Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.     

Critical study of preanalytical and analytical phases of adenine and pyridine nucleotide assay in human whole blood

Intracellular redox and energetic status play a crucial role in cardiovascular diseases and metabolic disorders. The physiological status of reducing agents, such as NADPH and NADH, and of high-energy molecules, such as ATP, is required for antioxidant system activity. For these reasons, an accurate measurement of adenine and pyridine nucleotides is fundamental. In this study we examined the preanalytical phase of reduced pyridine (RPN) and adenine and oxidized pyridine (AOPN) nucleotide assay in human whole blood. Different experimental conditions were applied to RPN alkaline and AOPN acid extracts to find the best analytical performance. Our results show that a good RPN and AOPN linearity (r from 0.994 to 0.999), recovery (near to 100%), and precision (coefficient of variation < 5%) were obtained when supernatant from acid and ultrafiltrate from alkaline extracts were neutralized, frozen, and thawed just before HPLC injection. Since NADH decays rapidly at -80 degrees C, RPN levels must be assayed within 72 h while AOPN can be stored for 1 month at the same temperature. An accurate and quantitative method for nucleotide determination can be obtained by applying the preanalytical conditions proposed in this study.     

Legume nodule senescence: roles for redox and hormone signalling in the orchestration of the natural aging process

Research on legume nodule development has contributed greatly to our current understanding of plant-microbe interactions. However, the factors that orchestrate root nodule senescence have received relatively little attention. Accumulating evidence suggests that redox signals contribute to the establishment of symbiosis and senescence. Although degenerative in nature, nodule senescence is an active process programmed in development in which reactive oxygen species (ROS), antioxidants, hormones and proteinases have key roles. Nodules have high levels of the redox buffers, ascorbate and glutathione, which are important in the nodulation process and in senescence. These metabolites decline with N-fixation as the nodule ages but the resultant decrease in redox buffering capacity does not necessarily lead to enhanced ROS or oxidative stress. We propose models by which ROS and antioxidants interact with hormones such as abscisic acid in the orchestration of nodule senescence.     

Interaction of intracellular signalling and metabolic pathways at inhibition of mitochondrial aconitase by fluoroacetate

Mitochondrial aconitase has been shown to be inactivated under the effects of many compounds and critical states. Fluoroacetate (FA) is the best-known aconitase-inhibiting toxic agent. The biochemistry of the toxic action of FA has been rather well studied; however, no effective therapy has been developed over the past six decades. To search for new approaches to the development of possible antidotes, experiments were carried out in vitro with rat liver mitochondria, Ehrlich ascite tumor (EAT) cells, and cardiomyocytes exposed to FA or fluorocitrate (FC). FA produced its effects at much higher concentrations as compared with FC; in experiments with mitochondria these effects depended on respiratory substrates: with pyruvate, FA induced a slow oxidation and/or a leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides (PN) was prevented by the incubation of mitochondria with cyclosporin A. Studies of the PN level and dynamics of Ca²⁺ in EAT cells during activation by ATP also revealed the PN leak from mitochondria, which led to a shift in the balance of mitochondrial and cytosolic NAD(P)H under action of FA. Moreover, an increase of cytosolic Ca²⁺ was revealed in the cells exposed to FA, which could be explained by the activation of plasma membrane calcium channels. This mechanism could affect the amplitude and rate of calcium waves in cardiomyocytes under the effects of FA. We emphasize the reciprocal relationship between intracellular PN dynamics and calcium balance and discuss possible pathways of metabolic modulation in the context of development of effective therapy of poisoning with FA and other aconitase inhibitors.     

Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress

Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.     

