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Plant and Cell Physiology Advance Access originally published online on February 10, 2009
Plant and Cell Physiology 2009 50(3):635-643; doi:10.1093/pcp/pcp021
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Control of Starch Synthesis in Cereals: Metabolite Analysis of Transgenic Rice Expressing an Up-Regulated Cytoplasmic ADP-Glucose Pyrophosphorylase in Developing Seeds

Yasuko S. Nagai1,2, Chotipa Sakulsingharoj3, Gerald E. Edwards2,4, Hikaru Satoh5, Thomas W. Greene6, Beth Blakeslee6 and Thomas W. Okita1,*

1Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA
2Graduate Program in Molecular Plant Sciences, Washington State University, WA 99164-6340, USA
3Department of Biology, Faculty of Science, Maejo University, Sunsai, Chiang Mai 50290, Thailand
4School of Biological Sciences, Washington State University, Pullman, WA 99164-6340, USA
5Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581 Japan
6Dow Agroscience LLC, Indianapolis, IN 46268, USA

*Corresponding author: E-mail, okita{at}wsu.edu; Fax, +1-509-335-7643.


  Abstract

We had previously demonstrated that expression of a cytoplasmic-localized ADPglucose pyrophosphorylase (AGPase) mutant gene from Escherichia coli in rice endosperm resulted in enhanced starch synthesis and, in turn, higher seed weights. In this study, the levels of the major primary carbon metabolites were assessed in wild type and four transgenic CS8 rice lines expressing 3- to 6-fold higher AGPase activity. Consistent with the increase in AGPase activity, all four transgenic CS8 lines showed elevated levels of ADPglucose (ADPglc) although the extent of increases in this metabolite was much higher than the extent of increases in starch as measured by seed weight. Surprisingly, the levels of several other key intermediates were significantly altered. Glucose 1-phosphate (Glc 1-P), a substrate of the AGPase reaction, as well as UDPglucose and Glc 6-P were also elevated to the same relative extent in the transgenic lines compared with the wild-type control. Analysis of metabolite ratios showed no significant differences between the wild type and transgenic lines, indicating that the reactions leading from sucrose metabolism to ADPglc formation were in near equilibrium. Moreover, glucose and fructose levels were also elevated in three transgenic lines that showed the largest differences in metabolites and seed weight over the wild type, suggesting the induction of invertase. Overall, the results indicate that the AGPase-catalyzed reaction is no longer limiting in the transgenic lines, and constraints on carbon flux into starch are downstream of ADPglc formation, resulting in an elevation of precursors upstream of ADPglc formation.

Keywords: ADPglucose pyrophosphorylase - Endosperm - glgC - Mass spectrometry - Rice - Starch.

Abbreviations: ADPglc, ADPglucose; AGPase, ADPglucose pyrophosphorylase; ANOVA, analysis of variance; DTT, dithiothreitol; Glc 1-P, glucose 1-phosphate; LC-MS/MS, liquid chromatography–tandem mass spectrometry; 3-PGA, 3-phosphoglyceric acid; TM, triple mutant; UDPglc, UDP glucose; UGPase, UDPglucose pyrophosphorylase.

(Received January 7, 2009; Accepted January 29, 2009)
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