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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #4)

Current understanding of the factors regulating methionine content in vegetative tissues of higher plants by Rachel Amir (pp. 917-931).

Methionine is a nutritionally essential, sulfur-containing amino acid found in low levels in plants, which often limits its value as a source of dietary protein to humans and animals. Methionine is also a fundamental metabolite in plant cells since, through its first metabolite, S-adenosylmethionine (SAM), it controls the level of several key metabolites, such as ethylene, polyamines and biotin. SAM is also the primary methyl group donor that regulates different processes in plants. Despite its nutritional and regulatory significance, the factors regulating methionine content in plants are not fully known. In this review, we summarize the current knowledge and recent progress made in our understanding of the methionine metabolism. The enzymes and substrates that regulate methionine synthesis were described, as well as the influences of the catabolic pathways of methionine on its content. The current effort to tailor an improvement of methionine content in vegetative tissues with minimal interference in plant growth and productivity is described as well. The accumulated knowledge has provided new insights into the control of methionine level in plants and, in some cases, has resulted in significant improvements in the nutritional value of plants.

Keywords: Aspartate family; Methionine metabolism; Methionine-rich storage proteins; Nutritional improvement; Regulation; S-adenosylmethionine

Interdependence of threonine, methionine and isoleucine metabolism in plants: accumulation and transcriptional regulation under abiotic stress by Vijay Joshi; Je-Gun Joung; Zhangjun Fei; Georg Jander (pp. 933-947).

Pathways regulating threonine, methionine and isoleucine metabolism are very efficiently interconnected in plants. As both threonine and methionine serve as substrates for isoleucine synthesis, their synthesis and catabolism under different developmental and environmental conditions also influence isoleucine availability. Together, methionine gamma-lyase and threonine deaminase maintain the isoleucine equilibrium in plants under varied substrate availabilities. Isoleucine and the two other branched-chain amino acids (BCAAs) (leucine and valine) share four common enzymes in their biosynthesis pathways and thus are coordinately regulated. Induction of free amino acids as osmolytes in response to abiotic stress is thought to play a role in plant stress tolerance. In particular, the accumulation of BCAAs is induced many-fold during osmotic stress. However, unlike in the case of proline, not much research has been focused on understanding the function of the response involving BCAAs. This review describes pathways influencing branched-chain amino acid metabolism and what is known about the biological significance of their accumulation under abiotic stress. A bioinformatics approach to understanding the transcriptional regulation of the genes involved in amino acid metabolism under abiotic stress is also presented.

Keywords: Methionine; Threonine; Isoleucine; Abiotic stress; Regulation

Proline metabolism and transport in plant development by Silke Lehmann; Dietmar Funck; László Szabados; Doris Rentsch (pp. 949-962).

Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plants.

Keywords: Proline; Plant; Metabolism; Transport; Regulation; Development

From sulfur to homoglutathione: thiol metabolism in soybean by Hankuil Yi; Geoffrey E. Ravilious; Ashley Galant; Hari B. Krishnan; Joseph M. Jez (pp. 963-978).

Sulfur is an essential plant nutrient and is metabolized into the sulfur-containing amino acids (cysteine and methionine) and into molecules that protect plants against oxidative and environmental stresses. Although studies of thiol metabolism in the model plant Arabidopsis thaliana (thale cress) have expanded our understanding of these dynamic processes, our knowledge of how sulfur is assimilated and metabolized in crop plants, such as soybean (Glycine max), remains limited in comparison. Soybean is a major crop used worldwide for food and animal feed. Although soybeans are protein-rich, they do not contain high levels of the sulfur-containing amino acids, cysteine and methionine. Ultimately, unraveling the fundamental steps and regulation of thiol metabolism in soybean is important for optimizing crop yield and quality. Here we review the pathways from sulfur uptake to glutathione and homoglutathione synthesis in soybean, the potential biotechnology benefits of understanding and modifying these pathways, and how information from the soybean genome may guide the next steps in exploring this biochemical system.

Keywords: Soybean; Sulfur; Cysteine; Glutathione; Homoglutathione; Metabolism; Seed; Biotechnology; Food and feed

High-lysine maize: the key discoveries that have made it possible by R. A. Azevedo; P. Arruda (pp. 979-989).

