Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance

Abstract

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

Quantification of Compartmented Metabolic Fluxes in Developing Soybean Embryos by Employing Biosynthetically Directed Fractional 13C Labeling, Two-Dimensional [13C, 1H] Nuclear Magnetic Resonance, and Comprehensive Isotopomer Balancing

“…Since fluxes provide a quantitative depiction of carbon flow through competing metabolic pathways (Ratcliffe and Shachar-Hill, 2001), they are an important physiological characteristic akin to levels of transcripts, proteins, and metabolites (Sauer, 2004). Flux measurements and comparisons of fluxes between phenotypes can provide insights toward selection of appropriate metabolic engineering targets (Stephanopoulos, 1999(Stephanopoulos, , 2002Glawischnig et al, 2002) and toward the construction of predictive models of plant metabolism (Ratcliffe and Shachar-Hill, 2001), the necessity for which has been emphasized recently (Girke et al, 2003;Katagiri, 2003;Raikhel and Coruzzi, 2003).…”

“…For example, Wheeler et al (1998) delineated the pathway of ascorbic acid synthesis in higher plants from 14 C labeling data, and Krook et al (1998) showed that two separate oxidative pentose phosphate pathways (oxPPP) operate in the cytosol and the plastid, using 13 C enrichment data from Daucus carota cell suspensions. More recently, a suite of articles by Eisenreich and co-workers (Bacher et al, 1999;Glawischnig et al, 2000Glawischnig et al, , 2001Glawischnig et al, , 2002 reported the abundances of isotopomers of several isolated sink metabolites in maize (Zea mays) kernels. Although these articles demonstrated advances in label measurement technology, the inferences from them were either qualitative or semiquantitative.…”

Isotopolog perturbation techniques for metabolic networks: Metabolic recycling of nutritional glucose inDrosophila melanogaster

“…Such a perturbation will spread through the entire metabolic network of an organism via hundreds to thousands of enzyme-catalyzed reactions operating simultaneously. The spreading of the perturbation is best described as a relaxation process which can be used to study dynamic processes in complex metabolic networks with considerable accuracy (18).…”

“…18. Coupling constants and isotope shifts were determined from 13 C NMR spectra of commercially available 13 C-labeled glucoses (Table 1).…”

“…Coupling constants and isotope shifts were determined from 13 C NMR spectra of commercially available 13 C-labeled glucoses (Table 1). The analysis of 13 C enrichment and isotopolog composition was performed as described (18,25). Briefly, absolute 13 C abundances for C-1 were obtained from 13 C coupling satellites of H-1␣ (5.26 ppm) in the 1 H NMR spectrum.…”