Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O₂ evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O₂ uptake with methyl viologen as acceptor.

Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H₂O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.

The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O₂ evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O₂ evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

     

Participation of β-carotene in reactivation of PSI of heptane-extracted spinach chloroplasts

A carotenoid requirement for photosystem I activity in spinach chloroplasts using extraction-reconstitution technique has been investigated. The transfer of electron from N,N,N,N-tetramethyl-p-phenylene diamine through the chloroplast photosystem to methyl viologen dye or to NADP⁺ was used as an assay of photosystem I activity. Extraction of lyophilized spinach chloroplasts with heptane at near 0°C removed almost all -carotene and reduced photochemical activities associated with photosystem I to a low level (about 15% of the original activity). Reconstitution of the extracted chloroplasts with -carotene completely restored photosystem I activity. The maximum rate of methyl viologen photoreduction in reconstituted chloroplasts occurred at an -carotene/chlorophyll molar ratio of 0.5. Cyclic phosphorylation mediated by phenazine methosulphate was partially restored. Xanthophylls (lutein, neoxanthin, violaxanthin), as components of chloroplast membranes, were not able to replace -carotene in reconstitution of chloroplasts and had essentially no effect on restoring photoreactions. On the basis of the P700/total chlorophyll ratio it can be assumed that extraction of lyophilized chloroplasts with heptane do not affect photosystem I reaction centre. Therefore it is possible that -carotene, removed during heptane extraction and belonging mainly to the antenna pigment pool of photosystem I, is effective in the restoration of photosystem I activity.Abbreviations chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - MV methyl viologen - PMS phenazine methosulphate - PQA plastoquinone A - PS I photosystem I - PS II photosystem II - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - Tricine N-tris(hydroxymethyl)methyglycine. D-1, D-10, D-50, D-144 represent chloroplast subfractions sedimented at 1000 × g, 10,000 g, 50,000 × g and 144,000 × g - s supernatant This paper is a partial fulfillment of the requirements for the Ph.D. degree of A.T. at Maria Curie-Skodowska University, Lublin.     

The effects of photooxidative stress on photosystem I measured in vivo in Chlamydomonas

The effects of different photooxidative stresses on the function of photosystem I were measured in vivo in Chlamydomonas reinhardtii. Pholooxidative stresses included strong light, light combined with chilling to 0 °C, and light combined with several concentrations of methyl viologen. Photosystem I function was measured in vivo using the absorbance change at 820 nm associated with P₇₀₀ oxidation. Photosystem II function was measured in vivo using chlorophyll fluorescence. Strong light or light combined with chilling caused inhibition of photosystem II function earlier than inhibition of photosystem I function. When photosystem I was inhibited, however, it did not recover. Light combined with 5 mmol m^?3 methyl viologen caused inhibition of photosystem I function earlier than inhibition of photosystem II. If the methyl viologen concentration was reduced to 1 mmol m^?3, the damage to PSI was accelerated by addition of 90 mmol m^?3 chloramphenicol. This effect of chloroamphenicol suggests a role for chloroplast-encoded proteins in protecting photosystem I against photooxidative damage caused by methyl viologen.     

Photosynthetic activity and membrane polypeptide composition of supergranal chloroplasts from plant tissue cultures containing a viruslike particle

Tissue culture cells of Streptanthus tortuosus (Kell.) var. orbiculatus (Greene) Hall (Cruciferae), having a viruslike particle in their nucleoli, the STV cell line, contain “supergranal” chloroplasts. Freeze-fracture studies of chloroplasts of a control cell line, which lacks the viruslike particles, reveal two complementary faces similar to those observed in spinach chloroplasts. Replicas of freeze-fractured STV supergranal chloroplasts, however, show that one membrane face (B) contains widely spaced 80 Å particles and the other face (C) is essentially smooth. Isolated STV supergranal chloroplasts lack photosystem II activity as indicated by their inability to reduce dichlorophenolindophenol and are unable to reduce NADP with electrons from photosystem II or from ascorbate-reduced dichlorophenolindophenol. However, partial photosystem I activity is indicated by the reduction of methyl viologen with electrons from dichlorophenolindophenol-ascorbate. This supports the concept that there is not a direct correspondence between grana formation and photosystem II activity. Electrophoresis shows that all of the major polypeptide bands present in the STV supergranal chloroplasts are also present in the control chloroplast membranes. One band, molecular weight 33,000, is present in a greatly increased amount in the STV supergranal chloroplast membranes and may be associated with grana stacking.     

