New insights on glyphosate mode of action in nodular metabolism: Role of shikimate accumulation

The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.     

Effect of dinoseb on nitrogen fixation of red clover (Trifolium pratense)

The effect of the herbicide dinoseb (2-sec-butyl-4,6-dinitrophenol) on the symbiotic nitrogen fixation of red clover (Trifolium pratense cv. Venla) was studied in field, greenhouse and laboratory experiments. In the field, the herbicide reduced the levels of nitrogenase (C₂H₂) activity of the plants up to 18 days after treatment, but increased the yields compared to hand-weeded and unweeded controls. Rhizobium strains isolated from root nodules of plants treated with dinoseb showed resistance to the herbicide when grown on laboratory media containing dinoseb. However, in a pot experiment, inoculation with a strain which grew in the presence of 200 μg dinoseb ml^?1 did not improve the performance of red clover treated with dinoseb. In the pot experiment dinoseb inhibited nitrogenase activity when sprayed on the leaves, but not when added to the growth medium. Application of starter nitrogen did not influence the effect of dinoseb. The results suggest that dinoseb did not act directly on the nodules, but affected nitrogen fixation by damaging the photosynthetic parts of the plant.     

缺硼、不同光强及外源葡萄糖对大豆根瘤固氮活性的影响

以大豆超结瘤突变体nts382及其亲本Braggcv．为试验材料，研究了正常供硼和缺硼条件下，不同光强和外源葡萄糖对大豆根瘤固氮活性的影响。结果表明，缺硼时根瘤固氮酶活性显著下降，此时降低光强并没有使根瘤固氮酶活性进一步下降，但使根瘤数减少；外源施用葡萄糖也不能提高根瘤固氮酶活性。在正常供硼情况下，低光强降低了根瘤固氮酶活性，并且使根瘤数和根瘤干重减少，外源施用葡萄糖也增加根瘤固氮酶活性。这说明了在缺硼条件下，根瘤固氮酶活性的下降并不是由于碳水化合物供应不足所造成的。     

Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.)

Horseweed (Conyza canadensis) is a cosmopolitan weed that commonly grows throughout North America. Horseweed that is not completely controlled by normal applications of glyphosate has been reported in western Tennessee. This research had three objectives: (1) to develop and validate an analytical procedure for the quantitative determination of shikimate, an important indicator of glyphosate activity in plants; (2) to confirm resistance to glyphosate in a horseweed population; and (3) to examine the accumulation of shikimate in both glyphosate-resistant and glyphosate-susceptible horseweed plants. The analytical procedure to determine shikimate used extraction with 1 M HCl for 24 h, followed by liquid chromatography using photodiode array detection, and shikimate recoveries were >or=82%. Glyphosate applications of both 0.84 kg ae/ha (the standard application rate) and 3.8 kg ae/ha to susceptible plants caused complete plant death. The same glyphosate applications to putative resistant populations caused less than 15% growth reduction as determined by visual evaluations, and fresh weights of these resistant plants 17 days after glyphosate treatment (DAT) were reduced an average of 45% in one population and were not affected in a different population. This direct comparison conclusively confirms that horseweed plants collected in western Tennessee in 2002 are resistant to 4 times the normal application dosage of glyphosate. The glyphosate-resistant horseweed biotypes still exhibited some herbicidal effects from the glyphosate, such as yellowing in the most actively growing, apical shoot meristems. The yellowing in the shoot apexes was transitory, and the plants recovered from this damage. Shikimate concentrations in all untreated horseweed plants were less than 100 microg/g, which was significantly less than that in all plants which had been treated with 0.84 kg ae/ha of glyphosate. Unexpectedly, shikimate accumulated (>1000 microg/g) in both resistant populations and the susceptible population. However, there were differences in shikimate accumulation patterns between resistant and susceptible horseweed biotypes. Shikimate concentrations in resistant populations declined about 40% from 2 to 4 DAT, while shikimate concentrations in the susceptible horseweed plants increased about 35% from 2 to 4 DAT. The confirmed resistance of a widespread weed implies that alternative control strategies for glyphosate-resistant horseweed will be needed in those no-tillage production systems where it commonly occurs.     