Forty-five years ago, a paper published by Mertz et al. (Science 145:279–280, 1964) initiated a revolution in the history of plant protein quality and affected dramatically the study of cereal crop storage proteins. The observation of the high lysine content of the endosperm of the opaque-2 (o2) maize mutant was a key factor in bringing about a new concept in the production of cereal seeds with a high nutritional value. It has been a long and very interesting road with astonishing results over these 45 years. We are now probably about to see the release of commercially engineered high-lysine maize lines. We have decided to pinpoint some key contributions to the science behind high-lysine plants and concentrated on the research done on maize, which is possibly the most complete and simple example to illustrate the advances achieved. However, studies on other plant species such as barley and model species such as tobacco are totally relevant and will be briefly addressed.

Keywords: Aspartic acid; Corn; Isoleucine; Lysine; Maize; Methionine; Threonine

Identification of genes regulated by ammonium availability in the roots of maritime pine trees by Javier Canales; Arantxa Flores-Monterrosso; Marina Rueda-López; Concepción Avila; Francisco M. Cánovas (pp. 991-1001).

Conifers have a preference for ammonium over nitrate as the main inorganic nitrogen source. However, it is unknown how changes in nitrogen nutrition may affect transcription profiles. In this study, microarray analysis and suppressive subtraction hybridization were used to identify differentially expressed genes in the roots of maritime pine exposed to changes in ammonium availability. A total of 225 unigenes that were differentially regulated by changes in ammonium nutrition were identified. Most of the unigenes were classified into seven functional categories by comparison with sequences deposited in the databases. A significant proportion of these genes were encoded for ammonium-regulated proteins of unknown functions. The differential expression of selected candidate genes was further validated in plants subjected to ammonium excess/deficiency. The transcript levels of representative genes were compared in maritime pine roots, 1, 15 and 35 days after nutritional treatments. Gene expression patterns suggest the existence of potential links between ammonium-responsive genes and genes involved in amino acid metabolism, particularly in asparagine biosynthesis and utilization. Functional analyses and exploration of the natural variability in maritime pine populations for a number of relevant genes are underway.

Keywords: Pinus; Conifers; Ammonium nutrition; Ammonium-responsive genes; Amino acid biosynthesis; Glutamine; Asparagine

Reverse genetics approach to characterize a function of NADH-glutamate synthase1 in rice plants by Wataru Tamura; Yusuke Hidaka; Mayumi Tabuchi; Soichi Kojima; Toshihiko Hayakawa; Tadashi Sato; Mitsuhiro Obara; Mikiko Kojima; Hitoshi Sakakibara; Tomoyuki Yamaya (pp. 1003-1012).

Rice plants grown in anaerobic paddy soil prefer to use ammonium ion as an inorganic nitrogen source for their growth. The ammonium ions are assimilated by the coupled reaction of glutamine synthetase (GS) and glutamate synthase (GOGAT). In rice, there is a small gene family for GOGAT: there are two NADH-dependent types and one ferredoxin (Fd)-dependent type. Fd-GOGAT is important in the re-assimilation of photorespiratorily generated ammonium ions in chloroplasts. Although cell-type and age-dependent expression of two NADH-GOGAT genes has been well characterized, metabolic function of individual gene product is not fully understood. Reverse genetics approach is a direct way to characterize functions of isoenzymes. We have isolated a knockout rice mutant lacking NADH-dependent glutamate synthase1 (NADH-GOGAT1) and our studies show that this isoenzyme is important for primary ammonium assimilation in roots at the seedling stage. NADH-GOGAT1 is also important in the development of active tiller number, when the mutant was grown in paddy field until the harvest. Expression of NADH-GOGAT2 and Fd-GOGAT in the mutant was identical with that in wild-type, suggesting that these GOGATs are not able to compensate for NADH-GOGAT1 function.

Keywords: Ammonium assimilation; Glutamate synthase; Glutamine synthetase; Oryza sativa L.; Retrotransposon; Rice

Comparative metabolomics charts the impact of genotype-dependent methionine accumulation in Arabidopsis thaliana by Miyako Kusano; Atsushi Fukushima; Henning Redestig; Makoto Kobayashi; Hitomi Otsuki; Hitoshi Onouchi; Satoshi Naito; Masami Yokota Hirai; Kazuki Saito (pp. 1013-1021).

