Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster

We have characterized biochemical effects of Idh ^GB1 in Drosophila melanogaster. This is a null-activity allele for NADP⁺-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The K _m values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP⁺ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.Research supported by an Alberta Heritage Foundation for Medical Research Establishment Grant to MMB and a Natural Sciences and Engineering Research Council Operating Grant to JHW.     

Intensity of free radical processes and regulation of cytoplasmic NADP-isocitrate dehydrogenase in rat cardiomyocytes under normal and ischemic conditions

The intensity of free radical processes and the regulation of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) activity have been studied in the cytoplasmic fraction of normal and ischemized rat myocardium. Chemiluminescence parameters, such as the light sum (S) of slow flash and the tangent of the kinetic curve slope angle (tan₁), which characterize the intensity of free radical processes, were increased in ischemia 2.1- and 20.0-fold, respectively. The slow flash intensity (I_max) was increased 22-fold. The contents of lipid peroxidation products–diene conjugates and malonic dialdehyde–were increased 11.9- and 4.7-fold, respectively, suggesting pronounced oxidative stress. Using homogenous enzyme preparations of NADP-IDH isolated from the normal and experimentally ischemized rat myocardium, a number of catalytic properties of the enzyme were characterized for normal and pathologic conditions. NADP-IDH from the normal and ischemized myocardium had the same electrophoretic mobility and was regulated similarly by Fe²⁺, Cu²⁺, Zn²⁺, and also with succinate and fumarate. However, under normal and pathologic conditions NADP-IDH was different in the affinity for substrates and in the sensitivity to inhibitory effects of hydrogen peroxide, reduced glutathione, and of Ca²⁺. The degree of synergy in the enzyme inhibition with Fe²⁺ and H₂O₂ was less pronounced in ischemia. The inhibitory effect of the reaction product 2-oxoglutarate was higher under normal conditions than in ischemia (the K _i values were 0.22 and 0.75 mM, respectively). The specific features of the NADP-IDH regulation in ischemia are suggested to promote the stimulation of the enzyme functioning during increased level of free radical processes, and this seems to be important for NADPH supplying for the glutathione reductase/glutathione peroxidase antioxidant system of cardiomyocytes.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

Hydrogen peroxide-induced salt tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis> salicylate-deficient transformants <Emphasis Type="Italic">NahG</Emphasis>

The effect of hydrogen peroxide treatment on the salt tolerance of wild-type Arabidopsis thaliana L. plants (Col-0) and plants transformed with the bacterial salicylate hydroxylase gene (NahG) was studied. The base tolerance to salt stress caused by 200 mM of NaCl in solution culture was higher in plants with the NahG genotype in comparison with the wild-type plants. Growth inhibition was observed for wild-type plants under the action of exogenous hydrogen peroxide, which was not observed for the NahG transformants; salt tolerance increased in the both types of plants after treatment, which was assessed based on the growth indicators and the ability to preserve the chlorophyll pool following NaCl treatment. The content of endogenous Н2О2 in the leaves of wild-type plants increased significantly following exogenous hydrogen peroxide treatment and salt stress, while it practically did not change in the leaves of the NahG genotype. The SOD activity increased in both genotypes after treatment with exogenous hydrogen peroxide, and remained at an elevated level after salt stress in comparison with the nontreated plants. Furthermore, the catalase activity increased in leaves of the salicylate-deficient genotype but not in the Col-0 genotype. The guaiacol peroxidase activity increased in plants of both genotypes under the action of hydrogen peroxide and salt stress, with the NahG plants demonstrating a higher degree of increase. The Н₂О₂ treatment facilitated the increase of the proline content in leaves of the plants of both genotypes under conditions of salt stress. It was concluded that there were hydrogen peroxide signal transduction pathways in Arabidopsis plants that were salicylic acid independent and that the antioxidant system functioned more effectively in salicylate-deficient Arabidopsis plants.     

