Transport of Metabolites across Isolated Envelope Membranes of Spinach Chloroplasts

Isolated envelope membranes of spinach chloroplasts (Spinacia oleracea L. var. Viroflay) exhibited selective permeability. Metabolites such as 3-phosphoglycerate, bicarbonate, glyoxylate, and acetate were transported rapidly; 6-phosphogluconate, glycolate, glycine, l-malate, and succinate were intermediate; whereas glucose 6-phosphate, fructose 1,6-diphosphate, and sucrose were hardly transported. Transport rates, metabolite accumulations within the membrane vesicles, and the internal water volume of isolated and in situ envelope membranes were compared and found to show similar trends.     

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.     

Isolation and Characterization of Phosphatase from Thylakoid Membranes of Spinach Chloroplasts

A phosphatase from thylakoid membrane of spinach (Spinacia oleracea L. ) chloroplasts was isolated with the methods of extraction with n-ButanoL centrifugation at 100000 g for 30 min and chromatographic separation through DEAE-Cellulose (DE 52) column.The phosphatase catalyzed hydrolysis of phosphate monoesters (4-nitrophenyl phosphate). The optimal pH for enzyme catalysis was below 7. The peak rate of the enzyme reaction was obtained when it was incubated at 60℃ for 15 min. The phosphatase was inhibited by ATP and phosphate. The results from SDS-PAGE showed that the preparation of enzyme was composed of two proteins.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.     

菠菜叶绿体的光抑制部位

有氧条件下,叶绿体的光抑制部位不是专一的。强光可使PSⅡ氧化侧、PSⅡ反应中心、PSⅡ还原侧,PSⅡ及类囊体膜透性都有不同程度的破坏。这种非专一性可能与类囊体膜蛋白在强光下的降解有关。无氧条件下,叶绿体的光抑制部位只是在PSⅡ反应中心及Q_B蛋白上。     

Electron Transport Reactions in Grana Preparations from Spinach Chloroplasts

Fraction 2 (grana-stack) particles prepared with the French press showed absorbance changes, at room temperature and with sodium ascorbate and methyl-viologen, that were produced by the oxidation of cytochrome b-559. This oxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and sensitized by system II of photosynthesis. The oxidation is too slow to account for the rates of the Hill reaction that have been observed with nicotinamide-adenine dinucleotide phosphate (NADP⁺). It appears that this cytochrome is not functioning in the main pathway of electron transport. In the presence of 2,3,5,6-tetramethyl-p-phenylene-diamine (DAD) and ascorbate, light-induced oxidation of cytochrome f took place within 3 msec (or faster) in the grana-stack particles. Treatment with the detergent Triton X-100 disrupted this rapid cytochrome f oxidation as well as the oxidation of cytochrome b-559. Subsequent plastocyanin addition did not restore the rapid oxidation of cytochrome f (nor of cytochrome b-559) but only slow changes of cytochrome f. In view of the fact that these particles contain almost no plastocyanin, it is unlikely that plastocyanin functions in electron transport between cytochrome f and P-700 in the particles derived from the grana-stack regions of the chloroplast.     

Reconstitution of the Functional Membranes from Chloroplasts with the Deficient Crista Membranes from Mitochondria

The basic mechanisms of phosphorylation of chloroplasts and mitochondria are identical. The identity may be proved by membrane combination. There are two ways to get the combination as shown in figure 1. One way is, as previously reported, to combine deficient membranes from chloroplasts with ctista membranes from mitochondria and the reconstituted membranes thus obtained greatly enhance photophosphorylated activities. The other way, i.e., to combine deficient crista membranes with thylakoid membranes, has also been successful, as shown in this paper. The reconstituted membranes obtained in this way can carry out oxidative phosphorylation in the dark as well as shown in Table 2. There are some relationship between the ATP formation from oxidative phosphorylation of reconstituted membranes and the protein of deficient crista membranes added, as shown in Table 3. When the quantity of combined chloroplast membranes is kept constant, the amount of ATP formation varies, within certain limits, with the amount of deficient crista membranes as shown in Table 3. But the reconstituted oxidative phosphorylation activity of membranes formed by combinating thylakoid with deficient crista membranes is lower than reconstituted photophosphorylation activity of combination in the opposite direction, i.e. by combinating deficient thylakoid membranes and crista membranes of mitochondria (compare Table 4 and 3).     