Photosynthetic activities of cadmium-treated tomato plants

Tomato plants (Lycopersicum esculentum Mill. cv. Moneymaker) grown on nutrient medium containing cadmium exhibit reduced net photosynthesis and reduced contents of chlorophyll and accessory pigments. In chloroplasts isolated from cadmiumtreated plants photosystem II activity, as measured by 2,6-dichlorophenolindophenol photoreduction, and photosystem II + I activity (H₂O → methyl viologen) were both inhibited to about 60%. When 1,5-diphenylcarbazide was used as artificial electron donor, no significant cadmium effect was observed. Photosystem I activity was not affected by cadmium. The fine structure of chloroplasts in cadmium-treated plants was degenerated, similarly to senescence response. The principal symptom of cadmium action was the occurrence of large plastoglobules and a disorganization of the lamellar structure, mainly grana stacks. Transfer of cadmium-treated plants into a medium with increased manganese level caused grana stacking and restoration of photosystem II activity.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.     

Absorption and Transfer of Light and Photoreduction Activities of Spinach Chloroplasts under Calcium Deficiency: Promotion by Cerium

Chloroplasts were isolated from spinach cultured in calcium-deficient, cerium-chloride-administered calcium-present Hoagland’s media or that of calcium-deficient Hoagland’s media and demonstrated the effects of cerium on distribution of light energy between photosystems II and I and photochemical activities of spinach chloroplast grown in calcium-deficient media. It was observed that calcium deprivation significantly inhibited light absorption, energy transfer from LHCII to photosystemII, excitation energy distribution from PSI to PSII, and transformation from light energy to electron energy and oxygen evolution of chloroplasts. However, cerium treatment to calcium-deficient chloroplasts could obviously improve light absorption and excitation energy distribution from photosystem I to photosystem II and increase activity of whole chain electron transport, photosystems II and I DCPIP photoreduction, and oxygen evolution of chloroplasts. The results suggested that cerium under calcium deficiency condition could substitute for calcium in chloroplasts, maintain the stability of chloroplast membrane, and improve photosynthesis of spinach chloroplast, but the mechanisms still need further study.     

Sedimentation equilibrium in reacting systems. VII. The temperature-dependent self-association of beta-lactoglobulin A at pH 2.46

The polyene antibiotic filipin inhibits the activities of both photosystem I and photosystem II in maize mesophyll chloroplasts and pea chloroplasts. Maximum inhibition of photosystem II activity was observed at a filipin concentration of about 0.4 mm in maize mesophyll chloroplasts and 1.0 mm in pea chloroplasts. Inhibition of photosystem II activity was temperature dependent, being much less if the antibiotic and chloroplasts were incubated at 0 °C compared to 25 °C. The inhibition of photosystem I activity of both maize mesophyll and pea chloroplasts caused by filipin, could be overcome by the addition of the soluble electron transfer protein, plastocyanin. It is concluded that the inhibition of photochemical activity caused by filipin is a secondary effect resulting from a change in membrane conformation induced by the antibiotic.     

Superoxide formation in pea chloroplasts by a dioxathiadiaza-2,5-pentalene derivative,a new lipophilic: Photosystem I acceptor

The dioxathiadiaza-2,5-pentalene derivative, HEP II, has herbicidal effects similar to those of methyl viologen. HEP II promotes superoxide formation when added to illuminated pea chloroplasts. Superoxide dismutase, but not catalase, diminished formation of the Superoxide whereas cyanide and azide enhanced its formation, presumably by inhibiting the endogenous superoxide dismutase activity. DCMU, which inhibits photosynthetic electron transport, inhibited Superoxide formation. Rates of superoxide formation and oxygen uptake were very similar when equal concentrations of methyl viologen or HEP II were added. At subsaturating concentrations of electron acceptor, Mg²⁺ decreased the rate of oxygen uptake with methyl viologen but not with HEP II, probably reflecting differences in their interactions with the Photosystem I electron donation site. It is likely that HEP II, by analogy with methyl viologen, is reduced by chloroplast Photosystem I and reoxidised by molecular oxygen, generating superoxide.     

Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra

Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O₂ uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O₂ evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as ¹⁴C fixation or O₂ evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.     

Redox Reactions between Kaempferol and Illuminated Chloroplasts

Bleaching of kaempferol by illuminated chloroplasts was observed at 380 nanometers. The photobleaching was stimulated by methyl viologen and suppressed by superoxide dismutase indicating the participation of O₂⁻ in the reaction. An electron transfer inhibitor on the oxidizing side of photosystem II, carbonylcyanide m-chlorophenylhydrazone (CCCP), stimulated the photobleaching and 3-(3,4-dichlorophenyl)-1,1-dimethylurea partially suppressed it. The stimulation by CCCP suggests that kaempferol is also bleached on the oxidizing side of photosystem II. The spectrum of kaempferol bleaching in the presence of methyl viologen was the same as that in the presence of CCCP having a maximum in absorbance decrease at around 380 nanometers. When kaempferol was oxidized by KMnO₂ or KO₂, the oxidized minus reduced difference spectra had also a negative peak at about 380 nanometers. The results suggest that kaempferol was oxidized by illuminated chloroplasts.