The roles of redox processes in pea nodule development and senescence

Nodule senescence is triggered by developmental and environmental cues. It is orchestrated through complex but poorly characterized genetic controls that involve changes in the endogenous levels of reactive oxygen species (ROS) and antioxidants. To elucidate the importance of such redox control mechanisms in pea root nodule senescence, redox metabolites were analysed throughout nodule development in a commercial pea variety ( Pisum sativum cv. Phoenix) inoculated with a commercial rhizobial strain ( Rhizobium leguminosarum bv. viciae ). Although a strong positive correlation between nitrogenase activity and nodule ascorbate and glutathione contents was observed, the progressive loss of these metabolites during nodule senescence was not accompanied by an increase in nodule superoxide or hydrogen peroxide. These oxidants were only detected in nodule meristem and cortex tissues, and the abundance of superoxide or hydrogen peroxide strongly declined with age. No evidence could be found of programmed cell death in nodule senescence and the protein carbonyl groups were more or less constant throughout nodule development. Pea nodules appear to have little capacity to synthesize ascorbate de novo . l -galactono-1, 4-lactone dehydrogenase (GalLDH), which catalyses the last step of ascorbate synthesis could not be detected in nodules. Moreover, when infiltrated with the substrates l -galactono-1, 4-lactone or l -gulonolactone, ascorbate did not accumulate. These data suggest that ROS, ascorbate and glutathione, which fulfil well recognized, signalling functions in plants, decline in a regulated manner during nodule development. This does not necessarily cause oxidative stress but rather indicates a development-related shift in redox-linked metabolite cross-talk that underpins the development and aging processes.     

Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context

While the chemical nature of reactive oxygen species (ROS) dictates that they are potentially harmful to cells, recent genetic evidence suggests that in planta purely physicochemical damage may be much more limited than previously thought. The most potentially deleterious effect of ROS under most conditions is that at high concentrations they trigger genetically programmed cell suicide events. Moreover, because plants use ROS as second messengers in signal transduction cascades in processes as diverse as mitosis, tropisms and cell death, their accumulation is crucial to plant development as well as defence. Direct ROS signal transduction will ensue only if ROS escape destruction by antioxidants or are otherwise consumed in a ROS cascade. Thus, the major low molecular weight antioxidants determine the specificity of the signal. They are also themselves signal-transducing molecules that can either signal independently or further transmit ROS signals. The moment has come to re-evaluate the concept of oxidative stress. In contrast to this pejorative or negative term, implying a state to be avoided, we propose that the syndrome would be more usefully described as 'oxidative signalling', that is, an important and critical function associated with the mechanisms by which plant cells sense the environment and make appropriate adjustments to gene expression, metabolism and physiology.     

Oxidative stress and antioxidants in tomato (Solanum lycopersicum) plants subjected to boron toxicity

BACKGROUND AND AIMS: Boron (B) toxicity triggers the formation of reactive oxygen species in plant tissues. However, there is still a lack of knowledge as to how B toxicity affects the plant antioxidant defence system. It has been suggested that ascorbate could be important against B stress, although existing information is limited in this respect. The objective of this study was to analyse how ascorbate and some other components of the antioxidant network respond to B toxicity. METHODS: Two tomato (Solanum lycopersicum) cultivars ('Kosaco' and 'Josefina') were subjected to 0.05 (control), 0.5 and 2 mm B. The following were studied in leaves: dry weight; relative leaf growth rate; total and free B; H(2)O(2); malondialdehyde; ascorbate; glutathione; sugars; total non-enzymatic antioxidant activity, and the activity of superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate oxidase and l-galactose dehydrogenase. KEY RESULTS: The B-toxicity treatments diminished growth and boosted the amount of B, malondialdehyde and H(2)O(2) in the leaves of the two cultivars, these trends being more pronounced in 'Josefina' than in 'Kosaco'. B toxicity increased ascorbate concentration in both cultivars and increased glutathione only in 'Kosaco'. Activities of antioxidant- and ascorbate-metabolizing enzymes were also induced. CONCLUSIONS: High B concentration in the culture medium provokes oxidative damage in tomato leaves and induces a general increase in antioxidant enzyme activity. In particular, B toxicity increased ascorbate pool size. It also increased the activity of l-galactose dehydrogenase, an enzyme involved in ascorbate biosynthesis, and the activity of enzymes of the Halliwell-Asada cycle. This work therefore provides a starting point towards a better understanding of the role of ascorbate in the plant response against B stress.     