Assessment of the preventive effect of vermicompost on salinity resistance in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> cv. Ailsa Craig)

To determine the effects of vermicompost leachate (VCL) on resistance to salt stress in plants, young tomato seedlings (Solanum lycopersicum, cv. Ailsa Craig) were exposed to salinity (150 mM NaCl addition to nutrient solution) for 7 days after or during 6 mL L^??1 VCL application. Salt stress significantly decreased leaf fresh and dry weights, reduced leaf water content, significantly increased root and leaf Na⁺ concentrations, and decreased K⁺ concentrations. Salt stress decreased stomatal conductance (g_s), net photosynthesis (A), instantaneous transpiration (E), maximal efficiency of PSII photochemistry in the dark-adapted state (F_v/F_m), photochemical quenching (qP), and actual PSII photochemical efficiency (ΦPSII). VCL applied during salt stress increased leaf fresh weight and g_s, but did not reduce leaf osmotic potential, despite increased proline content in salt-treated plants. VCL reduced Na⁺ concentrations in leaves (by 21.4%), but increased them in roots (by 16.9%). VCL pre-treatment followed by salt stress was more efficient than VCL concomitant to salt stress, since VCL pre-treatment provided the greatest osmotic adjustment recorded, with maintenance of net photosynthesis and K⁺/Na⁺ ratios following salt stress. VCL pre-treatment also led to the highest proline content in leaves (50 µmol g^??1 FW) and the highest sugar content in roots (9.2 µmol g^??1 FW). Fluorescence-related parameters confirmed that VCL pre-treatment of salt-stressed plants showed higher PSII stability and efficiency compared to plants under concomitant VCL and salt stress. Therefore, VCL represents an efficient protective agent for improvement of salt-stress resistance in tomato.     

Effect of salinity and waterlogging on growth,anatomical and antioxidative responses in <Emphasis Type="Italic">Mentha aquatica</Emphasis> L.

Salinity and waterlogging are two stresses which in nature often occur simultaneously. In this work, effects of combined waterlogging and salinity stresses are studied on the anatomical alteration, changes of enzymatic antioxidant system and lipid peroxidation in Mentha aquatica L. plants. Seedlings were cultured in half-strength Hoagland medium 50 days after sowing, and were treated under combination of three waterlogging levels (well drained, moderately drained and waterlogging) and NaCl (0, 50, 100, 150 mM) for 30 days. Moderately drained and waterlogging conditions induced differently aerenchyma formation in roots of M. aquatica salt-treated and untreated plants. Moreover, stele diameter and endodermis layer were also affected by salt stress and waterlogging. Salt stress significantly decreased growth, relative water content (RWC), protein level, catalase (CAT) and polyphenol oxidase (PPO) activities, and increased proline content, MDA content, H₂O₂ level and activities of superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX). Waterlogging in salt-untreated plants increased significantly growth parameters, RWC, protein content, antioxidant enzyme activity, and decreased proline content, H₂O₂ and MDA levels. In salt-treated plant, waterlogging caused strong induction of antioxidant enzymes activities especially at severe stress condition. These results suggest M. aquatica is a waterlogging tolerant plant due to significant increase of antioxidant activity, membrane stability and growth under water stress. High antioxidant capacity under waterlogging can be a protective strategy against oxidative damage, and help to salt stress alleviation.     

A rice mutant defective in antioxidant-defense system and sodium homeostasis possesses increased sensitivity to salt stress

Screening salt-sensitive mutants is a powerful method to identify genes associated with salt tolerance. We used forward genetic screening with sodium azide-mutated rice (Oryza sativa L. cv. Tainung 67) to identify mutants showing hypersensitivity to salt stress. A new mutant line, named salt hypersensitive 1 (shs1) and exhibiting a severe salt-sensitivity when grown under a high NaCl concentration, was identified; the salt hypersensitivity was caused by duplicate recessive epistasis with mutations likely in two different loci. The shs1 salt sensitive phenotypes included a decreased seed germination rate, reduced shoot height and root length, severe and quick wilting, and overaccumulation of sodium ions in shoots as compared with wild-type plants. In addition, shs1 showed a decreased photosynthetic efficiency and enhanced hydrogen peroxide (H₂O₂) production under the salt stress. An increased superoxide dismutase activity and decreased catalase activity were responsible for the hyperaccumulation of H₂O₂ in shs1. The hypersensitivity of shs1 to the salt stress might be caused by an impaired antioxidant machinery and cellular Na⁺ homeostasis.     

