An Improved Method for the Isolation of Spinach Chloroplast Envelope Membranes

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A three-phase discontinuous sucrose gradient yielded two fractions of chloroplast envelope membranes from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L., mesophyl and undifferentiated chloroplasts). These species were selected to represent plants with fast photorespiration and slow net photosynthesis, fast photorespiration yet fast net photosynthesis, and slow photorespiration and fast net photosynthesis, respectively. (2).A very few exceptions to this classification exist. Sunflower leaves have the morphology and photorespiratory rates indicative of species with less efficient photosynthesis, yet their photosynthetic rates approach those of the more photosynthetically efficient species (28). Clearly, sunflower has some mechanism to compensate for a fast photorespiration.One site of this greater efficiency in sunflower may reside in the chloroplast envelope membrane, which regulates the uptake of metabolites required for photosynthesis, including CO2 (7,20,21,25). Studies with chloroplasts involving transport and release of permeants in the presence of anions have indicated the presConnecticut Agricultural Experiment Station, ence of specific carriers ("translocators") in the envelope membrane. These translocators facilitate the exchange of phosphate, dicarboxylic acids, and ATP (7,25). A comparison of the biochemical properties of chloroplast envelope membranes from species with different photosynthetic rates might show differences in the permeability properties and help elucidate the operations of translocators. To this end chloroplast envelope membranes from leaves of the less efficient species, spinach and broad bean, have been isolated and partially characterized in several laboratories (4,11,12,19,22). No descriptions of the properties of isolated chloroplast envelope membranes from more efficient species, sunflower or maize, have previously been reported. This report describes the preparation and establishes the purity of isolated chloroplast envelope membranes from sunflower and maize and shows large differences in the activities of Mg2+-dependent nonlatent ATPase among chloroplast envelope membranes of plant species with different rates of net photosynthesis. These activities were compared with differences in the transport of bicarbonate.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Isolation and Bicarbonate Transport of Chloroplast Envelope Membranes from Species of Differing Net Photosynthetic Efficiency

Poincelot¹,

Day²

1976

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“…For comparison the quantity and fatty acid composition of the complete and incomplete envelope fractions prepared from intact chloroplasts by the method of Poincelot and Day (12) As a further check on the distribution of chloroplast envelope fragments in our homogenates, the centrifugal fractions were analyzed for Mg2"-dependent DCCD-insensitive ATPase, a high specific activity of which has been previously shown to be characteristic of envelopes (4,11,14). This enzyme was found to be present at a high specific activity in the 10,000g, 40,000g, and 144,000g fractions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The intactness of the chloroplasts was determined by phase contrast microscopy, the intact fraction containing 90 to 95% bright and highly refractile chloroplasts, and the broken fraction containing only chloroplasts with distinct grana visible. Chloroplast envelopes were prepared from intact chloroplasts by osmotic shock and sucrose gradients as described by Poincelot and Day (12). They obtained two fractions (complete and incomplete envelopes) although no electron microscope study was made in this case. Lipids from all of these fractions were extracted, separated, and analyzed quantitatively and for radioactivity as previously described (19)(20)(21) (1).…”

Section: Methodsmentioning

confidence: 99%

Galactolipid Synthesis in Vicia faba Leaves

Williams¹,

Simpson²,

Chapman³

1979

Leaves of Vicis faba were fed 14CO2 in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained "broken" and "intact" chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactoUpid was transferred from the envelope to the thylakoidcontaining fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids.Many reports have now appeared which indicate that the two major lipids in chloroplasts (mono-and digalactosyl diglyceride, MGDG and DGDG) are synthesized by galactosylation of a diglyceride and MGDG, respectively. Recently attention has focused on the site of the galactosyl transferase within the cell. In reports by Douce (3), Joyard and Douce (6) and van Hummel et al. (18) the chloroplast envelope has been shown to be a major site of galactosyl transferase activity in spinach leaves. These experiments were conducted on cell-free fractions and the rate of galactosyl transferase activity was determined by measuring the incorporation of galactose from UDP- ['4CJGa1.into the lipids. Similar experiments have been carried out by Liedvogel and Kleinig (7) using daffodil chromoplasts with essentially similar results. Although these reports indicate the envelope of the plastid to be a major site of galactolipid synthesis, they do not show that it is the only site or that lipid is transferred from the cnvelope to chloroplast thylakoids. We undertook this study to determine the site of incorporation of "'CO2 into galactolipid and phospholipids in vivo and to determine whether there was any redistribution of radioactivity and lipid within the cells with time. In this paper we present our data on galactose labeling of galactolipids in Vicia faba cellular fractions; in the next paper we will present our data on the labeling pattem of fatty acids in both galactolipids and phospholipids. MATERIALS AND METHODSBroad bean plants ( Viciafaba L. 'Giant Windsor') were grown at 21 C under fluorescent lights (1,100 ft-c) for 3 to 4 weeks. (800 ACi, 5.8 mCi/mmol) for 10 min as previously described (20).The feeding was followed by '2CO2 chase periods of 0, 0.25, 1, and 6 h under continuous light (1,100 ft-c).Cell Fractionation. The leaves were homogenized in sucrose phosphate buffer (0.3 M sucrose in 0.15 M sodium-potassium phosphate buffer, pH 7.2) for 15 s in a Waring Blendor. The homogenate was filtered through eight layers of cheesecloth and one layer of nylon bolting cloth (average pore size about 20 ,im). The filtrate was centrifuged at 5,500g for 60 s (RB-2, Sorvall). The 5,500g pellet was resuspended and used for the p...

“…St. John et al (30) have shown that BASF 13.338 treatment reduced the tolerance of cotton to chilling injury, prevented frost hardening in wheat, barley, and rye and induced greater resistance in Sorghum and corn to high temperature stress. Contrary to these observations, Ashworth et aL (3) (21). The chloroplast pellet was suspended in 20 mM with swirling and homogenized by giving three complete passes in a Teflon homogenizer.…”

mentioning

confidence: 99%

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)

Mannan¹,

Bose²

1986

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 ofelectron transport (H20 -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-dichlorophenol indophenolreduced -_ 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 (H20 -_ 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 (1C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55C; this decrease was biphasic in the control and monophasic in the treated plants.Like most growth processes, photosynthesis is strongly affected by temperature (5) and the temperature dependence of dry matter production is closely linked to the temperature dependence of photosynthetic rate (7). The observation that high temperature decreases the quantum yield of CO2 fLxation suggests a damage in the primary processes of photosynthesis taking place in the thylakoid membranes (1,6