Altered Amino Acid Metabolism in Glutamine Dumper1 Plants

Abstract

Amino acid metabolism lies at the crossroad between nitrogen assimilation, carbon fixation and secondary metabolism. Because of this central position in plant metabolism, amino acid metabolism is tightly regulated by numerous factors to match both demand from the organs and availability of reduced carbon and inorganic nitrogen. While the amino acid biosynthesis enzymes have been shown to be regulated at the transcriptional and protein levels, the genes involved in amino acid sensing, signal transduction and regulation have not yet been identified. The overexpression of Glutamine Dumper1 leads to a large increase in the amino acid content of the plant and, as we show here, to insensitivity to externally applied amino acids. This phenotype is reminiscent of that of the pig1-1 mutant proposed to display a deregulated metabolism. These data suggest that GDU1 is involved in the regulation of amino acid metabolism and transport. As published previously, the analysis of deletion mutants proves that GDU1's VIMAG domain is important for the function of the protein. The present data show furthermore that other regions participate to this function.

Key Words:

• amino acid metabolism
• Arabidopsis thaliana
• regulation
• structure-function relationships
• biosynthesis enzyme
• feedback regulation

This article refers to:

Regulation of Amino Acid Metabolism in Plants

Contrary to animals, plants are able to synthesize all amino acids using ammonium and intermediaries from the carbohydrate metabolism. Amino acid synthesis is thus intimately linked to nitrogen absorption efficiency and photosynthetic activity. The integration of these two parameters is achieved at the level of the regulation of the activity of the nitrate reductase which provides the metabolism with ammonium.1

The various pathways leading to the synthesis of amino acids in plants are quite well described.2 Six main tracks can be identified depending on the starting carbohydrate molecule, which comes from the glycolysis, the tricarboxylic acid cycle or the pentose phosphate pathway (Fig. 1). The regulation of the activity of the biosynthesis branches of the branched-chain amino acids (deriving from oxaloacetate) and of the aromatic amino acids (deriving from phosphoenolpyruvate) has been extensively studied. Importantly, some biosynthetic enzymes have been shown to be positively or negatively regulated by end-products (Fig. 1). As a consequence, plant or cell growth is inhibited by external application of an amino acid that drives a negative feedback effect: this amino acid inhibits the activity of one pathway, resulting in the depletion in the other amino acid(s) produced by this branch.3 Transcriptional regulation by different stresses has been shown for some enzymes of the aromatic amino acid pathway4,5 and the enzymes involved in the production and degradation of proline.6 Furthermore, experimental evidence shows that the activities of the different pathways are adjusted to one another, and the modification of the activity of one pathway leads to the modification of the content in most of the other amino acids.7,8 Guyer et al.9 also showed a clear enhancement of the accumulation of transcripts for enzymes of several pathways in response to an inhibition of histidine synthesis. A cross-pathway regulation of the metabolism seems then to exist in plant, but it is not yet clear whether this regulation takes place at the transcriptional level or/and at the post-translational level. It can be assumed that this cross-pathway regulation depends on amino acid sensing and signal transduction activities. However, the underlying mechanisms and the genes involved remain to be identified.

Resistance of Two Arabidopsis Mutants to High Concentrations of Amino Acids

Until recently, the mutations or manipulations that lead to modifications of amino acid content in plants concerned genes that encode enzymes of amino acid metabolism. In 2004, two papers described the isolation of mutants showing an increase in amino acid content: pig1-1 and gdu1-1D.10,11 Unfortunately the gene responsible for the recessive pig1-1 mutation has not been identified. The Pig1⁻ plants present an increased amino acid metabolism leading to amino acid over-accumulation and insensitivity to elevated amino acid concentrations in the growth medium. The gdu1-1D mutation is caused by the overexpression of a small membrane protein, GDU1. Gdu1D⁻ plants present higher amino acid content and transport within the plant compared to the wild-type and secrete glutamine by the hydathodes.