Characteristics of Prompt and Delayed Fluorescence from Spinach Chloroplasts

Effects of ferricyanide, dichlorophenyldimethylurea (DCMU), and uncouplers of phosphorylation on the prompt and delayed fluorescences from spinach chloroplasts are described. Any factor that affects the yield of prompt fluorescence will similarly influence the intensity of delayed fluorescence. This idea, recently investigated by Lavorel, should be expressed in terms of a “live” component of fluorescence; that is, the component from chlorophyll associated with the photochemical traps of System II. Some of the effects of ferricyanide and DCMU on delayed fluorescence can then be explained in terms of effects on the yield of prompt fluorescence. From the internal consistency of the explanation, applied to various observations, a judgment can be made that most of the prompt fluorescence observed initially when dark-adapted chloroplasts are first illuminated is “dead,” coming from chlorophyll not associated with trap II. The live fluorescence is represented almost entirely by the time-varying component that develops during illumination. The observed intensity of delayed fluorescence can be divided by the yield of live prompt fluorescence to give an intrinsic delayed fluorescence. This intrinsic delayed fluorescence is proportional to the square root of exciting light intensity (as long as the excitation is not saturating) and decays with second order kinetics. This behavior may reflect the photochemical formation and second order dissipation of an oxidized product of Photosystem II.     

Polypeptide Composition of Envelope Membranes Isolated from Chloroplasts of C3, C4, and CAM Plants

Chloroplast envelopes were isolated from chloroplasts purifiedfrom Spinacea oleracea L. (C₃), Panicum miliaceum L. (NAD-malicenzyme-type C₁), Digitaria sanguinalis (L.) Scop. (NADP-malicenzyme-type C₄), Kalanchoe daigremontiana Hamet et Perrier (constitutiveCAM), and from Mesembryanthemum crystallinum L. (inducible CAM)performing either C₃ photosynthesis or Crassulacean acid metabolism(CAM). For each species, methods were developed to isolate chloroplastenvelopes free of thylakoid contamination. The polypeptidesof ribulose bisphosphate (RuBP) carboxylase which has been consistentlyreported in envelope preparations of spinach were not foundin envelope preparations of C₄ mesophyll chloroplasts. Silverstaining of envelope polypeptides resolved electrophoreticallyon sodium dodecylsulfate polyacrylamide gradient slab gels produceda more complex profile than did Coomassie staining which haspreviously been used with C₃ envelope preparations, even thoughsilver reacted poorly with polypeptides corresponding to thesubunits of RuBP carboxylase. All of the plants examined possesseda major polypeptide of 27 to 29 kilodaltons (kD) which was previouslysuggested to be the phosphate translocator in spinach. WithC₃ M. crystallinum, the 29 kD polypeptide stained most intensely.After induction of CAM, a 32 kD polypeptide also stained intensely,giving a profile similar to that obtained with the constitutiveCAM species. A 32 kD polypeptide was also prominent in C₄ envelopepreparations, suggesting that a 32 kD polypeptide may be a translocatorprotein which is required in Crassulacean acid metabolism andC4 photosynthesis, but not in C₃ photosynthesis. (Received April 25, 1983; Accepted July 9, 1983)     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

红肉蜜柚果肉红色色素鉴定

红肉蜜柚是在琯溪蜜柚园中发现的一个红肉变异单株,经连续多年对无性子代(三代)系统观测,其红肉变异性状稳定。对其汁胞呈色色素测定结果表明,成分主要为番茄红素和β-胡萝卜素,含量分别为(55.45±1.13) μg/g·dw和(41.10±2.24) μg/g·dw;而叶黄素含量极微。