Molecular and Functional Analyses Support a Role of Ornithine-δ-Aminotransferase in the Provision of Glutamate for Glutamine Biosynthesis during Pine Germination

Abstract

We report the molecular characterization and functional analysis of a gene (PsdOAT) from Scots pine (Pinus sylvestris) encoding Orn-d-aminotransferase (d-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other d-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsdOAT encodes a functional d-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsdOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of d-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsdOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsdOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that d-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.

Inorganic Nitrogen Form Determines Nutrient Allocation and Metabolic Responses in Maritime Pine Seedlings

“…Curiously, excess nitrate is mainly stored in the stem, an organ with a less important N assimilatory role than needles and roots, as suggested by the PpNR and PpNiR expression levels ( Figure 5). This observation, along with the observed content of L-arginine in the stem ( Figure S2) and the accumulation of L-asparagine in the seedling hypocotyl during the postgermination phase [38,45], suggests that the pine stem has a role as a store of N that accumulates not only vegetative storage proteins (VSPs) in the bark [46], but also free metabolites such as nitrate, L-arginine or L-asparagine. It is known that trees are able to transiently accumulate N in free amino acids [47]; in the future, it will be interesting to analyze the role of adult pine stems in the storage of N through the accumulation of small metabolites such as nitrate or free amino acids.…”

“…L-asparagine is an amino acid that is synthesized from L-glutamine and L-aspartate and is employed as a temporal N reserve when C is depleted [37]. Similarly, the accumulation of L-ornithine could suggest the active catabolism of L-arginine to mobilize the stored N and produce L-glutamate [38]. Interestingly, in the roots of the ammonium-fed seedlings, a greater amount of choline (4 times) was observed than in the roots of nitrate-fed plants (Table S1).…”

The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana

“…There are two proline synthetic pathways in higher plants (Charest and Phan 1990). One is the conversion of glutamate via the action of pyrroline-5-carboxylate synthetase (P5CS) (Voetberg and Sharp ⎯⎯⎯⎯ 1991, Turchetto-Zolet et al 2009, Thippeswamy et al 2010, while the other is the conversion of ornithine (Canas et al 2008, Funck et al 2008) via ornithine aminotransferase (OAT). Conversely, proline can be catabolized to glutamate via the action of proline dehydrogenase (PDH; Kiyosue et al 1996, Peng et al 1996, Verbruggen et al 1996.…”

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

“…It has been well‐documented that glutamate, glutamine, argine, proline are important components of proteins and 49 genes involved in their metabolism (Osband and Cashon, 1990; Ogata et al, 1999; Doniger et al, 2003; Wang et al, 2007). GLS2 (glutaminase 2), OAT (ornithine aminotransferase) and ALDH4A1 (aldehyde dehydrogenase 4 family member A1), PRODH (proline dehydrogenase) catalysed glutamine, ornithine and proline into glutamate, respectively (Crabtree and Newsholme, 1970; James et al, 1998; Cañas et al, 2008). GLUD1 (glutamate dehydrogenase 1), GOT1 (glutamic‐oxaloacetic transaminase 1), GOT2 (glutamic‐oxaloacetic transaminase 2), GPT (glutamic‐pyruvate transaminase) or GPT2 (glutamic‐pyruvate transaminase 2) catabolized glutamate to ketoglutarate, which was completely degraded through TCA (tricarboxylic acid) cycle (Zhang et al, 2004).…”