids particularly in the 33, 23 and 17 kDa proteins of photosystem 2.     

Seasonally Induced Changes in Acyl Lipids and Fatty Acids of Chloroplast Thylakoids of Pinus silvestris: A CORRELATION BETWEEN THE LEVEL OF UNSATURATION OF MONOGALACTOSYLDIGLYCERIDE AND THE RATE OF ELECTRON TRANSPORT

Current-year needles were sampled regularly from an approximately 20-year-old natural stand of Pinus silvestris. Chloroplast thylakoids were isolated. The electron transport capacities of photosystem II + photosystem I, as well as of the partial photoreactions, were measured. The amounts and the fatty acid compositions of monogalactosyldiglyceride, digalactosyldiglyceride, and sulfolipid of the thylakoids were analyzed. The fatty acid composition of the phospholipids (total) was also determined.     

Thylakoid‐bound and stromal antioxidative enzymes in wheat treated with excess copper

Copper is a catalyst in the formation of reactive free radicals and its toxicity may be due, at least in part, to oxidative damage. The response of thylakoid‐bound and stromal antioxidative enzymes against the generation of superoxide radical was investigated in seedlings of wheat ( Triticum durum L. cv. Adamello) grown in hydroponic culture for 10 days and subjected to 10 and 50 µ M copper treatments. Electron spin resonance of roots evidenced a spectrum of copper, the intensity of which increased with the treatment, whereas the carbon‐centered free radical spectrum detected in the control leaves was not seen anymore in the treated samples. As well as thylakoids, photosystem II (PSII) particles were able to produce the superoxide radical. Increased superoxide production both by thylakoids and PSII was observed in the sample treated with 50 µ M Cu. Induction of thylakoid‐bound and stromal antioxidative enzymes, with the exception of dehydroascorbate reductase, was also detected in leaves treated with the highest copper concentration. No Mn‐superoxide dismutase (SOD, EC 1.15.1.1) was detected in thylakoids of wheat. Both stromal and thylakoid‐bound SOD were CuZn‐SOD with 16.2‐kDa subunits. Both western blotting and immuno‐electron microscopy showed that the SOD subunit was recognized by a polyclonal antibody against glyoxisomal CuZn‐SOD from watermelon cotyledon. In the stroma of wheat, ascorbate peroxidase showed at least three well‐resolved bands differently induced by copper treatments.     

Plastid Alternative Oxidase (PTOX) Promotes Oxidative Stress When Overexpressed in Tobacco

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b₆f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.     

Reduction of the chloroplast membrane system caused by disorders in early stages of chlorophyll biosynthesis

A chlorophyll-deficient xantha mutant of cotton (Gossypium hirsutum L.) was examined with respect to development and structural organization of the chloroplast membrane system as affected by disruption of early stages of chlorophyll biosynthesis in the light. The analysis of early chlorophyll precursors showed that the mutant is unable to synthesize 5-aminolevulinic acid (5-ALA) in the light. The disorders in early stages of chlorophyll biosynthesis arrested the development of chloroplast membrane system at the stage of vesicles and single thylakoids. The accumulation of 2–5% chlorophyll in the mutant was related to the formation of light-harvesting chlorophyll-a/b-protein complexes I and II, whereas pigment-protein complexes composing reaction centers of photosystem I and photosystem II were lacking. It is concluded that the chloroplast membrane system in the mutant with impaired 5-ALA synthesis is incapable of development and is even reduced upon long-term growing under light.