氮代谢参与植物逆境抵抗的作用机理研究进展

近年来,植物所受到的诸如干旱、盐、高温、低氧、重金属胁迫和营养元素缺乏等环境胁迫越来越多,严重影响了植物的生长发育及作物的质量和产量。氮素是植物生长发育所需的必需营养元素,同时也是核酸、蛋白质和叶绿素的重要组成成分,其代谢过程与植物抵抗逆境的能力息息相关。氮代谢是指植物对氮素的吸收、同化和利用的全过程,是植物体内基础代谢途径之一。氮代谢主要从氮素吸收、同化及氨基酸代谢等方面参与植物的抗逆性,并通过调节离子吸收和转运、稳定细胞形态和蛋白质结构、维持激素平衡和细胞代谢水平、减少体内活性氧(reactive oxygen species,ROS)生成以及促进叶绿素合成等生理机制来影响植物抵抗非生物胁迫的能力。因此,提高植物在逆境下的氮代谢水平是减轻外界胁迫对其损伤的一种潜在途径。该文从氮素同化的基本途径出发,分别阐述了氮代谢在干旱胁迫、盐胁迫和高温胁迫等多个方面的逆境抵抗过程中的作用机理,为氮代谢参与植物抗逆性研究提供了有利参考。     

The role of mitochondria in leaf nitrogen metabolism

For optimal plant growth and development, cellular nitrogen (N) metabolism must be closely coordinated with other metabolic pathways, and mitochondria are thought to play a central role in this process. Recent studies using genetically modified plants have provided insight into the role of mitochondria in N metabolism. Mitochondrial metabolism is linked with N assimilation by amino acid, carbon (C) and redox metabolism. Mitochondria are not only an important source of C skeletons for N incorporation, they also produce other necessary metabolites and energy used in N remobilization processes. Nitric oxide of mitochondrial origin regulates respiration and influences primary N metabolism. Here, we discuss the changes in mitochondrial metabolism during ammonium or nitrate nutrition and under low N conditions. We also describe the involvement of mitochondria in the redistribution of N during senescence. The aim of this review was to demonstrate the role of mitochondria as an integration point of N cellular metabolism.     

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.     

Effects of nitrogen availability on pigmentation and carbon assimilation in the cyanobacterium Synechococcus sp. strain SH-94–5

Because pigments of phototrophs can be involved either in photosynthesis or photoprotection, pigmentation changes in response to nutrient availability can affect how cells interact with their solar environment. We investigated the impact of nitrogen availability both on pigmentation of the cyanobacterium Synechococcus sp. strain SH-94-5 and on carbon assimilation by this strain in the presence or absence of UV radiation. Pigmentation changes in strain SH-94-5 due to ammonium exhaustion included phycobiliprotein degradation, an exponential decline in chlorophyll a content, and a net increase in beta-carotene. Following its replenishment, ammonium stimulated non-photosynthetic carbon assimilation for several hours prior to the resumption of photosynthesis and growth. Carbon fixation during this lag phase was concurrent with the metabolism of glycogen reserves, and it is likely that inorganic carbon was incorporated into glycogen-derived carbon skeletons primarily for amino acid synthesis. In contrast, carbon fixation was almost exclusively photosynthetic during exponential growth. UV-A radiation (320-400 nm) inhibited photosynthetic but not non-photosynthetic carbon assimilation. Only growing cells were inhibited, and the disappearance of inhibition following nitrogen depletion appeared to result from the reduction of cellular photosensitizing targets below a threshold level rather than from the inactivation of photosynthesis.     

Impairment of brain mitochondrial function by reactive nitrogen species: the role of glutathione in dictating susceptibility

Mitochondrial enzymes involved in energy metabolism display varying degrees of sensitivity towards reactive nitrogen species such as peroxynitrite (ONOO-). With regards to the electron transport chain, cytochrome oxidase appears particularly sensitive. Inhibition of this component may lead to an increase in mitochondrial superoxide formation, exacerbation of cellular oxidative stress and further mitochondrial damage. Impairment of the electron transport chain may lead to a loss of membrane potential, ATP deficiency, opening of the permeability transition pore and the release of factors capable of initiating apoptosis. Reduced glutathione will react, via a number of diverse reactions, with reactive nitrogen species and hence is capable of limiting mitochondiral damage. Loss of brain glutathione may therefore be an important factor in those neurological conditions in which there is evidence of excessive nitric oxide formation and mitochondrial damage.     

