The multifaceted role of aspartate-family amino acids in plant metabolism

Plants represent the major sources of human foods and livestock feeds, worldwide. However, the limited content of the essential amino acid lysine in cereal grains represents a major nutritional problem for human and for livestock feeding in developed countries. Optimizing the level of lysine in cereal grains requires extensive knowledge on the biological processes regulating the homeostasis of this essential amino acid as well as the biological consequences of this homeostasis. Manipulating biosynthetic and catabolic enzymes of lysine metabolism enabled an enhanced accumulation of this essential amino acid in seeds. However, this approach had a major effect on the levels of various metabolites of the tricarboxylic acid (TCA) cycle, revealing a strong interaction between lysine metabolism and cellular energy metabolism. Recent studies discussed here have shed new light on the metabolic processes responsible for the catabolism of lysine, as well as isoleucine, another amino acid of the aspartate-family pathway, into the TCA cycle. Here we discuss progress being made to understand biological processes associated with the catabolism of amino acids of the aspartate-family pathway and its importance for optimal improvement of the nutritional quality of plants.     

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD⁺/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.     

Metabolic engineering and profiling of rice with increased lysine

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback‐insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down‐regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12‐fold in leaves and ~60‐fold in seeds, substantially greater than the 2.5‐fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.     

A T-DNA insertion knockout of the bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase gene elevates lysine levels in Arabidopsis seeds

Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds.     

Citric Acid Cycle and Role of its Intermediates in Metabolism

The citric acid cycle is the final common oxidative pathway for carbohydrates, fats and amino acids. It is the most important metabolic pathway for the energy supply to the body. TCA is the most important central pathway connecting almost all the individual metabolic pathways. In this review article, introduction, regulation and energetics of TCA cycle have been discussed. The present study was carried out to review literature on TCA cycle.     

Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds

To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained approximately 12-fold or approximately 5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic approximately 80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to approximately 38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.     

Improving the levels of essential amino acids and sulfur metabolites in plants

Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.     

Nitric oxide, γ‐aminobutyric acid,and mannose pretreatment influence metabolic profiles in white clover under water stress

Nitric oxide (NO), γ‐aminobutyric acid (GABA), and mannose (MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside (SNP, a NO donor), GABA, and MAS improved plant growth under water‐sufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo‐inositol content; the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose, and glycerol under water deficit. In addition, citric acid was uniquely up‐regulated by SNP associated with tricarboxylic acid (TCA) cycle under water stress. GABA specially induced the accumulation of GABA, glycine, methionine, and aconitic acid related to GABA shunt, amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D‐pinitol in association with amino acids and sugars metabolism under water stress. SNP‐, GABA‐, and MAS‐induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover.     

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with ¹³C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes.     

Unraveling Physiological and Metabolomic Responses Involved in Phlox subulata L. Tolerance to Drought Stress

Metabolic responses are important for plant adaptation to abiotic stress. To investigate the responses of Phlox subulata L. to drought stress, we analyzed its physiological and metabolic changes using gas chromatography-mass spectrometer. Based on the physiological indices, P. subulata L. has tolerance to drought to some degree. Our results showed that there were a total of 30 key metabolites induced by drought stress, including amino acids, organic acids, sugars and sugar alcohols, nucleic acid and its derivatives, and other organic compounds. The glutamic acid-mediated proline biosynthesis pathway is continuously upregulated under drought stress, which could regulate osmotic pressure and maintain intracellular environmental stability. More secondary metabolites are used to increase glycolysis and tricarboxylic acid cycle, to accelerate energy production and to enhance the glutamic acid-mediated proline biosynthesis pathway, which are necessary to increase osmotic regulation. Prolonged drought stress induced progressive accumulation of compatible osmolytes, such as proline and inositol, sugars, and amino acids. Therefore, drought caused systemic alterations in metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolism of pyrimidine. These data suggest that plants may utilize these physiological and metabolomic adjustments as adaptive responses in the early stages of drought stress. These results deepen our understanding of the mechanisms involved in P. subulata L. drought tolerance, which will help improve the understanding of drought’s effects on plant systems.

