Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-13 C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [ 13 C, 1 H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 13 C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain SDs. A high flux was found through the oxidative pentose phosphate pathway (19.99 6 4.39 mmol d 21 cotyledon 21 , or 104.2 carbon mol 6 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 6 5.00 mmol d 21 cotyledon 21, or 113.2 carbon mol 626.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

Seed tissues (endosperm, embryo, and pericarp) are often separated into tissue‐enriched fractions by wet‐ or dry‐milling methods for use in food, feed, and industrial products. Seed tissue markers that are sensitive and readily quantifiable would be useful to optimize fractionation processes. To meet this need for tissue markers, we set out to produce and characterize different transgenic maize lines, each containing green fluorescent protein (GFP) in either endosperm or embryo. We examined mRNA transcripts using expressed sequence tag (EST) profiles of several major seed proteins and selected several with strong seed tissue preferences. Stably transformed maize lines were produced, and visual observation of fluorescence confirmed the presence of GFP in the desired tissues. To establish the utility of this grain for evaluating the effectiveness or separation efficiencies of fractionation processes, transgenic kernels were hand‐dissected into pericarp, endosperm, and embryo fractions and the GFP concentration in each fraction was determined. The GFP distribution between fractions of each transgenic event was calculated from GFP concentration and mass balance, which enabled the determination of GFP yield based on the hand‐dissection fractionation data and the amount of tissue contamination in each fraction. Our transgenic lines exhibited strong tissue preference for either embryo or endosperm. These lines should be useful for assessing separation efficiencies in maize fractionation processes.

Mutations in the Opaque2 (O2) gene of maize (Zea mays L.) improve the nutritional value of maize by reducing the level of zeins in the kernel. The phenotype of o2 grain is controlled by many modifier genes and is therefore strongly dependent on genetic background. We propose two hypotheses to explain differences in phenotypic severity in different genetic backgrounds: (i) Specific genes are differentially (o2 vs. wild‐type) expressed only in certain genotypes, and (ii) A set of genes are differentially expressed in all backgrounds, but the degree of differential expression differs in different backgrounds. To determine the extent to which these two hypotheses contribute to determining the severity of o2 in different genetic backgrounds, we identified transcripts likely to be differentially expressed in several genetic backgrounds by transcript profile comparison of endosperm RNA pools from eight o2 inbred lines and their wild‐type counterparts. The inbred line B46 was identified as having severe o2 phenotypes while the line M14 was identified as having minimal o2 phenotypes. The degree of wild‐type vs. o2 differential expression of transcripts was determined for these two lines. We found that most genes that are downregulated by o2 tend to be differentially expressed to a greater degree in B46 than in M14, while upregulated genes tend to be more highly differentially expressed in one genetic background or the other. Thus, hypothesis one functions more prominently for upregulated genes while hypothesis two functions most prominently for downregulated genes.

The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue‐specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry‐milling fractionation process. The two lines were dry‐milled to produce three fractions (bran‐, endosperm‐, and germ‐rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry‐milling procedure and our GFP‐containing grain, we determined that the endosperm‐rich fraction contained 4% germ tissue, the germ‐rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran‐rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP‐containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies.

In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein α-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of α-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29-47% greater in endosperm from transgene positive kernels. The content of some other amino acids was changed to a lesser extent. Taken together, these changes resulted in the transgenic endosperms having an improved amino acid balance relative to non-transgenic endosperms produced on the same ear. Kernel appearance, weight, density and zein content did not exhibit substantial differences in kernels expressing the transgene when compared to non-expressing siblings. Assessment of the antigenicity and impacts on animal health will be required in order to determine the overall value of this technology. Abstract In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein a-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of a-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29-47% greater in endosperm from transgene positive kernels. The content of some other...