Inhibition of Light-Dependent Development of Chloroplasts by 5-Fluorouracil

The uracil analogue, 5-fluorouracil, inhibited the developmentof chloroplasts in Euglena gracilis, strain Z. Chlorophyll synthesiswas inhibited when dark-grown cells were illuminated in thepresence of 5-fluorouracil, but only if the 5-fluorouracil waspresent during the lag phase of chlorophyll synthesis. Ribonucleaseshowed a similar inhibition. Equimolar concentrations of uracilreleased inhibition by 5-fluorouracil, but if cells were incubatedin the light with 5-fluorouracil before addition of uracil,the ability of uracil to effect rapid reversal of 5-fluorouracilinhibition was decreased. In contrast, prior incubation with5-fluorouracil in the dark did not affect reversibility by uracil.The synthesis of a chloroplast-localized protein, cytochromec (552, Euglena), was also inhibited by 5-fluorouracil, whereasthe light-stimulated synthesis of a number of cytoplasmic enzymeswas enhanced. The results suggest that addition of 5-fluorouracilat the beginning of the illumination period preferentially interfereswith the synthesis of chloroplast protein compared with thesynthesis of cytoplasmic protein by inhibiting the formationof a ribosomal system, presumably localized in the chloroplast,that functions in the synthesis of chloroplast protein. Thedata also suggest that in uninhibited cells, the formation ofthis ribosomal system was largely completed within the first10 to 14 h of illumination and before the main period of synthesisof chloroplastproteins.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.     

Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride

Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl₂ (less than 1 μmole Hg²⁺/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl₂ concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl₂ to these sites.     

The Photo-oxidation of Epinephrine by Spinach Chloroplasts and Its Inhibition by Superoxide Dismutase: Evidence for the Formation of Superoxide Radicals in Chloroplasts

The thiobarbituric acid (TBA) test for detecting lipid hydroperoxides does not require for fomation of TBA reacting compounds from hydroperoxides, but oxygen has an unfavorable effect, that is, it forms new hydroperoxides during the reaction when unoxidized lipids co-exist. Therefore, a method using a vacuum reaction tube was proposed.     

Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts

Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H₂O₂. The in vitro activation by H₂O₂ was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H₂O₂ of SH groups on the enzyme which were reduced in the light by the light effect mediator system.     

Nano-Anatase Relieves the Inhibition of Electron Transport Caused by Linolenic Acid in Chloroplasts of Spinach

Linolenic acid is an inhibitor of electron transport in chloroplasts of higher plants. It has obvious effects on the structure and function of chloroplasts. In the present paper, we investigated the nano-anatase relieving the inhibition of photoreduction activity and oxygen evolution caused by linolenic acid in spinach chloroplasts. The results showed that linolenic acid in various concentrations could obviously reduce the whole chain electron transport and the photoreduction activity of two photosystems, especially on the oxidative reside and reduce reside of photosystem II (PS II). After adding nano-anatase to chloroplasts treated by linolenic acid, the whole chain electron transport rate, the photoreduction activity of two photosystems, and the oxygen evolution rate were increased significantly, indicating that nano-anatase could obviously decrease the inhibition of linolenic acid on the electron transport, photoreduction activity, and oxygen evolution of spinach chloroplasts.     

Interaction of Chloroplasts with Inhibitors: Location of Carotenoid Synthesis and Inhibition during Chloroplast Development

The inhibitor SAN 6706 [4-chloro-5-(dimethylamino)-2-(α,α,α,-trifluoro- m-tolyl-3(2H)-pyridazinone] has been used to study the synthesis of carotenes and xanthophylls during the conversion of etioplasts to chloroplasts in developing barley (Hordeum vulgare) shoots. SAN 6706 inhibits carotenoid synthesis and causes an accumulation of phytoene, but it is also a potent inhibitor of chloroplast electron transport. When developing barley is treated with SAN 6706, carotenoid synthesis is inhibited but total photosynthesis is unaffected. The ability of SAN 6706 to inhibit carotenoid synthesis becomes progressively less if etiolated shoots are illuminated for increasing lengths of time before treatment. During the greening of treated barley shoots only light-induced β-carotene synthesis is immediately inhibited; xanthophyll synthesis is not affected until after about 8 hours. The hypothesis that SAN 6706 cannot enter the chloroplast but inhibits carotenoid synthesis from the cytoplasm is discussed, and the question as to whether there are not two separate groups of enzymes for the synthesis of carotenes and xanthophylls is considered.     

Photooxidation by Photosystem II of Tris-washed Chloroplasts

Irradiation of tris-washed chloroplasts with moderate intensities of red light caused a partial bleaching of chloroplast pigments and an inhibition of the hydroquinone-supported photoreduction of NADP. The presence of an electron donor for photosystem 2 (PS2) during the irradiation prevented the bleaching and inhibition. It is concluded that the strong oxidant produced by PS2 accumulates in tris-washed chloroplasts during irradiation and an electron donor for PS2 protects against the photooxidation reactions.     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg²⁺ concentration ([Mg²⁺]) in chloroplast. Stromal [Mg²⁺] was increased by dark incubation of chloroplasts in the K⁺-gluconate medium (pH 8.0), or by NH₄Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg²⁺] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H⁺ efflux inhibited the alkalinization-induced increase in [Mg²⁺].     

Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites

The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H₂O→methyl viologen, H₂O→silicomolybdic acid H₂O→ferricyanide, and H₂O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H₂O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.     

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O₂ evolution (mean of 13 preparations, 17.5 μmole per mg chlorophyll per hour, sd 3.64). The optimum concentration of nitrite was 0.5 mm; 0.12 mm nitrite supported V_max/2. The reaction was accompanied by the consumption of nitrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. In short experiments (less than 10 minutes) the O₂ to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O₂ evolution with chloroplasts from plants grown without added nitrate but such chloroplasts could assimilate ammonia at about the usual rate. The results are consistent with the reduction of nitrite to ammonia involving nitrate-induced nitrite reductase and a reductant generated by the chloroplast electron transport chain.     

Improvement of antagonistic capability of Trichoderma harzianum by UV-Irradiation for management of Macrophomina phaseolina

Antagonistic capability of Trichoderma harzianum was improved through UV-irradiation. Four different type of mutants, T. harzianum - M_a (Th-M_a), T. harzianum - M_b(Th-M_b), T. harzianum - M_c (Th-M_c), T. harzianum - M_d (Th-M_d) of T. harzianum and the parent strain (Th-P) were selected for further studies. Th-M_a and Th-M_b showed more antagonistic capability against Macrophomina phaseolina than its parent strain Th-P in dual culture. Biochemical analysis of these four mutants and the parent strain showed that Th-M_a releases higher level of two lytic enzymes i.e. chitinases and cellulases and Th-M_b produces more β-1,3-glucanase activity than the parent strain. Culture filtrate of Th-M_a also showed antifungal properties. Study of the competitive saprophytic ability (CSA) of these four mutants and the parent strain were also made. Th-M_a exhibited higher CSA than the parental isolate while Th-M_d had less CSA than all other mutants and the parent strain of T. harzianum.     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.