     

Nitric oxide,g-aminobutyric acid,and mannose pretreatment influence metabolic profiles in white clover under water stress

Nitric oxide(NO), g-aminobutyric acid(GABA),and mannose(MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside(SNP, a NO donor),GABA, and MAS improved plant growth under watersufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo-inositol content;the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose,and glycerol under water deficit. In addition, citric acid was uniquely up-regulated by SNP associated with tricarboxylic acid(TCA) cycle under water stress. GABAspecially induced the accumulation of GABA, glycine,methionine, and aconitic acid related to GABA shunt,amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D-pinitol in association with amino acids and sugars metabolism under water stress. SNP-, GABA-, and MAS-induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover.     

Reappraisal of the 20th-century version of amino acid metabolism

In this article, we advocate the radical revision of the 20th-century version of amino acid metabolism as follows. (1) Classic studies on the incorporation of [15N]ammonia into glutamate, once considered to be an epoch-making event, are not distinctive proof of the ability of animals to utilize ammonia for the synthesis of alpha-amino nitrogen. (2) Mammalian glutamate dehydrogenase has been implicated to function as a glutamate-synthesizing enzyme albeit lack of convincing proof. This enzyme, in combination with aminotransferases, is now known to play an exclusive role in the metabolic removal of amino nitrogen and energy production from excess amino acids. (3) Dr. William C Rose's "nutritionally nonessential amino acids" are, of course, essential in cellular metabolism; the nutritional nonessentiality is related to their carbon skeletons, many of which are intermediates of glycolysis or the TCA cycle. Obviously, the prime importance of amino acid nutrition should be the means of obtaining amino nitrogen. (4) Because there is no evidence of the presence of any glutamate-synthesizing enzymes in mammalian tissues, animals must depend on plants and microorganisms for preformed alpha-amino nitrogen. This is analogous to the case of carbohydrates. (5) In contrast, individual essential amino acids, similar to vitamins and essential fatty acids, should be considered important nutrients that must be included regularly in sufficient amounts in the diet.     

Improvement of the nutritional quality of legume seed storage proteins by molecular breeding

Legume seeds contain a large amount of proteins and are one of the essential protein sources for humans and animals. However, the protein, in legume seeds is usually poor in sulfur-containing amino acids, and its nutritional value is lower than the protein from animal sources. Recently plant breeding has become available by the introduction of molecular biology, and a technique, called molecular breeding, was applied to the production of legume seeds that contain proteins with high nutritional quality. This review describes the expression of legume seed protein genes and the transformation of legume plants. Approaches to improve the legume seed storage protein will be discussed.     

Metabolic engineering of a complex biochemical pathway: The lysine and threonine biosynthesis as an example

The nutritional quality of crop plants is determined by their content in essential amino acids provided in food for humans or in feed for monogastric animals. Amino acid composition of crop–based diets can be improved via manipulation of the properties of key enzymes of amino acid biosynthetic pathways by mutation and transformation. We focused on the aspartate-derived amino acid pathway producing four essential amino acids: lysine, threonine, isoleucine and methionine. Genes encoding aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) that operate as key genes of the aspartate pathway have been cloned from Arabidopsis. Genetic and molecular studies revealed that at least five different ak genes are represented. Some of them were characterized in terms of gene and promoter structure, developmental expression and regulatory properties. In the case of dhdps, two quite identical genes have been identified and characterized at expression level. Mutated genes encoding a fully feedback-insensitive form of the DHDPS enzyme were obtained from Nicotiana sylvestris and Arabidopsis. Several chimeric constructs harbouring this mutated allele under the control of constitutive or seed-specific promoters were transferred via Agrobacterium or biolistics in various plant species. In all cases, lines with significant increase of free lysine content were obtained in vegetative organs, but the impact of the transgene in seeds is limited due to the presence of an active catabolic enzyme, lysine ketoreductase. These results show that, although dealing with a complex, highly regulated pathway, the overexpression of a single gene encoding a feedback-insensitive form of the key enzyme DHDPS exerts a significant effect on the carbon flux through the aspartate pathway towards lysine production.     