The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation

The redox level and compartmentation of pyridine nucleotides was studied under photorespiratory and non-photorespiratory conditions using rapid fractionation of barley ( Hordeum vulgare L. cv. Gunilla, Svalöv) leaf protoplasts. From comparative measurements of the NADPH/NADP⁺ ratio and the ATP/ADP ratio one acidic and one alkaline extraction medium was chosen which quenched the metabolism very efficiently. The mitochondrial NADH/NAD⁺ was higher under photorespiratory conditions than under non-photorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, lowered the mitochondrial NADH/NAD⁺ ratio. This supports the hypothesis that glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. The chloroplastic NADPH/NADP⁺ as well as the NADH/NAD⁺ ratios were quite stable in saturating and limiting CO₂ as well as in the presence of aminoacetonitrile, although the triosephosphate/phosphoglycerate ratios changed. Thus, the redox level in the stroma seems to be tightly regulated.     

Effect of salt stress on lipid peroxidation and superoxide dismutase and peroxidase activities of Lycopersicon esculentum and L. pennellii

In this study, a relationship between lipid peroxidation, the antioxidant defense system and salt stress in salt-sensitive cultivated tomato (Lycopersicon esculentum) and its salt-tolerant wild relative (L. pennellii) was established. Superoxide dismutase (SOD) activities were significantly higher in the leaves of L. pennellii than those of L. esculentum after 12 and 84 d. POX activity showed a gradual increase in both cultivars under 70 mM NaCl. POX activity in L. pennellii significantly increased after 6 and 84 d whereas showed no remarkable change in leaves of L. esculentum under 140 mM NaCl. A higher salinity tolerance of L. pennellii was also correlated with a lower lipid peroxidation, which might be due to a higher content of antioxidant enzymes studied.     

The significance of antioxidants in the aphid-plant interaction: The redox hypothesis

The hypothesis is advanced that a redox system controls oxidation rates during the responses of plants to attack by sucking insects, that soluble antioxidants, such as ascorbate and glutathione, enhance the effectiveness of the plant's defensive system, and that oxidising enzymes in the saliva of aphids (and other phytophagous sucking insects) serve to counter it. Plants typically respond to wounding, including that caused by sucking insects, by mobilising and oxidising phenolic compounds. The initial phenolic monomers, and especially the monomerico-quinones to which many give rise on oxidation, are generally deterrent to insects. Their final oxidation products, however, are polymers and phenol-protein conjugates, which are non-toxic, but serve to seal off damaged cells. It is suggested that effective defence by the plant requires oxidation of phenolics at a controlled rate that maintains a deterrent titre of the monomers, while allowing a well ordered deposition of sealants. It is also suggested that the salivary oxidases of the insects hasten oxidation in the affected tissues, thereby decreasing concentrations of monomeric phenols and quinones. It is further suggested that sedentary species may also thereby disorganise the sealing off of affected tissues. A kinetic model is developed to show that inclusion of a reductive cycle in the sequential oxidation of phenolics increases the steady state concentrations of monomeric phenols for a given throughput. Conversely, an increase in oxidation rates diminishes the titre of monomers for the same throughput. In initial biological tests of the hypothesis, infiltration of stems of lucerne cultivars with ascorbate or glutathione reduced the reproductive rate of spotted alfalfa aphid,Therioaphis trifolii maculata (Buckton), and of blue-green aphid,Acyrthosiphon kondo Shinji, feeding thereon. Intrinsically non-deterrent concentrations of ascorbate synergised the deterrence of the plant phenolics chlorogenic acid and catechin to the apple aphid,Aphis pomi (de Geer), and the spotted alfalfa aphid,T. t. maculata, respectively.     

NAD: Not just a pawn on the board of plant-pathogen interactions

Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections.     

Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development

Nonselective cation channels (NSCCs) catalyse passive fluxes of cations through plant membranes. NSCCs do not, or only to a small extent, select between monovalent cations, and several are also permeable to divalent cations. Although a number of NSCC genes has been identified in plant genomes, a direct correlation between gene products and in vivo observed currents is still largely absent for most NSCCs. In this review, physiological functions and molecular properties of NSCCs are critically discussed. Recent studies have demonstrated that NSCCs are directly involved in a multitude of stress responses, growth and development, uptake of nutrients and calcium signalling. NSCCs can also function in the perception of external stimuli and as signal transducers for reactive oxygen species, pathogen elicitors, cyclic nucleotides, membrane stretch, amino acids and purines.     

