Auxin biosynthesis was analyzed in a maize (Zea mays) kernel culture system in which the seeds develop under physiological conditions similar to the in vivo situation. This system was modified for precursor feeding experiments. Tryptophan (Trp) is efficiently incorporated into indole-3-acetic acid (IAA) with retention of the 3,3Ј bond. Conversion of Trp to IAA is not competed by indole. Labeling with the general precursors [U-13 C 6 ]glucose and [1,2-13 C 2 ]acetate followed by retrobiosynthetic analysis strongly suggest that Trp-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. The synthesis of IAA from indole glycerol phosphate and IAA formation via condensation of indole with an acetyl-coenzyme A or phosphoenolpyruvate derived metabolite can be excluded.

Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) Plant metabolism is a complex network of many interconnected reactions and metabolites (Fien et al., 2000). For the analysis of metabolic networks, it is important to study metabolic pathways not only on the level of isolated genes or enzymes but also to quantify metabolite flux, which is involved in the formation of sink metabolites, such as starch.The biosynthesis of starch in the storage tissue of monocotyledonous plants has been studied in detail (for review, see Neuhaus and Emes, 2000). In maize (Zea mays), Suc from source leaves is imported into the developing cob tissue and converted into a mixture of Fru and UDP-Glc in the cytosol of endosperm cells (Chourey and Nelson, 1976;Chourey et al., 1998). UDP-Glc is converted into activated hexoses (i.e. Glc-1-P and Glc-6-P), which have been reported as starch precursors in various species. In maize, Glc-1-P is converted to the starch precursor ADP-Glc, which is transported into the plastid (Shannon et al., 1998). In contrast to the well-characterized import of activated hexoses into the amyloplasts as starch precursors, there is little evidence for the incorporation of trioses into starch (Neuhaus and Emes, 2000). It is therefore conceivable that starch is formed from intact C 6 units derived from cleaved Suc. However, in different systems, redistribution between C-1 and C-6 of Glc moieties of starch was observed, indicating metabolic cycling between trioses and hexoses in the cytosol (Hatzfeld and Stitt, 1990;Viola et al., 1991;Dieuaide-Noubhani et al., 1995;Krook et al., 1998). This phenomenon is also observed in starchaccumulating organs of cereals. In wheat (Triticum aestivum), 15% to 20% redistribution of 13 C-label between C-1 and C-6 of Glc recovered from starch was observed (Keeling et al., 1988). In maize, randomization of the carbon moieties of starch was detected using [1-14 C]Glc that was injected into developing kernels (Hatzfeld and Stitt, 1990).In this paper, we determine the metabolic history of monosaccharide units before their incorporation into starch by retrobiosynthetic NMR analysis (Eisenreich et al., 1993), a technique that is nonintrusive and nondestructive (Szyperski, 1995;Schmidt et al., 1998;Fiaux et al., 1999;Park et al., 1999;Eisenreich and Bacher, 2000;Glawischnig et al., 2001 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi