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Fractionation of cell-wall preparations from grass leaves by centrifuging in non-aqueous density gradients.

ABSTRACT: 1. Dried preparations of cell walls from perennial-ryegrass (Lolium perenne) and Italian-ryegrass (L. multiflorum) leaves were suspended in mixtures of carbon tetrachloride with light-petroleum (b.p. 45--50 degrees C) or alcohols and layered on density gradients formed from the same solvents. 2. On centrifugation, the cell walls become distributed throughout a suitably chosen gradient. Fractions corresponding to various regions of the gradient were separated, examined under the microscope and analysed. 3. Cell-wall preparations made from leaf material ground in liquid N2, or in a triple roll mill, showed considerable heterogeneity in particle size, and their behaviour in the density gradient was variable, although there was a general indication that walls derived from vascular bundles were less dense than those from sclerenchyma. 4 Treatment in a vibratory ball mill decreased the size of the particles and produced a more uniform material, but made it impossible to distinguish the origins of the particles. This material behaved more reproducibly in the density gradient. 5. Some fractionations were also made by successive centrifugation in media of increasing relative density. 6. Analyses of the fractions obtained by each method indicated that the less dense had a greater proportion of xylose in the polysaccharide components, and higher contents of acetyl groups and lignin, confirming the close relationship between these components in plant cell walls. 7. The results show that there are differences in polysaccharide composition between the cell-wall types in the grass leaf, the vascular tissue being richer in hemicellulose relative to cellulose than the sclerenchyma.

SUBMITTER: Gordon AH

PROVIDER: S-EPMC1162665 | biostudies-other | 1981 Mar

REPOSITORIES: biostudies-other

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Project description:Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

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Fractionation of cell-wall preparations from grass leaves by centrifuging in non-aqueous density gradients.

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