Metabolite Fingerprinting in Transgenic Nicotiana tabacum Altered by the Escherichia coli Glutamate Dehydrogenase Gene

Abstract

With about 200 000 phytochemicals in existence, identifying those of biomedical significance is a mammoth task. In the postgenomic era, relating metabolite fingerprints, abundances, and profiles to genotype is also a large task. Ion analysis using Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR-MS) may provide a high-throughput approach to measure genotype dependency of the inferred metabolome if reproducible techniques can be established. Ion profile inferred metabolite fingerprints are coproducts. We used FT-ICR-MS-derived ion analysis to examine gdhA (glutamate dehydrogenase (GDH; EC 1.4.1.1)) transgenic Nicotiana tabacum (tobacco) carrying out altered glutamate, amino acid, and carbon metabolisms, that fundamentally alter plant productivity. Cause and effect between gdhA expression, glutamate metabolism, and plant phenotypes was analyzed by 13NH4+ labeling of amino acid fractions, and by FT-ICR-MS analysis of metabolites. The gdhA transgenic plants increased 13N labeling of glutamate and glutamine significantly. FT-ICR-MS detected 2 012 ions reproducible in 2 to 4 ionization protocols. There were 283 ions in roots and 98 ions in leaves that appeared to significantly change abundance due to the measured GDH activity. About 58% percent of ions could not be used to infer a corresponding metabolite. From the 42% of ions that inferred known metabolites we found that certain amino acids, organic acids, and sugars increased and some fatty acids decreased. The transgene caused increased ammonium assimilation and detectable ion variation. Thirty-two compounds with biomedical significance were altered in abundance by GDH including 9 known carcinogens and 14 potential drugs. Therefore, the GDH transgene may lead to new uses for crops like tobacco.

Tobacco Isoenzyme 1 of NAD(H)-Dependent Glutamate Dehydrogenase Catabolizes Glutamate in Vivo

“…GS2 is crucial for the reassimilation of this NH 4 1 , because mutants lacking this enzyme accumulate NH 4 1 and die (Wallsgrove et al, 1987). Although GDH activity is usually very low in mesophyll cells compared with GS2 (Bechtold et al, 1998), several studies have provided evidence that mesophyll GDH may reassimilate small amounts of photorespiratory NH 4 1 (Hartmann and Ehmke, 1980;Neeman et al, 1985;Yamaya et al, 1986 1 ] produce more labeled Glu and Gln than controls (Ameziane et al, 2000;Mungur et al, 2005). Combined, these results provide strong evidence that the pool of E. coli GDH in these transformants reassimilates photorespiratory NH 4 1 .…”

Nuclear Magnetic Resonance Spectroscopy-Based Metabolite Profiling of Transgenic Tomato Fruit Engineered to Accumulate Spermidine and Spermine Reveals Enhanced Anabolic and Nitrogen-Carbon Interactions

“…When high-resolution NMR methods are coupled to multivariate statistical analysis, the resulting partitions give unambiguous information about influences of nutritional and genetic backgrounds (Ratcliffe and Shachar-Hill, 2001;Mannina et al, 2003;Amato et al, 2004;Krishnan et al, 2005). Metabolite profiling, metabolomics, offers a powerful approach to monitor complexity of genetically modified crops and document any unintended consequences of a modified gene introduced in crops (Kuiper et al, 2001;Sumner et al, 2003;Tretheway, 2004;Mungur et al, 2005). We have used high-resolution NMR methods to generate an analysis of the principal, soluble constituents of wildtype and polyamine-accumulating transgenic tomato.…”

Transgenic Tomato Plants that Overexpress a Gene for NADH-dependent Glutamate Dehydrogenase (legdh1)

“…In transgenic tobacco plants with the gdhA transgene, 13 N labeling of glutamate and glutamine increased significantly (Mungur et al 2005). We also reported that the glutamate content of the fruits of the transgenic tomato plants that harbored a gdhA gene for NADPH-dependent GDH from Aspergillus nidulans was approximately twice that of the wild type (Kisaka and Kida 2003).…”