The GDU1 protein contains two conserved domains: a membrane segment and the plant-specific “VIMAG” domain, present in the C-terminal part of the protein. We showed recently that the VIMAG domain is necessary for GDU1 to cause the Gdu1D⁻ phenotype when overexpressed in plants.12 The striking similarities between the phenotypes of both pig1-1 and gdu1-1D plants prompted us to compare the resistance of these mutants to toxic concentrations of amino acids. The growth of mutants overexpressing various mutated or truncated versions of GDU1 was also tested in that assay. The log1 plants bear a suppressor G to R mutation in the VIMAG domain of GDU1 in the gdu1-6D background, the ΔGDU1 plants over-express GDU1 lacking the VIMAG domain, and the C-GDU1 plants overexpress only the C-terminal domain of GDU1. These three genotypes do not present the Gdu1⁻ phenotype.12

The growth of wild type plants (WS and Col-7 ecotypes) is severely inhibited by the presence of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine (Fig. 2). pig1-1 plants were only susceptible to the branched-chain amino acids leucine, lysine and methionine. This is in good agreement with the fact that this mutant was identified by its resistance to phenylalanine, an aromatic amino acid.10 Strikingly, line gdu1-6D overexpressing the intact GDU1 protein presents no growth inhibition by any of the amino acids in the media, showing that these plants are even less susceptible to amino acids than pig1-1. A reason for this phenotype can be that the plants are less sensitive to the negative feedback effect of amino acids on the metabolic enzymes, or that they present like pig1-1 an increased rate of breakdown and synthesis of amino acids. This is supposed to be a consequence of a deregulation of amino acid homeostasis.10 It is important to note that mutations affecting amino acid transporters can lead to an amino acid resistant phenotype.13–15 Nevertheless, this tolerance is not accompanied by the dramatic changes in amino acid content of the plants seen for pig1-1 and gdu1-6D.10,11

The C-GDU1 plants display no Gdu1⁻ phenotype on soil.12 In good agreement with this fact, these plants behave exactly as the wild type on amino acid containing media. On the contrary, while the plants overexpressing a modified GDU1 protein (log1 and DGDU1) presented no visible phenotype on soil,12 they were found to be resistant to some amino acids (histidine, isoleucine, and valine, and also lysine for the log1 mutants). Despite the loss of most of its functionality by the mutation or the deletion of the VIMAG domain, GDU1 is thus still able to confer resistance to some exogenously applied amino acids. This shows that the VIMAG domain is not the only functional domain of GDU1. The sequence of the transmembrane domain is particularly conserved among GDU proteins,12 suggesting that it could bear part of the function of the protein. The involvement of less conserved, C-terminal stretches of amino acids in the function of GDU1 can however not be excluded.

The first conclusion from the experiment presented here is that although the VIMAG domain is necessary for many aspects of Gdu1⁻ phenotype, other regions of GDU1 are important for the function of the protein. But more importantly, this experiment emphasizes another component of the Gdu1D⁻ phenotype, namely the resistance to externally applied amino acids, which reveals a probable disturbance in the regulation of amino acid metabolism. We propose that the membrane protein GDU1 is involved in the regulation of the amino acid metabolism. GDU1 would be the first gene identified as involved in this process and the study of GDU1 function will help to shed light on an uncharacterized, but central, process of plant physiology.

Figures and Tables

Figure 1 Biochemical pathways for synthesis of amino acids in plants. Starting carbohydrate metabolites are boxed. Thick lines correspond to biochemical pathways and may correspond to more than one reaction. Dotted lines correspond to feedback inhibition (T bar) or activation (arrow) by a metabolite on an enzyme (black dots). (1) chorismate mutase; (2) anthranilate synthase; (3) aspartate kinase; (4) homoserine dehydrogenase; (5) dihydrodipicolinate synthase; (6) threonine synthase; (7) cystathionine gamma-synthase; (8) threonine deaminase; (9) acetohydroxy acid synthase; (10) 2-isopropylmalate synthase. αKG, α-ketoglutarate; PEP, phosphoenolpyruvate; PYR, pyruvate; OAA, oxaloacetate; 3-PG, 3-phosphoglycerate; PRPP, 5-phospho-α-D-ribosyl-1-pyrophosphate; SAM, S-adenosylmethionine. Standard three-letter code is used for amino acids. Adapted from reference 2.

Figure 2 Effect of externally applied amino acids on the growth of pig1-1 and GDU1 overexpressing plants. Seeds were surface sterilized and grown for three weeks on Murashige and Skoog medium (MS, Duchefa) containing 10 mmol/l of the various amino acids (except lysine: 5 mmol/l). (A) Example of growth test for five amino acids. (B) Qualitative summary of the growth on the various media. WS (Wassilewskija) is the wild type corresponding to the pig1-1 mutant while Col-7 is the wild type for all other lines.

Acknowledgements

The authors would like to thank Dr. A. Weber for kindly providing the pig1-1 mutant. The authors are supported by grants from the Deutsche Forschungsgemeinschaft to G.P. (PI 607/1-1 and PI 607/2-1).