Metabolic responses of wheat roots to alkaline stress

Aims The aim of this study was to investigate the effects of alkaline stress on primary, secondary metabolites and metabolic pathways in the roots of wheat (Triticum aestivum). The results were used to evaluate the physiological adaptive mechanisms by which wheat tolerated alkali stress.Methods A pot experiment was carried out in the greenhouse. For each plastic pot, five wheat seeds were planted. After germination, seedlings were allowed to grow under controlled water and nutrient conditions for two months, then seedlings were exposed to alkaline stress (NaHCO₃-Na₂CO₃) for 12 days. The relative growth rate (RGR), absolute water content (AWC), metal elements, free cations and metabolites were measured.Important findings The alkaline stress caused the reduction of RGR and AWC. Alkaline stress caused a rapid increase of Na content with the concurrent decrease in K and Cl content, resulting in inhibited metal element accumulation and an ionic imbalance. In the present study, alkaline stress strongly enhanced Ca accumulation in wheat roots, suggesting that an increased Ca concentration can immediately trigger the salt overly sensitive (SOS)-Na exclusion system and reduce Na-associated injuries. Also, 70 metabolites, including organic acids, amino acids, sugars/polyols and others, behaved differently in the alkaline stress treatments according to a GC-MS analysis. The metabolic profiles of wheat were closely associated with alkaline-stress conditions. Alkaline stress caused the accumulation of organic acids, accompanied by the depletion of sugars/polyols and amino acids. Organic acids could play a central role in the regulation of intracellular pH by accumulating vacuoles to neutralize excess cations. Glycolysis and amino acid synthesis in roots were inhibited under salt stress while prolonged alkaline stress led to a progressive tricarboxylic acid (TCA) cycle. The severe negative effects of alkaline stress on sugar synthesis and storage may reflect the toxic levels of Na⁺ accumulating in plant cells in a high-pH environment, implying that the reactive oxygen species detoxification capacity was diminished by the high pH. A lack of NO₃^- in wheat roots can decrease synthase enzyme activities, limiting the synthesis of amino acids. Under salt stress, the TCA cycle and organic acid accumulation increased, but glycolysis and amino acid synthesis were inhibited in roots. Thus, energy levels and high concentrations of organic acids may be the key adaptive mechanisms by which wheat seedlings maintain their intracellular ion balance under alkaline stress.     

Growth inhibition by selenium is associated with changes in primary metabolism and nutrient levels in Arabidopsis thaliana

Although Selenium (Se) stress is relatively well known for causing growth inhibition, its effects on primary metabolism remain rather unclear. Here, we characterized both the modulation of the expression of specific genes and the metabolic adjustments in Arabidopsis thaliana in response to changes in Se level in the soil. Se treatment culminated with strong inhibition of both shoot and root growth. Notably, growth inhibition in Se‐treated plants was associated with an incomplete mobilization of starch during the night. Minor changes in amino acids levels were observed in shoots and roots of plants treated with Se whereas the pool size of tricarboxylic acid (TCA) cycle intermediates in root was not altered in response to Se. By contrast, decreased levels of organic acids involved in the first part of the TCA cycle were observed in shoots of Se‐treated plants. Furthermore, decreased expression levels of expansins and endotransglucosylases/endohydrolases (XHTs) genes were observed after Se treatment, coupled with a significant decrease in the levels of essential elements. Collectively, our results revealed an exquisite interaction between energy metabolism and Se‐mediated control of growth in Arabidopsis thaliana to coordinate cell wall extension, starch turnover and the levels of a few essential nutrients.     

Dynamic metabonomic responses of tobacco (Nicotiana tabacum) plants to salt stress

Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry.