The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.
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"DAHP synthase, the first committed step in the pathway seems to be under various control mechanisms which differ greatly among species. For instance, it has been shown in Arabidopsis leaves that the enzyme requires Mn2+ and reduced thioredoxin to be active (Entus et al., 2002), which would mean decreased activity under non-reducing conditions. Although regulatory mechanisms acting on DAHP synthase seem to be extensive and not understood, a study by Pinto et al. (1988) provides, to some extent, insight into the observed phenotype in U-IN-2. "
[Show abstract][Hide abstract]ABSTRACT: Cytosolic (U-IN-2) or apoplasmic (U-IN-1) targeting of yeast invertase in potato tubers leads to reduced sucrose and increased glucose, but specific phenotypical changes are dependent on the subcellular targeting of the enzyme. U-IN-2 has a more severe phenotype with the most striking aspects being reduced starch and increased respiration. Despite extensive research, the regulatory mechanisms leading to these changes remain obscure. Technological advancements regarding potato transcriptional and genomic research presented us with the opportunity to revisit these lines and perform detailed gene expression analysis, in combination with metabolic profiling, to identify regulatory networks underlying the observed changes. Our results indicate that in both genotypes reduced UDP-glucose production is associated with a reduced expression of cell wall biosynthetic genes. In addition, U-IN-1 are characterised by elevated expression of senescence-associated genes, coupled to reduced expression of genes related to photosynthesis and the cytoskeleton. We provide evidence that increased respiration, observed specifically in U-IN-2, might be due to sugar signalling via released trehalose-6-phosphate inhibition of the SnRK1 complex. In both genotypes, expression of the plastidic G6P transporter (GPT) is significantly down-regulated, leading to a shift in the cytosolic to plastidic G6P ratio and hence might limit starch synthesis but also the oxidative pentose phosphate pathway. This might explain the observed changes in several additional plastidic pathways, most notably reduced expression of fatty acid biosynthetic genes and an accumulation of shikimate. A strict negative correlation between invertase and GPT expression could be observed in a wide range of potato tubers. This reciprocal regulation may be part of a more general switch controlling energy versus storage metabolism, suggesting that the fate of assimilate utilisation is coordinated at the level of sucrose degradation.
Full-text · Article · Feb 2012 · Frontiers in Plant Science
"Transcriptional regulation of the shikimate pathway and aromatic amino acid metabolism in plants has so far not been studied extensively . The expression of DAHPS encoding the fi rst enzyme of the shikimate pathway (Fig. 2) is induced by physical wounding and methyl-jasmonate (Devoto et al., 2005; Yan et al., 2007), infi ltration with pathogenic Pseudomonas syringae strains (Keith et al., 1991), redox state (Entus et al., 2002) and abscisic acid (Leonhardt et al., 2004; Catala et al., 2007). The expression of the gene encoding EPSPS is induced in response to infection by the necrotrophic fungal pathogen Botrytis cinerea (Ferrari et al., 2007) and by sulfate starvation (Nikiforova et al., 2003). "
[Show abstract][Hide abstract]ABSTRACT: The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three enzymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allosterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identified in Arabidopsis and other plant species.
Full-text · Article · Jan 2010 · The Arabidopsis Book
"Thus, exogenous amino acids may restore the growth of ntrc plants by altering the homeostasis among biochemical pathways in chloroplasts. Attempts to identify thiol-redox-regulated enzymes have revealed putative targets for the thioredoxin systems both in the shikimate pathway and in the biosynthesis of aromatic amino acids, including 3-deoxy-D- arabino-heptulosonate 7-phosphate synthase and Trp synthase b (Entus et al., 2002; Balmer et al., 2006; Kolbe et al., 2006). Currently, we are exploring whether these enzymes are subjects for thiol-redox regulation by NTRC. "
[Show abstract][Hide abstract]ABSTRACT: Chloroplast NADPH-thioredoxin reductase (NTRC) belongs to the thioredoxin systems that control crucial metabolic and regulatory pathways in plants. Here, by characterization of T-DNA insertion lines of NTRC gene, we uncover a novel connection between chloroplast thiol redox regulation and the control of photoperiodic growth in Arabidopsis (Arabidopsis thaliana). Transcript and metabolite profiling revealed severe developmental and metabolic defects in ntrc plants grown under a short 8-h light period. Besides reduced chlorophyll and anthocyanin contents, ntrc plants showed alterations in the levels of amino acids and auxin. Furthermore, a low carbon assimilation rate of ntrc leaves was associated with enhanced transpiration and photorespiration. All of these characteristics of ntrc were less severe when plants were grown under a long 16-h photoperiod. Transcript profiling revealed that the mutant phenotypes of ntrc were accompanied by differential expression of genes involved in stomatal development, chlorophyll biosynthesis, chloroplast biogenesis, and circadian clock-linked light perception systems in ntrc plants. We propose that NTRC regulates several key processes, including chlorophyll biosynthesis and the shikimate pathway, in chloroplasts. In the absence of NTRC, imbalanced metabolic activities presumably modulate the chloroplast retrograde signals, leading to altered expression of nuclear genes and, ultimately, to the formation of the pleiotrophic phenotypes in ntrc mutant plants.