Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

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103

The aim of this work was to investigate whether sucrose synthesis in the cytosol of leaf cells is regulated in response to the supply of energy and organic carbon from the chloroplast. Fluxes into sucrose and metabolite levels in wheat (Triticum aestivum var Timmo) leaf protoplasts were compared in a range of light intensities and CO2 concentrations, showing that sucrose-phosphate synthase and the cytosolic fructose-l,6-bisphosphatase are inhibited in situ when the supply of trioseP from the chloroplasts decreases. Such a regulation might aid CO2 fixation in limiting conditions by permitting stromal metabolites to be maintained at higher levels than would otherwise be possible. pools are depleted, or whether the enzymes of sucrose synthesis are actually regulated by smaller fluctuations of selected metabolites. This would promote CO2 fixation in limiting conditions by permitting higher levels of metabolites to be maintained in the stroma.There is already evidence that protoplasts and leaves can contain substantial levels of metabolites in the dark (25). We have now studied in more detail the fluxes and metabolite levels in wheat leaf protoplasts during photosynthesis in varying light intensities and CO2 concentrations. The results show that both the cytosolic FBPase and sucrose P synthase are involved in regulating the rate of sucrose synthesis in response to the capacity of the chloroplast to provide trioseP.The trioseP produced in the stroma during photosynthesis represents an important branch point in metabolism. Some is metabolized further in the stroma to regenerate RuBP2 which is needed to support more CO2 incorporation, while some is exported via the Pi translocator in exchange for Pi. The two processes must be balanced to achieve rapid CO2 fixation. If export of trioseP should exceed the rate at which new trioseP is being synthesized from CO2 and Pi, the levels of metabolites in the stroma would decrease and the operation of the Calvin cycle would be impaired or eventually stopped (see 26). With isolated chloroplasts, this balance is artificially achieved by optimizing the Pi concentration used in the medium. When CO2 fixation is restricted by low pH, C02, or light, the maximal rates of CO2 fixation in the new conditions are attained only when the Pi in the medium is lowered (6), decreasing the rate at which carbon is withdrawn from the chloroplasts, so that it matches the lower rate of CO2 fixation. This minimizes the depletion of the stromal metabolite levels, which would otherwise lead to an additional inhibition ofphotosynthesis.In situ, most of the exported trioseP is converted to sucrose in the cytosol, releasing Pi which reenters the chloroplast. In analogy to isolated chloroplasts, there will be a marked depletion of the stromal metabolite pools during photosynthesis in limiting light or CO2, unless the rate of sucrose synthesis is readjusted to match the rate of CO2 fixation. The question arises whether photosynthetic cells undergo large fluctuations in the levels of metabolites because ...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Sucrose Synthesis by Cytoplasmic Fructosebisphosphatase and Sucrose Phosphate Synthase during Photosynthesis in Varying Light and Carbon Dioxide

Stitt¹,

Wirtz²,

Heldt³

1983

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103

“…The concentrations of malate and pyruvate in endosperm tissue was determined using standard techniques (4,24). Endosperm tissue was frozen in liquid N2, ground in a mortar and pestle, extracted in 10% HC104, and neutralized with 5 N KOH/1 M triethanolamine.…”

Section: Metabolite Analysesmentioning

confidence: 99%

Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm

Smith¹,

Gauthier²,

Dennis³

et al. 1992

153

102

Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoil gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.or leucoplast glycolytic enzymes are more than adequate to account for the triacylglycerol synthesis of castor endosperm (17). However, glycolytic intermediates have not been well characterized as carbon sources for fatty acid synthesis (18). In this study, we present data regarding the kinetics and exogenous cofactor requirements for fatty acid synthesis in isolated leucoplast preparations using pyruvate, malate, and acetate as carbon sources. We show that malate and pyruvate support much higher rates of fatty acid synthesis than does acetate and the metabolism of both these substrates alleviates any requirement for externally added reductant. Based on the measurement of leucoplast-associated NADP+-malic enzyme activity, we suggest that cytosolic malate may be a source of carbon for fatty acid synthesis in vivo. MATERIALS AND METHODS Plant MaterialCastor plants (Ricinus communis L. cv Baker 296) were greenhouse grown under natural light supplemented with 16 h fluorescent light.Storage lipids make up 45 to 50% of the dry weight of castor bean (Ricinus communis) endosperm (9, 15). During development, the conversion of sucrose to triacylglycerols is the major metabolic activity of this tissue (9). Dennis (11) presented a model indicating the possible pathways of triacylglycerol synthesis from sucrose. The model is primarily based on enzyme compartmentation studies (12,17,22,23,27) and suggests that glycolytic intermediates may cross the leucoplast membrane and serve as carbon skeletons for fatty acid synthesis. Acetate is another possible precursor.Acetate incorporation into fatty acids has been used to study the cofactor requirements for fatty acid biosynthesis (6,16,18,25,26). It is unlikely that acetate is the in vivo carbon precursor of fatty acid synthesis because of its relatively low cellular concentration and the low efficiency of acetate utilization for de novo fat...

“…This demands a procedure for tissue fractionation during which the total content and the subcellular distribution of metabolites in these compartments is not altered. Recently, methods have been developed for the assay of subcellular metabolite levels in plant protoplasts in which the protoplasts were ruptured by passage through a nylon net or a capillary tube followed by immediate filtration of the particles either through a layer of silicone oil (8,15,18) or a combination of membrane filters (12). The measurement of subcellular metabolite levels in whole leaves is more difficult; one possible approach is to use a nonaqueous fractionation procedure similar to that developed in 1932 by Behrens (2) for the fractionation of nuclei.…”

mentioning

confidence: 99%

Measurement of Subcellular Metabolite Levels in Leaves by Fractionation of Freeze-Stopped Material in Nonaqueous Media

Gerhardt

Heldt²

1984

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244

188

ABSTRAC1This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia okracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifuption.Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated.As a frst application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole fails from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately I millimolar at the end of the night.An understanding of the metabolism of a plant cell requires information about the metabolite levels in the various metabolic compartments, such as the chloroplast, the mitochondria, the vacuole, and the cytosol. This demands a procedure for tissue fractionation during which the total content and the subcellular distribution of metabolites in these compartments is not altered. Recently, methods have been developed for the assay of subcellular metabolite levels in plant protoplasts in which the protoplasts were ruptured by passage through a nylon net or a capillary tube followed by immediate filtration of the particles either through a layer of silicone oil (8, 15, 18) or a combination of membrane filters (12). The measurement of subcellular metabolite levels in whole leaves is more difficult; one possible approach is to use a nonaqueous fractionation procedure similar to that developed in 1932 by Behrens (2) for the fractionation of nuclei. In this method the tissue is frozen and lyophilized, the dried material homogenized in nonaqueous solvents, and the resulting homogenate is fractionated by centrifugation. The absence of water prevents the enzymic interconversion of metabolites, and the polar enzymes and metabolites are not extracted during the treatment of the cell material with the nonpolar ' Supported by the Deutsche Forschungsgemeinschaft.solvents. This method has also been successfully applied to the separation of chloroplasts (9, 17). The general principle was to purify a chloroplast fraction by several centrifugation steps. Such procedure involves the loss of a large portion ofchloroplasts...