Deciphering the Role of Aspartate and Prephenate Aminotransferase Activities in Plastid Nitrogen Metabolism

Abstract

Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development.

Transgenic poplar expressing the pine GS1a show alterations in nitrogen homeostasis during drought

“…3). Interestingly, Maeda et al (2011) and Graindorge et al (2010) report that plant prokaryotictype AATs display not only AAT activity but also prephenate aminotransferase activity, which is a key step in the biosynthesis of aromatic amino acids in plastids, a metabolic link between glutamate and phenylpropanoid metabolism (de la Torre et al, 2014).…”

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

“…Unlike the major effect of ADT suppression on the levels of Phe and Phe-derived compounds (Maeda et al, 2010, Corea et al, 2012a, 2012b, PPA-AT suppression led to a minor reduction in Phe levels (Maeda et al, 2011;de la Torre et al, 2014). However, this was due to compensatory operation of Phe biosynthesis via alternative phenylpyruvate pathway mediated by a separate phenylpyruvate aminotransferase (Yoo et al, 2013), which is homologous to tyrosine aminotransferase and has a broad substrate specificity (Gelfand and Steinberg, 1977;Gonda et al, 2010;Lee and Facchini, 2011;Riewe et al, 2012).…”

“…However, this was due to compensatory operation of Phe biosynthesis via alternative phenylpyruvate pathway mediated by a separate phenylpyruvate aminotransferase (Yoo et al, 2013), which is homologous to tyrosine aminotransferase and has a broad substrate specificity (Gelfand and Steinberg, 1977;Gonda et al, 2010;Lee and Facchini, 2011;Riewe et al, 2012). Thus, besides having a role in plastidic aspartate metabolism owing to its Asp-AT activity (de la Torre et al, 2014), the identified plant PPA-ATs are indeed involved in Phe biosynthesis (Maeda et al, 2011;Yoo et al, 2013;de la Torre et al, 2014). The strict substrate specificity of plant PPA-ATs toward prephenate among the three aromatic keto acid substrates in Phe and Tyr biosynthesis (Graindorge et al, 2010;Maeda et al, 2011) also suggest that plant PPA-AT directs carbon flow specifically from prephenate toward arogenate and hence serves as an ideal marker to investigate the molecular and biochemical evolution of the plant arogenate pathway.…”

Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions

“…Lignin levels were determined by the thioacidolysis method [35] and adapted as previously described [36]. …”