Nitrogen assimilation studies using 15N in soybean plants treated with imazethapyr, an inhibitor of branched-chain amino acid biosynthesis

The pattern of nitrogen assimilation in soybean plants treated with a herbicide that inhibits branched-chain amino acid biosynthesis was evaluated by (15)N isotopic analysis. The herbicide imazethapyr caused a strong decrease in nitrate uptake by roots, partly due to a reduced stomatal conductance. The inhibition of (15)N uptake was accompanied by a decrease in the (15)N content in the plant and, concomitantly, an inhibition of translocation to the shoot. Imazethapyr inhibited nitrate reductase activity in leaves and roots. Among all parameters studied, "de novo" synthesis of proteins was the first parameter of the N assimilation metabolism affected by the herbicide. These results show that this class of herbicides totally damages N metabolism and indicates a regulatory effect on N uptake and translocation that would be mediated by the increase in free amino acid pool provoked by the inhibition of branched-chain amino acid biosynthesis.     

Effects of long‐term nitrogen dioxide fumigation and nitrate supply on nitrite and Leghemoglobin concentrations,pH, and nitrogenase activity of soybean root nodules

Exposing 12‐day‐old soybean plants to 0.2 ppm nitrogen dioxide (NO₂) for four weeks increased the nitrite concentration and acidity, and decreased the Leghemoglobin (LHb) concentration and the nitrogenase activity of root nodules. The supply of 1 mol.m^‐3 nitrate to the roots intensified the nitrite accumulation, decreased the acidity of the nodules, and alleviated the inhibition of nitrogenase activity by NO₂ fumigation. These results suggested that the inhibition of nitrogen (N₂) fixation by N fertilizer supply might relate to the acid‐alkali balance in nodules.     

Isoflavone,glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated,glyphosate-resistant soybean

The estrogenic isoflavones of soybeans and their glycosides are products of the shikimate pathway, the target pathway of glyphosate. This study tested the hypothesis that nonphytotoxic levels of glyphosate and other herbicides known to affect phenolic compound biosynthesis might influence levels of these nutraceutical compounds in glyphosate-resistant soybeans. The effects of glyphosate and other herbicides were determined on estrogenic isoflavones and shikimate in glyphosate-resistant soybeans from identical experiments conducted on different cultivars in Mississippi and Missouri. Four commonly used herbicide treatments were compared to a hand-weeded control. The herbicide treatments were (1) glyphosate at 1260 g/ha at 3 weeks after planting (WAP), followed by glyphosate at 840 g/ha at 6 WAP; (2) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied preemergence (PRE), followed by glyphosate at 1260 g/ha at 6 WAP; (3) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by glyphosate at 1260 g/ha at full bloom; and (4) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by acifluorfen at 280 g/ha plus bentazon at 560 g/ha plus clethodim at 140 g/ha at 6 WAP. Soybeans were harvested at maturity, and seeds were analyzed for daidzein, daidzin, genistein, genistin, glycitin, glycitein, shikimate, glyphosate, and the glyphosate degradation product, aminomethylphosphonic acid (AMPA). There were no remarkable effects of any treatment on the contents of any of the biosynthetic compounds in soybean seed from either test site, indicating that early and later season applications of glyphosate have no effects on phytoestrogen levels in glyphosate-resistant soybeans. Glyphosate and AMPA residues were higher in seeds from treatment 3 than from the other two treatments in which glyphosate was used earlier. Intermediate levels were found in treatments 1 and 2. Low levels of glyphosate and AMPA were found in treatment 4 and a hand-weeded control, apparently due to herbicide drift.     

Tolerance and accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant and nonglyphosate-resistant cotton (Gossypium hirsutum L.).

Measurement of shikimic acid accumulation in response to glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase is a rapid and accurate assay to quantify glyphosate-induced damage in sensitive plants. Two methods of assaying shikimic acid, a spectrophotometric and a high-performance liquid chromatography (HPLC) method, were compared for their accuracy of recovering known amounts of shikimic acid spiked into plant samples. The HPLC method recovered essentially 100% of shikimic acid as compared with only 73% using the spectrophotometric method. Relative sensitivity to glyphosate was measured in glyphosate-resistant (GR) and non-GR cotton leaves, fruiting branches, and squares (floral buds) by assaying shikimic acid. Accumulation of shikimic acid was not observed in any tissue, either GR or non-GR, at rates of 5 mM glyphosate or less applied to leaves. All tissues of non-GR plants accumulated shikimic acid in response to glyphosate treatment; however, only fruiting branches and squares of GR plants accumulated a slight amount of shikimic acid. In non-GR cotton, fruiting branches and squares accumulated 18 and 11 times, respectively, more shikimic acid per micromolar of translocated glyphosate than leaf tissue, suggesting increased sensitivity to glyphosate of reproductive tissue over vegetative tissue. GR cotton leaves treated with 80 mM of glyphosate accumulated 57 times less shikimic acid per micromolar of translocated glyphosate than non-GR cotton but only 12.4- and 4-fold less in fruiting branches and squares, respectively. The increased sensitivity of reproductive structures to glyphosate inhibition may be due to a higher demand for shikimate pathway products and may provide an explanation for reports of fruit abortion from glyphosate-treated GR cotton.     