Induction of capsaicinoid accumulation in placental tissues of Capsicum chinense Jacq. requires primary ammonia assimilation

The activities of primary ammonia assimilation enzymes were analyzed in isolated placentas of habanero peppers (Capsicum chinense Jacq.). The placentas were cultured in vitro and exposed to conditions promoting capsaicinoid accumulation, such as treatments with salicylic acid (SA) and methyl jasmonate (MeJa). Although exposure to both inducers resulted in increased accumulation of capsaicinoids, the induction by SA was more pronounced. Glutamine synthetase (GS) activity, which incorporates ammonia into glutamine, increased more than six fold under such conditions, suggesting GS participation in fulfilling the demand for amino acids required to support the increase in capsaicinoid synthesis. Glutamate dehydrogenase (GDH), which has been involved in nitrogen assimilation in non-photosynthetic tissues such as placentas, was apparently not involved; its activity decreased in tissues exposed to the inducers. Thus, under the conditions tested, the activation of secondary metabolism required activation of basal nitrogen metabolism.     

GAMT joins the p53 network: Branching into metabolism

The p53 protein functions to prevent tumor development by restricting proliferation, motility and survival of abnormal or stressed cells. In addition to well-established roles, recent discoveries indicate a role for p53 in the regulation of pathways involved in energy metabolism. The metabolic functions of p53 can inhibit the shift to glycolysis that is characteristically seen in cancer cells, while favoring the energy production by mitochondrial oxidative phosphorylation. Identification of guanidinoacetate methyltransferase (GAMT) as a new p53 target connects p53 to creatine metabolism critical in the regulation of ATP homeostasis. The involvement of GAMT in both genotoxic and metabolic stress-induced apoptosis, as well as the requirement of p53-dependent up-regulation of GAMT in glucose starvation-mediated fatty acid oxidation (FAO), demonstrate a further role of p53 in coordinating stress response with changes in cellular metabolism. Such activities of p53 would help to bring a better understanding of how cancer cells acquire unique metabolic features to maintain their own survival and proliferation, and might provide interesting clues toward the development of novel therapies.     

Methionine supplementation stimulates mitochondrial respiration

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases.By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways.These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways.     

Potential metabolic mechanisms for inhibited chloroplast nitrogen assimilation under high CO2

Improving photosynthesis is considered a major and feasible option to dramatically increase crop yield potential. Increased atmospheric CO₂ concentration often stimulates both photosynthesis and crop yield, but decreases protein content in the main C₃ cereal crops. This decreased protein content in crops constrains the benefits of elevated CO₂ on crop yield and affects their nutritional value for humans. To support studies of photosynthetic nitrogen assimilation and its complex interaction with photosynthetic carbon metabolism for crop improvement, we developed a dynamic systems model of plant primary metabolism, which includes the Calvin–Benson cycle, the photorespiration pathway, starch synthesis, glycolysis–gluconeogenesis, the tricarboxylic acid cycle, and chloroplastic nitrogen assimilation. This model successfully captures responses of net photosynthetic CO₂ uptake rate (A), respiration rate, and nitrogen assimilation rate to different irradiance and CO₂ levels. We then used this model to predict inhibition of nitrogen assimilation under elevated CO₂. The potential mechanisms underlying inhibited nitrogen assimilation under elevated CO₂ were further explored with this model. Simulations suggest that enhancing the supply of α-ketoglutarate is a potential strategy to maintain high rates of nitrogen assimilation under elevated CO₂. This model can be used as a heuristic tool to support research on interactions between photosynthesis, respiration, and nitrogen assimilation. It also provides a basic framework to support the design and engineering of C₃ plant primary metabolism for enhanced photosynthetic efficiency and nitrogen assimilation in the coming high-CO₂ world.