The rate of kaempferol photooxidation increased as its concentration was increased from 1 to 100 micromolar. The rate of quercetin photooxidation also increased as its concentration was increased from 1 to 100 micromolar. Concentration of quercetin glycosides higher than 10 micromolar was required to detect their photobleaching by illuminated chloroplasts. From these results, it is postulated that flavonols function as antioxidants in chloroplasts.

     

Evidence for multiple sites of ferricyanide reduction in chloroplasts

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pH's. It was noted that at low pH ferricyanide reduction was not completely inhibited by orthophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyurea - MV methyl viologen - DBMIB 2,5-dibromothymoquinone - DMBQ 2,6-dimethyl benzoquinone - OP 1,10-orthophenanthroline - TMPD tetramethyl-p-phenylenediamine - PS 1 photosystem I - PS II photosystem II - SN sucrose-sodium chloride chloroplasts Supported by NSF Grant BMS 74-19689.     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O₂ evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.     

Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology

Tissue-specific effects of low growth temperature on maize chloroplast thylakoid protein accumulation were analysed using immunocytology. Sections of leaves from plants grown at 25 and 14°C were probed with antibodies to specific chloroplast thylakoid proteins from the four major protein multisubunit complexes of the thylakoid membrane followed by fluorescein-conjugated goat anti-rabbit antibodies. At a normal growth temperature of 25°C, the 32 kDa D1 protein of the photosystem II reaction centre and the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II are both accumulated to a greater degree in the mesophyll than in the bundle sheath chloroplasts. In contrast, subunit II of photosystem I, cytochrome f and the α- and β-subunits of ATP synthetase are predominant in the bundle sheath thylakoids at 25°C. A striking difference between the 25°C-grown and the 14°C-grown leaf tissue was the presence in the latter of (20–30%) cells whose chloroplasts apparently completely lack several of the thylakoid proteins. In plants grown at 14°C, the accumulation of the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II was apparently unchanged, but other thylakoid proteins showed a significant reduction. The uneven distribution of proteins between the bundle sheath and mesophyll chloroplasts observed at 25°C was also maintained at 14°C. Reduction in the fluorescence at 14°C was manifested either as an overall reduction in the diffuse fluorescence across the chloroplast profiles or less frequently as a reduction to small discrete bodies of intense fluorescence. The significance of these results to low-temperature-induced reduction in the photosynthetic productivity of maize is discussed.     

Detoxification of SO2 derivatives in chloroplasts ofHardwickia binata

Intact chloroplasts isolated from sulphur dioxide fumigatedHardwickia binata leaves showed inhibition of PS II electron transport activity without any significant effect on photosystem I. Sulphur dioxide exposed leaves accumulated more hydrogen peroxide than those from non-fumigated plants and this was caused by increase in superoxide radical production. Hydrogen peroxide formation was inhibited by addition of cytochrome C and superoxide disrnutase. In sulphur dioxide fumigated leaves, increase in superoxide dismutase activity showed resistance to sulphite toxicity. The localization of ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase activities in chloroplasts provide evidence for the photogeneration of ascorbate. The scavenging of hydrogen peroxide in chloroplast due to ascorbate regenerated from DHA by the system: PS I → Fd → NADP → glutathione. The system can be considered as a means for preliminary detoxification of sulphur dioxide by chloroplasts     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Sensitivity of photosynthesis by spinach chloroplast membranes to osmotic stress in vitro: Rapid inhibition of O2 evolution in presence of magnesium

Thylakoids prepared from spinach (Spinacea oleracea L.) chloroplasts were exposed to osmotic stress in vitro in the presence or absence of different inorganic salts. By an hour after incubation in 1.0 M sorbitol and 10 mM (or more) MgCl₂, the thylakoids lost approximately 80% of their photosystem (PS) II activity, but not PS I. The inhibition occurred only in presence of magnesium as indicated by the combinations of several cations/anions. The PS II activity was relatively insensitive to osmotic stress in the presence of diphenyl carbazide. We therefore conclude that under conditions of water stress in the presence of 10 mM or higher Mg²⁺, the oxygen evolving system in chloroplasts is rapidly inactivated.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - DPC diphenyl carbazide - MV methyl viologen - PS photosystem Part of this work was included in the thesis submitted by the first author of M.Phil.degree.