Methionine (Met) is an essential amino acid for all organisms. In plants, Met also functions as a precursor of plant hormones, polyamines, and defense metabolites. The regulatory mechanism of Met biosynthesis is highly complex and, despite its great importance, remains unclear. To investigate how accumulation of Met influences metabolism as a whole in Arabidopsis, three methionine over-accumulation (mto) mutants were examined using a gas chromatography–mass spectrometry-based metabolomics approach. Multivariate statistical analyses of the three mto mutants (mto1, mto2, and mto3) revealed distinct metabolomic phenotypes. Orthogonal projection to latent structures–discriminant analysis highlighted discriminative metabolites contributing to the separation of each mutant and the corresponding control samples. Though Met accumulation in mto1 had no dramatic effect on other metabolic pathways except for the aspartate family, metabolite profiles of mto2 and mto3 indicated that several extensive pathways were affected in addition to over-accumulation of Met. The pronounced changes in metabolic pathways in both mto2 and mto3 were associated with polyamines. The findings suggest that our metabolomics approach not only can reveal the impact of Met over-accumulation on metabolism, but also may provide clues to identify crucial pathways for regulation of metabolism in plants.

Keywords: mto ; Metabolite profiling; Polyamines; Multivariate statistical analysis

Principal transcriptional regulation and genome-wide system interactions of the Asp-family and aromatic amino acid networks of amino acid metabolism in plants by Hadar Less; Ruthie Angelovici; Vered Tzin; Gad Galili (pp. 1023-1028).

Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energy-associated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058–2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156–167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain branched pathways requiring extensive regulation of fluxes between the different branches. Additional views on the biochemistry, regulation and functional significance of the Asp-family and aromatic amino acid networks and some of their associated metabolites that are discussed in the present report, as well as the nutritional importance of Lys and Trp to human and farm animals, and attempts to improve Lys level in crop plants, can be obtained from the following reviews as examples (Radwanski and Last in Plant Cell 7:921–934, 1995; Halkier and Gershenzon in Annu Rev Plant Biol 57:303–333, 2006; Ufaz and Galili in Plant Physiol 147:954–961, 2008; Jander and Joshi in Mol Plant 3:54–65, 2010).

Keywords: Amino acids; Aspartate family pathway; Lysine; Aromatic amino acids; Bioinformatics; Plants

Impact of sulfur starvation on cysteine biosynthesis in T-DNA mutants deficient for compartment-specific serine-acetyltransferase by Stephan Krueger; Andrea Donath; M. Carmen Lopez-Martin; Rainer Hoefgen; Cecilia Gotor; Holger Hesse (pp. 1029-1042).

Sulfur plays a pivotal role in the cellular metabolism of many organisms. In plants, the uptake and assimilation of sulfate is strongly regulated at the transcriptional level. Regulatory factors are the demand of reduced sulfur in organic or non-organic form and the level of O-acetylserine (OAS), the carbon precursor for cysteine biosynthesis. In plants, cysteine is synthesized by action of the cysteine–synthase complex (CSC) containing serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL). Both enzymes are located in plastids, mitochondria and the cytosol. The function of the compartmentation of the CSC to regulate sulfate uptake and assimilation is still not clearly resolved. To address this question, we analyzed Arabidopsis thaliana mutants for the plastidic and cytosolic SAT isoenzymes under sulfur starvation conditions. In addition, subcellular metabolite analysis by non-aqueous fractionation revealed distinct changes in subcellular metabolite distribution upon short-term sulfur starvation. Metabolite and transcript analyses of SERAT1.1 and SERAT2.1 mutants [previously analyzed in Krueger et al. (Plant Cell Environ 32:349–367, 2009)] grown under sulfur starvation conditions indicate that both isoenzymes do not contribute directly to the transcriptional regulation of genes involved in sulfate uptake and assimilation. Here, we summarize the current knowledge about the regulation of cysteine biosynthesis and the contribution of the different compartments to this metabolic process. We relate hypotheses and views of the regulation of cysteine biosynthesis with our results of applying sulfur starvation to mutants impaired in compartment-specific cysteine biosynthetic enzymes.