Effects of flumioxazin herbicide on carbon nutrition of Vitis vinifera L

To evaluate the impact of the herbicide flumioxazin (fmx) on nontarget grapevines, its effects were assessed on fruiting cuttings and field-grown plants. The stress caused by the herbicide differed according to the grapevine model. In cuttings, leaf gas exchange and photosynthetic pigment levels as well as hexose contents decreased, whereas sucrose and starch accumulated, suggesting an inhibition of photosynthesis and an increase of carbohydrate reserves as a response to the fmx-induced stress. Paradoxically, in the field-grown grapevine leaves, fmx caused a stimulation of photosynthesis, an accumulation of photosynthetic pigments and monosaccharides, in parallel with a mobilization of sucrose and starch. These results suggest that fmx reaches grapevine leaves via root uptake and has prolonged effects. In cuttings, fmx generated a toxic effect related to its target, whereas in field-grown plants, fmx had rather positive physiological effects and acts as a signal further stimulating photosynthesis and related parameters.     

Effects of salt stress on nitrogen fixation,oxygen diffusion,and ion distribution in soybean,common bean,and alfalfa

The exposure of legume nodulated‐roots to 100 mM NaCl resulted in a rapid decrease in plant growth associated with a short‐term inhibition of both nodule growth and nitrogenase activity (C₂H₂ reduction=ARA). However, these NaCl effects varied among species, common bean being more sensitive than soybean and alfalfa. The higher sensitivity of common bean was associated with a higher accumulation of sodium (Na) and chlorine (Cl) in the nodules and only a small difference between salt‐treated and control plants of common bean in their responses of ARA to raising rhizosphere pO₂. By contrast, soybean and alfalfa plants showed a higher stimulation of ARA by pO₂ for the salt‐treatment than for the control. It is concluded that the intraspecific variation in short‐term inhibition of ARA by salt may involve the regulation of O₂ diffusion and the distribution of ions in nodules.     

Manganese in RR Cotton as Affected by Glyphosate

There is evidence that glyphosate application in soybean tolerant to herbicides could interfere in the manganese (Mn) nutrition of the crop, but there is no information on this effect in cotton plants. This study aimed at assessing manganese accumulation and distribution in cotton as affected by glyphosate application. The experiment was conducted in nutrient solution with four Mn concentrations and two cotton cultivars: conventional NuOpal and NuOpal tolerant to glyphosate (RR). Glyphosate was applied or not to the tolerant cultivar. The inclusion of the glyphosate resistance gene in cotton and herbicide, application increased shikimic acid (ShA) concentration in the plants. Glyphosate application decreased cotton leaf area and the dry matter production of the plant structures. The adverse effects of glyphosate were not overcome with higher Mn rates in the solution.     

Influence of glyphosate on amino acid composition of Egyptian broomrape

The parasitic plant broomrape is entirely dependent on its host for reduced carbon and nitrogen and is also susceptible to inhibition by glyphosate that is translocated to the parasite through a host. Studies were conducted to examine the effect of broomrape parasitism on amino acid concentrations of two hosts: common vetch that is tolerant of low levels of glyphosate and oilseed rape that has been genetically engineered for glyphosate resistance. The influence of glyphosate on the amino acid content of broomrape and the two hosts was also examined. Amino acid concentrations in leaves and roots of parasitized common vetch plants were generally similar to those of the corresponding tissues of nonparasitized plants. Amino acid concentrations in broomrape were lower than those of the parasitized common vetch root. For common vetch, glyphosate applied at rates that selectively inhibited broomrape growth did not alter individual amino acid concentrations in the leaves, but generally increased amino acid levels at 0.18 kg ha-1. Glyphosate application also increased the amino acid concentrations, with the exception of arginine, of broomrape growing on common vetch and did not generally influence concentrations in leaves or roots of common vetch. In oilseed rape, parasitization by broomrape generally led to higher amino acid concentrations in leaves but lower concentrations in roots of parasitized plants. Broomrape had higher amino acid concentrations than roots of the parasitized oilseed rape. Glyphosate applied at 0.25 and 0.5 kg ha-1 generally increased the amino acid concentrations in oilseed rape leaves, but the 0.75 kg ha-1 application caused the amino acid concentrations to decrease compared to those of untreated plants. In oilseed rape root the general trend was an increase in the concentration of amino acids at the two highest rates of glyphosate. Individual amino acid concentrations in broomrape attachments growing on oilseed rape were generally increased following glyphosate application of 0.25 kg ha-1. These results indicate that low rates of glyphosate alter amino acid profiles in both host and broomrape and raise questions about the regulation of amino acid metabolism in the parasite.     

