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  Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Tiessen, A., Nerlich, A., Faix, B., Hummer, C., Fox, S., Trafford, K., et al. (2012). Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method. Journal of Experimental Botany, 63(5), 2071-2087. doi:10.1093/Jxb/Err408.

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Tiessen, A.1, Author              
Nerlich, A.2, Author
Faix, B.2, Author
Hummer, C.2, Author
Fox, S.2, Author
Trafford, K.2, Author
Weber, H.2, Author
Weschke, W.2, Author
Geigenberger, P.1, Author              
1Storage Carbohydrate Metabolism, Department Stitt, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753336              
2External Organizations, ou_persistent22              


Free keywords: cereal seeds metabolic regulation non-aqueous fractionation starch subcellular metabolite analysis adp-glucose pyrophosphorylase potato solanum-tuberosum posttranslational redox-modification protein-protein interactions mays-l endosperm biosynthetic-enzymes cereal endosperm zea-mays molecular characterization transporter activity
 Abstract: 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.


Language(s): eng - English
 Dates: 2011-12-262012
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: ISI: ISI:000301297800022
DOI: 10.1093/Jxb/Err408
ISSN: 1460-2431 (Electronic) 0022-0957 (Linking)
URI: ://000301297800022 http://jxb.oxfordjournals.org/content/63/5/2071.full.pdf
 Degree: -



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Title: Journal of Experimental Botany
Source Genre: Journal
Publ. Info: -
Pages: - Volume / Issue: 63 (5) Sequence Number: - Start / End Page: 2071 - 2087 Identifier: -