蕾期土壤盐度降低后棉花叶片的生理功能恢复

试验于2011—2012年在江苏南京江苏省农业科学院经济作物研究所试验田进行,采用盆栽方法,以鲁棉研37号和苏棉22号为供试材料,设置土壤盐度降低试验(初始土壤含盐量为0.2%,棉花进入二叶期后每7d加入混合盐1次,每次增加0.1%,使土壤含盐量逐渐达到0.5%,蕾期进行盐度降低处理,使土壤含盐量降低到0.2%左右),研究蕾期土壤盐度降低后棉花叶片的生理代谢动态特征。结果表明:土壤盐度降低后,棉花叶片叶绿素(Chl)、类胡萝卜素(Car)含量和Chl/Car升高;净光合速率和气孔导度升高,且分别在土壤盐度降低后第14天和7天接近于低盐对照;土壤盐度降低后棉花叶片超氧化物歧化酶(SOD)和过氧化物酶(POD)活性升高,过氧化氢酶(CAT)活性和丙二醛(MDA)含量降低,MDA含量在土壤盐度降低后第14天接近于低盐对照;土壤盐度降低后棉花叶片中可溶性糖、游离氨基酸和脯氨酸含量降低,且接近于低盐对照。上述结果表明土壤盐度降低后,棉花叶片生理功能逐渐恢复,进而实现棉花生长发育的恢复补偿。棉花叶片生理功能在土壤盐度降低后的恢复能力存在品种间差异,鲁棉研37号较苏棉22号叶片生理功能表现出更强的恢复能力。     

Hydrogen Peroxide and Strigolactones Signaling Are Involved in Alleviation of Salt Stress Induced by Arbuscular Mycorrhizal Fungus in <Emphasis Type="Italic">Sesbania cannabina</Emphasis> Seedlings

The arbuscular mycorrhizal symbiosis can alleviate salt stress in plants by altering strigolactone levels in the host plant. The aim of this study was to investigate the mechanism by which strigolactones enhance salt stress tolerance in arbuscular mycorrhizal Sesbania cannabina seedlings. Strigolactone levels, as determined by means of germination bioassay, gradually increased with treatment time of NaCl applied. Inhibition of NADPH oxidase activity and chemical scavenging of H₂O₂ significantly reduced strigolactone-induced salt tolerance and decreased strigolactone levels. The H₂O₂-induced strigolactone accumulation was accompanied by increased tolerance to salt stress. These results strongly indicated that elevated H₂O₂ concentration resulting from enhanced NADPH oxidase activity regulated strigolactone-induced salt stress tolerance in arbuscular mycorrhizal S. cannabina seedlings.     

Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress

Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H₂O₂) accumulation. H₂O₂ acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.Key words: glucose-6-phosphate dehydrogenase, hydrogen peroxide, reduced glutathione, redox homeostasis, salt stressEnvironmental stresses inevitably induce the production of reactive oxygen species (ROS).¹ Reduced glutathione (GSH) is a key substance in the network of antioxidants that include ascorbate, glutathione, α-tocopherol and a serial of antioxidant enzymes,² which metabolizes H₂O₂ mainly via the ascorbate-glutathione cycle, the most important detoxifying system in plants.³ Thus, the regulatory ability to maintain the cellular GSH balance is crucial to confer the resistance to oxidative stress in plants. However, to our knowledge, the regulatory mechanism on the intracellular GSH-pool equilibrium under environmental stresses has been largely unknown in plants.A main source of GSH is regenerated from its oxidative form (GSSG) via glutathione cycling, which uses NADPH as the reductant.⁴ G6PDH is the key enzyme of pentose phosphate pathway that is responsible for the generation of NADPH.⁵ G6PDH has been shown to play a protective role against ROS in human and animal cells,^6,7 and the enhanced expression of G6PDH could enhance the GSH levels and the ability of resistance to oxidative stress.^5,8 In plants, it has been reported that oxidative stress induced by the elicitor stimulated G6PDH activity in tobacco cells,^9,10 and the GSH-biosynthesis inhibitor or GSH precursor could increase or suppressed G6PDH activity, respectively.¹⁰ Interestingly, after G6PDH activity was inhibited, not only GSH levels dramatically decreased, but the elicitor-induced H₂O₂ accumulation was also completely counteracted.^9,10 Thus, the functions of G6PDH under oxidative stress seem to be involved in these two contradictory courses in cells: the regeneration of GSH as well as H₂O₂ accumulation. The role of G6PDH under environmental stresses remained limited to clarify this, so we studied the G6PDH functions with a series of inhibitor or donor of GSH, H₂O₂ and G6PDH in reed calli under salt stress. Our recent studied clearly demonstrated that G6PDH activity was also simultaneously involved in intracellular GSH maintenance and H₂O₂ accumulation in salt stress. Further studies revealed that a plasma membrane (PM) NADPH oxidase, using NADPH as substrate mainly produced by G6PDH, was mainly responsible for the generation of H₂O₂. And H₂O₂, produced under salt stress, induced the increased G6PDH activity and the enzymes of glutathione cycle, which concomitantly resulted in an increased GSH contents. Foyer and Noctor (2005) suggested that the cellular “oxidative signaling” was made possibly by homeostatic regulation by antioxidant redox buffer.¹¹ Based on these, it can be speculated that G6PDH might play an important role in maintaining the cellular redox signals under salt stress in plants.Our recent work provides a new insight into G6PDH functions under environmental stresses in plants. Growing evidences suggest that PM NADPH oxidase is responsible for H₂O₂ accumulation under stresses,^12,13 and H₂O₂ is involved in various signaling pathways in plants, such as defense gene expression, stomatal closure, root growth, programmed cell death (PCD) and so on.¹¹ In addition, GSH, as a key antioxidant, also influences gene expression associated with biotic and abiotic stress responses to maximum defense.² Recent study also reported that G6PDH was involved in NR-dependent NO production, and thus played a pivotal role in establishing tolerance of red kidney bean roots to salt stress.¹⁴ Therefore, the research work is required to further clarify the regulatory mechanism underlying the roles of G6PDH in the cellular redox homeostasis as well as the related signals under environmental stresses in plants.Based on the results obtained so far, a model for G6PDH functions under salt stress is proposed (Fig. 1). In our model, the increased G6PDH activity is tightly correlated with GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase under salt stress in plants. Under salt stress, H₂O₂ activities the activities of G6PDH and the enzymes in glutathione recycle, which finally result in the enhanced glutathione cycling rate and thus the increased GSH levels. This enhanced antioxidant ability can facilitate to maintain a steady-state level of H₂O₂. Eventually, the properly intracellular redox state is established under salt stress and forms a metabolic interface for signals. Thus, we suggest that G6PDH plays a crucial role in establishing this cellular redox homeostasis under salt stress.Open in a separate windowFigure 1Hypothetical model for the roles of G6PDH under salt stress. Under salt stress, G6PDH activity is involved in both GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase. H₂O₂, as a signal, increases the activities of G6PDH, glutathione (GR) and glutathione peroxidase (GPX), which finally enhance glutathione cycle rate and result in the increased GSH levels. This enhanced antioxidant ability could facilitate to keep H₂O₂ in a steady state for signal in salt stress.     

Isolation and properties of an isocitrate dehydrogenase from Anacystis nidulans

A NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) was isolated and purified over 400-fold from Anacystis nidulans. The enzyme activity responded slowly to rapid changes in ligand (NADP⁺, isocitrate, Mg²⁺-ions) or enzyme concentration as well as to rapid changes in temperature. These are properties characteristic of the hysteretic enzymes. In addition, the enzyme activity was subject to product (-ketoglutarate) inhibition. ATP, ADP and CDP also inhibited the enzyme. Unlike several other cyanobacterial enzymes, the isocitrate dehydrogenase of Anacystis is not under redox control.     