Mathematical model for the analysis of irrigation water and fertilizer management for spring wheat in the semiarid area of West Liaoning,China

Soybean plants (Glycine max L. cv. Akisengoku) were grown in water culture in a greenhouse. At the pod-setting and pod-filling stages, plants were subjected to stem-ringing or treated with high concentration of nitrate. Respiration and N₂ fixation (acetylene reducing activity) were studied in individual nodules along with the concentrations of ATP and magnesium.

There was a high positive correlation between respiration activity and acetylene reduction in soybean nodules. The maintenance respiration in mature nodules corresponded to a CO₂ evolution of 5.5 µmol/g F.W., and the respiratory cost for nitrogen fixation was estimated at 2 mg C liberated/mg N fixed, though this value was probably underestimated due to CO₂ fixation by the nodules. For the nitrogenase activity there was a threshold value of ATP concentration at around 0.15 µmol/g F.W., and the activity increased up to around 0.35 µmol/g F.W., beyond which the ATP concentration did not increase unlike the nitrogenase activity. The values for the magnesium concentration in the nodules detected in the present experiments were above the optimum level.     

Aminomethylphosphonic acid accumulation in plant species treated with glyphosate

Aminomethylphosphonic acid (AMPA) is the most frequently detected metabolite of glyphosate in plants. The objective of this study was to determine if there is any correlation of metabolism of glyphosate to AMPA in different plant species and their natural level of resistance to glyphosate. Greenhouse studies were conducted to determine the glyphosate I 50 values (rate required to cause a 50% reduction in plant growth) and to quantify AMPA and shikimate concentrations in selected leguminous and nonleguminous species treated with glyphosate at respective I 50 rates. Coffee senna [ Cassia occidentalis (L.) Link] was the most sensitive ( I 50 = 75 g/ha) and hemp sesbania [ Sesbania herbacea (P.Mill.) McVaugh] was the most resistant ( I 50 = 456 g/ha) to glyphosate. Hemp sesbania was 6-fold and Illinois bundleflower [ Desmanthus illinoensis (Michx.) MacM. ex B.L.Robins. & Fern.] was 4-fold more resistant to glyphosate than coffee senna. Glyphosate was present in all plant species, and its concentration ranged from 0.308 to 38.7 microg/g of tissue. AMPA was present in all leguminous species studied except hemp sesbania. AMPA concentration ranged from 0.119 to 4.77 microg/g of tissue. Shikimate was present in all plant species treated with glyphosate, and levels ranged from 0.053 to 16.5 mg/g of tissue. Non-glyphosate-resistant (non-GR) soybean accumulated much higher shikimate than glyphosate-resistant (GR) soybean. Although some leguminous species were found to be more resistant to glyphosate than others, and there was considerable variation between species in the glyphosate to AMPA levels found, metabolism of glyphosate to AMPA did not appear to be a common factor in explaining natural resistance levels.     

Einfluß von Herbiziden auf die N2-Fixierung und Atmungsaktivität von Mikroorganismen in Ackerböden

Influence of herbicides on nitrogen fixation and respiration activity of microorganisms in arable soils The influence of pesticides on nitrogen fixation (acetylene reduction test) as well on respiration activity was determined in model experiments. The following soils were used: Chernozem from loess (Boroll), Luvisol from loess (Boralfs), Rendsina (Lithic Rendoll), Pelosol (Fine textured Cambisol) and Humic Podzol (Humod). The tested soils differed considerably in both parameters. The rendsina showed remarkably low fixation rates whereas the podsol reduced acetylene only at higher water contents. The soil herbicides chlortoluron, terbutryne, metabenzthiazuron and chloridazon did not affect the course and the magnitude of the tested parameters even not at higher doses. Only the leaf herbicide dinosebacetate revealed a distinct inhibition of nitrogenase activity in the podsol and in the luvisol from loess. The fungicide carbendazime caused a strong stimulation of the nitrogenase activity in all soils. The respiration activity could not been influenced significantly.     

