Targeted Enhancement of Glutamate-to-γ-Aminobutyrate Conversion in Arabidopsis Seeds Affects Carbon-Nitrogen Balance and Storage Reserves in a Development-Dependent Manner    

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 g-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.Efficient assimilation of carbon (C) and nitrogen (N) is essential for optimal plant growth, productivity, and yield (Stitt, 1999), particularly in seeds in which the content of essential amino acids is low. Hence, a comprehensive understanding of seed development and metabolism is central to the enhancement of crop yield and quality. Seed development can be divided into three phases: cell division, maturation (accumulation of food reserves), and desiccation (Weber et al., 2005). The shift from one phase to the other is affected by sugars (Suc, hexoses, trehalose) and abscisic acid. In the early stages of seed development, maternal regulation is maintained via assimilate unloading and supply of nutrients, thereby sustaining cell division. During maturation, seed metabolism changes and storage reserves accumulate in expanding cells. At this time, photosynthetic activity is initiated in the seed, which is believed to improve oxygen supply and the energy state in the seed, thus counteracting increasingly hypoxic conditions (Borisjuk and Rolletschek, 2009). The maturation stage is followed by a phase termed "maturation drying," in which the metabolism of Arabidopsis (Arabidopsis thaliana) seeds shifts from a gen...

Identifying Genotype-by-Environment Interactions in the Metabolism of Germinating Arabidopsis Seeds Using Generalized Genetical Genomics

“…It has been postulated that it has roles in herbivore deterrence, pH and redox regulation, energy production, and maintenance of carbon/nitrogen balance (Bouché and Fromm, 2004). In a recent study, GABA levels in seeds were shown to increase by expressing Glu decarboxylase under a seed maturationspecific phaseolin promoter (Fait et al, 2011). In accordance with our findings, this resulted in smaller seed size and reduced seed vigor in T3 plants.…”

Seed-Specific Expression of a Feedback-Insensitive Form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis Stimulates Metabolic and Transcriptomic Responses Associated with Desiccation Stress

“…Altogether, the results point at major changes in endogenous metabolic processes that occur in SSE seeds during the transition from late reserve accumulation to desiccation when the phaseolin promoter is active (Fait et al, 2011). This assumption is based on previous results showing that amino acids and sugars accumulate specifically during seed desiccation in Arabidopsis wildtype seeds (Fait et al, 2006;Angelovici et al, 2010).…”

“…This promoter is the most abundant seed storage protein in the common bean (Phaseolus vulgaris) and is stringently turned off during all vegetative stages of plant development (Sundaram et al, 2013). It was demonstrated that this promoter is induced constitutively during the maturation and desiccation stages of seed development (Fait et al, 2011). The fragment containing the phaseolin promoter-AtD-CGS without its stop codon was then digested from the vector using SmaI and introduced with the same enzyme into the binary Ti plasmid pZP111 (9,735 bp) carrying three in-frame copies of hemagglutinin epitope tag and an octopine synthase terminator (Hacham et al, 2006;Supplemental Fig.…”

“…This promoter is the most abundant seed storage protein in the common bean (Phaseolus vulgaris) and is stringently turned off during all vegetative stages of plant development (Sundaram et al, 2013). It was also demonstrated that this promoter is induced constitutively during the maturation and desiccation stages of seed development (Fait et al, 2011). Seeds from 20 kanamycin-resistant plants were screened by immunoblot, and the two transgenic lines, SSE 1 and SSE 2, exhibiting the highest expression levels of the AtD-CGS transgene ( Fig.…”

“…Developing seeds can serve as an excellent system for studying metabolic regulation, including both metabolic and transcriptional parameters, since during their developmental stages seeds induce a massive synthesis of reserve compounds, including storage proteins, starch, and oil (Angelovici et al, 2010;Fait et al, 2011). Moreover, during their development and maturation, the seed's metabolism is associated with temporally distinct metabolic switches that alter their metabolic and transcriptomic profiles (Fait et al, 2006;Angelovici et al, 2009).…”