La Dendrologia di Ulisse Aldrovandi

Abstract

Legume-Rhizobium symbiotic nitrogen (N₂) fixation plays a critical role in sustainable nitrogen management in agriculture. The nitrogen fixed by the root nodules not only affects the nitrogen cycle of nature, but is also of great economic importance. A number of physiological and biochemical processes in the nodules are affected by salt stress. The objective of this study was to evaluate the role of arbuscular mycorrhiza (AM) in moderating toxic effects of salt stress on nodular metabolism in Cajanus cajan (L.) Millspaugh (pigeonpea) cv. Manak. Exposure of plants to salinity stress (4, 6 and 8 dSm^?1) caused ionic imbalance, which resulted in increased Na⁺ and reduced K⁺ and Ca²⁺ contents in the nodules. Salinity induced increased synthesis and accumulation of proline and glycine betaine. Salt stress significantly increased the antioxidant enzyme activities in the nodules of all plants. Nodular growth suffered remarkably and a marked decline in nodule biomass was observed under salt stress. Leghemoglobin content and acetylene reduction activity (ARA) also declined under saline conditions. AM could significantly improve nodule dry mass, leghemoglobin content and nitrogenase activity, and phosphorus content under salt stress. Activities of antioxidant enzymes increased markedly in nodules of mycorrhizal-stressed plants. This study suggested a correlation between improved functional efficiency of nodules and higher osmolyte accumulation and enhanced antioxidant enzyme activities of AM plants under stressed conditions relative to the nodules of uninoculated plants.     

Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Superoxide dismutases (SODs) play important role in stress tolerance of plants. In this study, an MnSOD gene (TaMnSOD) from Tamarix androssowii, under the control of the CaMV35S promoter, was introduced into poplar (Populus davidiana × P. bolleana). The physiological parameters, including SOD activity, malondialdehyde (MDA) content, relative electrical conductivity (REC) and relative weight gain, of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that SOD activity was enhanced in transgenic plants, and the MDA content and REC were significantly decreased compared to WT plants when exposed to NaCl stress. In addition, the relative weight gains of the transgenic plants were 8- to 23-fold of those observed for WT plants after NaCl stress for 30 days. The data showed that the SOD activities that increased in transgenic lines are 1.3–4-folds of that increased in the WT plant when exposed to NaCl stress. Our analysis showed that increases in SOD activities as low as 0.15-fold can also significantly enhance salt tolerance in transgenic plants, suggesting an important role of increased SOD activity in plant salt tolerance.     

Growth-promoting bacteria and natural regulators mitigate salt toxicity and improve rapeseed plant performance

Salinity is a major environmental stress that limits plant production and portraits a critical challenge to food security in the world. In this research, the impacts of plant growth–promoting bacteria (Pseudomonas RS-198 and Azospirillum brasilense RS-SP7) and foliar application of plant hormones (salicylic acid 1 mM and jasmonic acid 0.5 mM) on alleviating the harmful effects of salt stress in rapeseed plants (Brassica napus cv. okapi) were examined under greenhouse condition. Salt stress diminished rapeseed biomass, leaf area, water content, nitrogen, phosphorus, potassium, calcium, magnesium, and chlorophyll content, while it increased sodium content, endogenous salicylic and jasmonic acids, osmolyte production, H₂O₂ and O₂^•− generations, TBARS content, and antioxidant enzyme activities. Plant growth, nutrient content, leaf expansion, osmolyte production, and antioxidant enzyme activities were increased, but oxidative and osmotic stress indicators were decreased by bacteria inoculation + salicylic acid under salt stress. Antioxidant enzyme activities were amplified by jasmonic acid treatments under salt stress, although rapeseed growth was not generally affected by jasmonic acid. Bacterial + hormonal treatments were superior to individual treatments in reducing detrimental effects of salt stress. The best treatment in rectifying rapeseed growth under salt stress was combination of Pseudomonas and salicylic acid. This combination attenuated destructive salinity properties and subsequently amended rapeseed growth via enhancing endogenous salicylic acid content and some essential nutrients such as potassium, phosphorus, and magnesium.

     

Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F_v/F_m relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mol photons m^–2 s^–1 under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mol photons m^–2 s^–1, under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F_v/F_m and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.This revised version was published online in October 2005 with corrections to the Cover Date.