Effects of salinity and nitrogen source on growth and nitrogen fixation in alfalfa

The Interaction between the effects of nitrate (NO₃) and sodium chloride (NaCl) concentration on growth) water relations, nitrogen (N) contents and N fixation were investigated in alfalfa (Medicago sativa L. cv. Magali). The plants were grown hydroponically in a growth chamber, in the presence or absence of 3 mM potassium nitrate (KNO₃) and exposed to various concentrations of NaCl. Increased salinity resulted in a significant decrease in shoot and root biomass, relative water content and water potential. Shoot growth was more inhibited by NaCl than root biomass. The plants grown in the presence of NO₃ were slightly less affected by NaCl than the plants dependent on N fixation for their N nutrition. Nitrogenase activity measured by acetylene reduction activity was substantially inhibited by NaCl, and this inhibition was significantly correlated to the inhibition of shoot growth and total N contents. The comparison of the curves of ARA response to oxygen (O₂) partial pressure showed that the salt‐induced inhibition of nitrogenase activity was associated with a significant increase in the critical O₂ pressure of the nodules exposed to NaCl. This result shows that NaCl decreases the nodule permeability to O₂ diffusion in undeterminate nodule of alfalfa, like previously shown with determinate nodules of soybean.     

草甘膦对烟草叶片超微结构的影响

草甘膦是农业生产中广泛使用的广谱非选择性除草剂,它抑制5-烯醇丙酮酸莽草酸-3-磷酸合成酶。该酶是莽草酸途径中的关键酶,广泛存在于微生物与植物中。用30mmol／L草甘膦处理烟草叶片2、6、12、24、48h,结果显示,处理12h内烟草叶片无明显变化,24、48h后叶片黄化,并出现坏死。对各处理时期烟草叶片的超微结构研究显示,草甘膦处理导致烟草叶片细胞的叶绿体基粒片层结构被破坏,线粒体电子密度下降。这些结果提示,草甘膦通过阻碍植物的光命作用和呼晖作用面导致叶片坏死。     

Inhibitory effects of plant phenols on the activity of selected enzymes

Selected enzymes (alpha-amylase, trypsin, and lysozyme) were allowed to react with some simple phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, m-, o-, and p-dihydroxybenzenes, quinic acid, and p-benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments showed that the enzymatic activity of the derivatives was adversely affected. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The decrease in the activity was accompanied by a reduction in the amount of free amino and thiol groups, as well as tryptophan residues, which resulted from the covalent attachment of the phenolic and related compounds to these reactive nucleophilic sites in the enzymes. The enzyme inhibition correlates well with the blocking of the mentioned amino acid side chains.     

Pool of resistance mechanisms to glyphosate in Digitaria insularis

Digitaria insularis biotypes resistant to glyphosate have been detected in Brazil. Studies were carried out in controlled conditions to determine the role of absorption, translocation, metabolism, and gene mutation as mechanisms of glyphosate resistance in D. insularis. The susceptible biotype absorbed at least 12% more (14)C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential (14)C-glyphosate translocation was observed at 12 HAT, so that >70% of the absorbed herbicide remained in the treated leaf in resistant biotypes, whereas 42% remained in the susceptible biotype at 96 HAT. Glyphosate was degraded to aminomethylphosphonic acid (AMPA), glyoxylate, and sarcosine by >90% in resistant biotypes, whereas a small amount of herbicide (up to 11%) was degraded by the susceptible biotype up to 168 HAT. Two amino acid changes were found at positions 182 and 310 in EPSPS, consisting of a proline to threonine and a tyrosine to cysteine substitution, respectively, in resistant biotypes. Therefore, absorption, translocation, metabolism, and gene mutation play an important role in the D. insularis glyphosate resistance.     

Changes in Phosphate Fractions Extracted From Different Organs of Phosphorus Starved Nitrogen Fixing Pea Plants

The present work investigates the impact of phosphorus (P) starvation on plant growth, symbiotic nitrogen fixation, and internal P status (determined as extracted P fractions) of leaves, roots, and nodules of 27-days–old pea (Pisum sativum L) plants inoculated with Rhizobium leguminosarum bv viciae strain D293. The procedure of separation of organic and inorganic P compounds in 10% perchloric acid (HCLO₄) and the absorption of nucleotides in active charcoal gave several fractions, containing different phosphorus compounds, which were extracted and determined as inorganic phosphate after combustion. These are acid soluble and insoluble P, sugar P, nucleotide P, and inorganic P. The P starvation of plants inhibited significantly plant dry mass accumulation, nodulation rate and specific nitrogenase activity of nodules. These results were accompanied with lower quantities of total P per plant, acid soluble and acid non-soluble P fractions in all plant organs. The inhibited accumulation of P in the acid soluble P fraction was associated with decrease of sugar, nucleotide and inorganic P in all plant organs. The most negatively affected were all P fractions extracted from nodules and leaves. The low content of inorganic P in the stressed plant tissues was regarded as primary reason for induced alterations in the content of analyzed P fractions.