Odorant-induced kinetics of Ca<Superscript>2+</Superscript>, NADH,and oxidized flavoproteins in frog olfactory mucosa

A study was made of the odorant-induced changes in the fluorescence of the Ca²⁺-chlortetracycline-membrane complex, NADH, and oxidized flavoproteins in the frog olfactory epithelium. Cineole and vanillin induce faster changes than camphor and pentanol. The different kinetics of NADH and membrane calcium evoked by these odorants are attributed to the heterogeneity of the molecular mechanisms involved in olfactory signal transduction. By contrast, ammonia and β-mercaptoethanol permeate the olfactory cells and without second messengers inhibit the mitochondrial respiratory chain and suppress the motility of olfactory cilia.     

Antioxidant status of the potato tuber and Ca2+ deficiency as a physiological stress

The antioxidant status of potato ( Solanum tuberosum L.) tubers of two genotypes, cv. Désirée and clone 10337de40 was investigated in relation to susceptibility to internal rust spot (IRS), a Ca²⁺-related physiological disorder. Concentrations of total calcium within the perimedulla tissue of tubers, grown with a restricted (1 m M CaCl₂) Ca²⁺ supply, were similar in cv. Désirée (IRS resistant) and clone 10337de40 (IRS susceptible). A range of antioxidants was assayed in order to assess antioxidant status in both genotypes under the two Ca²⁺ treatments. Although no appreciable differences were detected between low Ca²⁺ and control treatments, certain antioxidants were present at significantly higher levels in the IRS resistant genotype, cv. Désirée. These included dehydroascorbate reductase (EC 1.8.5.1) activity (more than 100% higher), total glutathione content (ca 40% higher), glutathione reductase (EC 1.6.4.2) activity (almost 50% higher), peroxidase (EC 1.11.1.7) activity (ca 60% higher) and superoxide dismutase (EC 1.15.1.1) activity (almost 80% higher). There was no difference in ascorbate content, ascorbate free radical reductase activity (EC 1.6.5.4), α-tocopherol levels and catalase activity (EC 1.11.1.6) between the two genotypes. The possible relationship between resistance to IRS and a superior antioxidant status, found in cv. Désirée, is discussed.     

黄土高原冰草叶片抗坏血酸和谷胱甘肽合成及循环代谢对干旱胁迫的生理响应

通过盆栽实验, 对干旱胁迫下黄土高原地区冰草(Agropyron cristatum)叶片的抗坏血酸和谷胱甘肽合成及循环代谢相关酶及物质含量进行了研究。结果表明: 冰草可以通过增强叶片的抗坏血酸和谷胱甘肽合成及循环代谢酶: 抗坏血酸过氧化物酶、谷胱甘肽还原酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、L-半乳糖酸-1, 4-内酯脱氢酶和γ-谷氨酰半胱氨酸合成酶活性, 维持植物体内抗坏血酸和谷胱甘肽水平及氧化还原状态, 从而抵御干旱造成的氧化胁迫。但叶片抗坏血酸和谷胱甘肽合成及循环代谢对不同水平干旱胁迫的响应, 随胁迫时间的延长而不同。在胁迫24天以前, 严重干旱下叶片的抗坏血酸和谷胱甘肽合成及循环代谢增强较显著; 在胁迫24天后, 由于该胁迫下植物所遭受的氧化胁迫较为严重, 叶片中上述6种酶的活性均呈降低趋势。而在中度干旱下叶片抗坏血酸和谷胱甘肽合成及循环代谢相关的6种酶在整个胁迫过程中均保持较高的活性。这说明, 冰草能够长时间有效地抵御中度干旱所造成的氧化胁迫, 但只能在一定时间范围内有效地抵御严重干旱所造成的氧化胁迫, 胁迫时间延长则会降低其抵御严重干旱的能力。