Abstract
In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca(2+)-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation.
Highlights
In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form g-aminobutyric acid (GABA)
Informative, radiolabeling studies are limited in their applicability to monitoring the degree of labeling across a range of metabolites; as a result, stable isotopes coupled with mass spectrometry (MS) or NMR have been increasingly used in the last decade for pathway elucidation and flux analysis (Sauer, 2006, and refs. therein)
To elucidate whether the GABA shunt operates as a nexus between C and N metabolism upon seed imbibition prior to seed germination, we evaluated the metabolic fate of Glu in Arabidopsis seeds freshly stratified and 1 d after stratification (DAS; when germination has been initiated); an adapted gas chromatography (GC)MS-based protocol (Roessner-Tunali et al, 2004) was used in combination with a feeding 13C-substrate
Summary
Glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form g-aminobutyric acid (GABA). Genes involved in the regulation of germination were up-regulated Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation. GABA is derived from Glu via the action of glutamate decarboxylase (GAD), the enzyme responsible for the first step in the GABA shunt metabolic pathway (Fig. 1; Bown and Shelp, 1997) In plant species such as petunia (Petunia hybrida; Chen et al, 1994; Arazi et al, 1995), soybean (Glycine max; Snedden et al, 1995), tobacco (Nicotiana tabacum; Baum et al, 1996), and Arabidopsis (Turano and Fang, 1998; Zik et al, 1998), the GAD polypeptides contain C-terminal extensions, ranging between 30 and 50 amino acids, that are not present in the bacterial GAD enzyme and. It has been suggested that GABA constitutes a readily accessible nontoxic reserve of C and N for amino acid metabolism and tricarboxylic acid (TCA) cycle activity, which is of particular relevance during
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