Keywords: Serine acetyltransferase; O-acetylserine-(thiol)-lyase; CSC complex; SAT mutants; Non-aqueous fractionation

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress by Marcio Rocha; Ladaslav Sodek; Francesco Licausi; Muhammad Waqar Hameed; Marcelo Carnier Dornelas; Joost T. van Dongen (pp. 1043-1053).

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

Keywords: Glycine max ; Soybean; Alanine aminotransferase; Hypoxic stress; Waterlogging; Nitrogen fertilisation

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth by Ronan Sulpice; Agata Sienkiewicz-Porzucek; Sonia Osorio; Ina Krahnert; Mark Stitt; Alisdair R. Fernie; Adriano Nunes-Nesi (pp. 1055-1066).

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F _v/F _m), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.

Keywords: Cytosolic isocitrate dehydrogenase; Solanum lycopersicum ; Mitochondria; TCA cycle; Nitrogen metabolism; Amino acid biosynthesis

Widely targeted metabolomics and coexpression analysis as tools to identify genes involved in the side-chain elongation steps of aliphatic glucosinolate biosynthesis by Doris Albinsky; Yuji Sawada; Ayuko Kuwahara; Mutsumi Nagano; Akiko Hirai; Kazuki Saito; Masami Yokota Hirai (pp. 1067-1075).

Amino acid and glucosinolate biosynthesis are two interdependent pathways; amino acid synthesis as a part of primary metabolism provides the precursors for glucosinolate biosynthesis in secondary metabolism. In our previous studies, the combination of coexpression analysis and metabolite profiling led to the identification of genes and key regulators involved in glucosinolate biosynthesis. Moreover, the integration of transcriptome and metabolome data of sulphur-deprived Arabidopsis plants revealed coordinate changes in the expression profiles of genes involved in glucosinolate and amino acid metabolism.This review provides an overview of our recent studies involving Arabidopsis mutant plants that exhibit impairment in the side-chain elongation process occurring during aliphatic glucosinolate biosynthesis by means of coexpression analysis and a novel metabolite profiling approach based on ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (UPLC-TQMS) (Sawada et al. 2009a). Thus, this review highlights the advantages of the omics-based approach in identifying genes involved in glucosinolate biosynthesis.

Keywords: Glucosinolate biosynthesis; Widely targeted metabolomics; Batch-learning self-organising map (BL-SOM); Side-chain elongation; Leucine biosynthesis

Enzymes of cysteine synthesis show extensive and conserved modifications patterns that include N^α-terminal acetylation by Markus Wirtz; Corinna Heeg; Arman Allboje Samami; Thomas Ruppert; Rüdiger Hell (pp. 1077-1086).

Biosynthesis of cysteine is a two-step process in higher plants subsequently catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which are present in cytosol, plastids and mitochondria. Recently, the distribution of SAT and OAS-TL in these subcellular compartments was shown to be crucial for efficient cysteine synthesis in Arabidopsis thaliana. In this study, the abundances of OAS-TLs were quantified independently by immunological detection in crude protein extracts and by SAT affinity purification (SAP) of OAS-TL. OAS-TL A and B were evidenced to be the most abundant isoforms in all analyzed tissues, which is consistent with micro array-based transcript analyses. Application of SAP to Arabidopsis revealed significant modification of the major OAS-TL isoforms present in cytosol, plastids and mitochondria into up to seven subspecies. Specific OAS-TL isoforms were found to be differentially modified in the leaves, roots, stem and cell culture. Sulphur deficiency did not alter modification of OAS-TL proteins purified from cell culture that showed the highest complexity of OAS-TL modifications. However, the pattern of OAS-TL modification was found to be stable within an analyzed tissue, pointing not only to a high reproducibility of SAP but likely biological significance of each subspecies. The most abundant OAS-TL subspecies in cytosol and plastids were subject of N-terminal processing followed by acetylation of the newly originated N-terminus. The mode of N^α-terminal acetylation of OAS-TL and its possible biological function are discussed.

Keywords: Sulphur metabolism; Cysteine synthesis; Post-translational modification; Co-translational modification

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #4)