Simulations with a dynamic systems model of C₃ primary metabolism show that the decreased supply of reducing equivalent and 2-oxoglutaric acid cause decreased nitrogen assimilation under elevated CO₂.     

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO₂ assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH₄ ⁺ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.     

Uncoupling proteins in modulation of mitochondrial functions--therapeutic prospects

Enormous interest in mitochondrial uncoupling proteins (UCPs) is caused by relevant impact of these energy-dissipating systems on cellular energy transduction. A key role of UCPs in regulation of mitochondrial metabolism is supported by existence of their different isoforms in various mammalian tissues. Recent studies have shown that UCPs have an important part in pathogenesis of various disorders, such as obesity, type-2 diabetes, cachexia, aging or tumor. The obscure roles of UCPs in normal physiology and their emerging role in pathophysiology, provide exciting potential for further investigation. However, neither the exact physiological nor biochemical roles of UCP homologues are well understood. Therefore, providing mechanistic explanation of their functions in cellular physiology may be the basis for potential farmacological targeting of UCPs in future on clinical scale.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation

Mitochondria are one of the most complex of subcellular organelles and play key roles in many cellular functions including energy production, fatty acid metabolism, pyrimidine biosynthesis, calcium homeostasis, and cell signaling. In recent years, we and other groups have attempted to identify the complete set of proteins that are localized to human mitochondria as a way to better understand its cellular functions and how it communicates with other cell compartment in complex signaling pathways such as oxidative stress and apoptosis. Indeed, there is an increasing interest in understanding the molecular details of oxidative stress and the mitochondrial role in this process, as well as assessing how mitochondrial proteins become damaged or posttranslationally modified as a consequence of a major change in a cell's redox status. In this review, we report on the current status of the human mitochondrial proteome with an emphasis towards understanding how mitochondrial proteins, especially the proteins that make up the respiratory chain or oxidative phosphorylation (OXPHOS) enzymes, are modified in various models of age-related diseases such as cancer and Parkinson's disease (PD).     

Prospects for improving nitrogen use efficiency: Insights given by 15N-labelling experiments

In higher plants, recent advances in plant molecular genetics, combined with modern physiological and biochemical studies, have expanded our understanding of the regulatory mechanisms controlling the primary steps of inorganic nitrogen assimilation and the subsequent biochemical pathways involved in nitrogen supply and recycling for higher plant metabolism, growth and development. In this presentation, we describe improvements in our understanding of the molecular controls of nitrogen assimilation through the use of transgenic plants and the study of genetic variability in model and crop species. To illustrate this research programme, the physiological impact of modified gene expression, using either transgenic plants or different genotypes, was studied using ¹⁵N-labelling experiments in order to monitor the influx of nitrate or ammonia and its subsequent incorporation into amino acids.     

Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase

Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part. Recent studies indicated that the number of mitochondrial phosphoproteins is higher than expected; however, the effect of reversible phosphorylation on mitochondrial structure and function has only been defined in a few cases. It is thus crucial to determine authentic protein phosphorylation sites from highly purified mitochondria in a genetically tractable organism. The yeast Saccharomyces cerevisiae is a major model organism for the analysis of mitochondrial functions. We isolated highly pure yeast mitochondria and performed a systematic analysis of phosphorylation sites by a combination of different enrichment strategies and mass spectrometry. We identified 80 phosphorylation sites in 48 different proteins. These mitochondrial phosphoproteins are involved in critical mitochondrial functions, including energy metabolism, protein biogenesis, fatty acid metabolism, metabolite transport, and redox regulation. By combining yeast genetics and in vitro biochemical analysis, we found that phosphorylation of a serine residue in subunit g (Atp20) regulates dimerization of the mitochondrial ATP synthase. The authentic phosphoproteome of yeast mitochondria will represent a rich source to uncover novel roles of reversible protein phosphorylation.     

