First published online August 29, 2002; 10.1104/pp.004275
Plant Physiol, September 2002, Vol. 130, pp. 347-361
Probing in Vivo Metabolism by Stable Isotope Labeling of
Storage Lipids and Proteins in Developing Brassica napus
Embryos1
Jörg
Schwender and
John B.
Ohlrogge*
Michigan State University, Department of Plant Biology, East
Lansing, Michigan 48824
Developing embryos of Brassica napus
accumulate both triacylglycerols and proteins as major storage
reserves. To evaluate metabolic fluxes during embryo development, we
have established conditions for stable isotope labeling of cultured
embryos under steady-state conditions. Sucrose supplied via the
endosperm is considered to be the main carbon and energy source for
seed metabolism. However, in addition to 220 to 270 mM
carbohydrates (sucrose, glucose, and fructose), analysis of endosperm
liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid. Therefore, a labeling approach with multiple
carbon sources is a precondition to quantitatively reflect fluxes of
central carbon metabolism in developing embryos. Mid-cotyledon stage
B. napus embryos were dissected from plants and cultured
for 15 d on a complex liquid medium containing
13C-labeled carbohydrates. The 13C enrichment
of fatty acids and amino acids (after hydrolysis of the seed proteins)
was determined by gas chromatography/mass spectrometry. Analysis
of 13C isotope isomers of labeled fatty acids and
plastid-derived amino acids indicated that direct glycolysis provides
at least 90% of precursors of plastid acetyl-coenzyme A (CoA).
Unlabeled amino acids, when added to the growth medium, did not reduce
incorporation of 13C label into plastid-formed fatty acids,
but substantially diluted 13C label in seed protein.
Approximately 30% of carbon in seed protein was derived from exogenous
amino acids and as a consequence, the use of amino acids as a carbon
source may have significant influence on the total carbon and energy
balance in seed metabolism. 13C label in the terminal
acetate units of C20 and C22 fatty acids that
derive from cytosolic acetyl-CoA was also significantly diluted by
unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more
complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the
growth medium did not dilute 13C label incorporation into
fatty acids or proteins and can be ruled out as a source of carbon for
the major storage components of B. napus embryos.
1
This work was supported by the Department of
Energy (grant no. DE-FG02-87ER13729), by the National Science
Foundation (grant no. MCB 98-17882), and by the Michigan Agricultural
Experiment Station.
*
Corresponding author; e-mail Ohlrogge{at}msu.edu; fax
517-353-1926.
© 2002 American Society of Plant Physiologists
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