Structure and Function of Plant-Type Ferredoxins

The plant-type ferredoxins (Fds) are the [2Fe-2S] proteins that function primarily in photosynthesis; they transfer electrons from photoreduced Photosystem I to ferredoxin NADP(+) reductase in which NADPH is produced for CO(2) assimilation. In addition, Fds partition electrons to various ferredoxin-dependent enzymes not only for assimilations of inorganic nitrogen and sulfur and N(2) fixation but also for regulation of CO(2) assimilation cycle. Although Fds are small iron-sulfur proteins with molecular weight of 11 KDa, they are expected to interact with surprisingly many enzymes. Several Fd isoforms were found in non-photosynthetic cells as well as Fds in photosynthetic cells, leading to the recognition that they have differentiated physiological roles. In a quarter of century, X-ray crystallography and NMR spectroscopy provided wealth of structural data, which shed light on the structure-function relationship of the plant-type Fds and gave structural basis for the biochemical and spectroscopic properties so far accumulated. Thus the structural studies of Fds have come to a new era in which different roles of Fds and interactions with various enzymes are clarified on the basis of the tertiary and quaternary structures, although they are premature at present. This article reviews briefly the structures of the plant-type Fds together with their functions, properties, and interactions with Fd related enzymes. Lastly the folding motif of Fd, that has grown to be a large family by including many functionally unrelated proteins, is noted.     

Mitochondrial respiratory chain dysfunction: implications in neurodegeneration

For decades mitochondria have been considered static round-shaped organelles in charge of energy production. In contrast, they are highly dynamic cellular components that undergo continuous cycles of fusion and fission influenced, for instance, by oxidative stress, cellular energy requirements, or the cell cycle state. New important functions beyond energy production have been attributed to mitochondria, such as the regulation of cell survival, because of their role in the modulation of apoptosis, autophagy, and aging. Primary mitochondrial diseases due to mutations in genes involved in these new mitochondrial functions and the implication of mitochondrial dysfunction in multifactorial human pathologies such as cancer, Alzheimer and Parkinson diseases, or diabetes has been demonstrated. Therefore, mitochondria are set at a central point of the equilibrium between health and disease, and a better understanding of mitochondrial functions will open new fields for exploring the roles of these mitochondrial pathways in human pathologies. This review dissects the relationships between activity and assembly defects of the mitochondrial respiratory chain, oxidative damage, and alterations in mitochondrial dynamics, with special focus on their implications for neurodegeneration.     

Carbon and nitrogen assimilation in relation to yield: mechanisms are the key to understanding production systems

Improved understanding of crop production systems in relation to N-supply has come from a knowledge of basic plant biochemistry and physiology. Gene expression leads to protein synthesis and the formation of metabolic systems; the ensuing metabolism determines the capacity for growth, development and yield production. This constitutes the genetic potential. These processes set the requirements for the supply of resources. The interactions between carbon dioxide (CO(2)) and nitrate () assimilation and their dynamics are of key importance for crop production. In particular, an adequate supply of, its assimilation to amino acids (for which photosynthesized carbon compounds are required) and their availability for protein synthesis, are essential for metabolism. An adequate supply of stimulates leaf growth and photosynthesis, the former via cell growth and division, the latter by larger contents of components of the light reactions, and those of CO(2) assimilation and related processes. If the supply of resources exceeds the demand set by the genetic potential then production is maximal, but if it is less then potential is not reached; matching resources to potential is the aim of agriculture. However, the connection between metabolism and yield is poorly quantified. Biochemical characteristics and simulation models must be better used and combined to improve fertilizer-N application, efficiency of N-use, and yields. Increasing N-uptake at inadequate N-supply by increasing rooting volume and density is feasible, increasing affinity is less so. It would increase biomass and N/C ratio. With adequate N, at full genetic potential, more C-assimilation per unit N would increase biomass, but energy would be limiting at full canopy. Increasing C-assimilation per unit N would increase biomass but decrease N/C at both large and small N-supply. Increasing production of all biochemical components would increase biomass and demand for N, and maintain N/C ratio. Changing C- or N-assimilation requires modifications to many processes to effect improvements in the whole system; genetic engineering/molecular biological alterations to single steps in the central metabolism are unlikely to achieve this, because targets are unclear, and also because of the complex interactions between processes and environment. Achievement of the long-term objectives of improving crop N-use and yield with fewer inputs and less pollution, by agronomy, breeding or genetic engineering, requires a better understanding of the whole system, from genes via metabolism to yield.