Glyphosate-induced metabolic changes in susceptible and glyphosate-resistant soybean (Glycine max L.) roots

Glyphosate (N-(phosphonomethyl)glycine) blocks the shikimate pathway, reducing the biosynthesis of aromatic amino acids, followed by the arrest interruption of protein production and a general metabolic disruption of the phenylpropanoid pathway. Glyphosate-resistance is conferred to soybean by incorporating a gene encoding a glyphosate-insensitive enzyme (CP4-EPSP synthase) that acts in the shikimate pathway. This paper evaluates the metabolic effects caused by this herbicide on the shikimate (shikimate dehydrogenase activity and shikimate content) and phenylpropanoid (phenylalanine ammonia-lyase activity, phenolic and lignin contents) pathways in BRS-133 (susceptible) and BRS-245RR (resistant) soybean (Glycine max L.) roots. In general, the results showed that in susceptible roots (1) glyphosate affects the shikimate pathway (massive shikimate accumulation and enhanced shikimate dehydrogenase activity) and the phenylpropanoid pathway (increase in PAL activity, production of benzoate derivatives and decrease of lignin) and (2) the metabolic disruption contributes to the production of p-hydroxybenzoate and vanillate, which likely originate from shikimate and/or cinnamate and their derivatives. No such changes were observed in the genetically modified soybean consistent with its resistance to glyphosate.     

Pesticide effects on secondary metabolism of higher plants

Secondary compounds serve both endogenous and exogenous functions in higher plants because they are involved in plant growth and development as well as intraspecies and interspecies interactions. Documentation of the effects of pesticides on secondary compound biosynthesis in higher plants is increasing. While several herbicides have been reported to reduce levels of secondary compounds by non-specific mechanisms, a few herbicides, such as alachlor and glyphosate, directly affect specific biosynthetic steps. Alachlor reduces flavonoid synthesis at a step late in the biosynthetic pathway, and glyphosate blocks synthesis of all cinnamate derivatives by inhibiting 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase. Inhibition of EPSP synthase also leads to the accumulation of high levels of shikimate, benzoic acids and benzoic acid derivatives. The sulfonylureas and p-nitro-substituted diphenylether (DPE) herbicides can cause increases in the level of cinnamatederived phenolic compounds and the DPEs can cause dramatic increases in terpenoid stress metabolites. Certain fungicides are thought to act through enhancing the capacity of plants to produce phytoalexins. These and other data suggest that sublethal effects of pesticides on target and non-target plants can significantly affect agricultural ecosystems by altering the synthesis of compounds important in inter- and intraspecies interactions.     

Glyphosate applied at low doses can stimulate plant growth

BACKGROUND: Glyphosate blocks the shikimic acid pathway, inhibiting the production of aromatic amino acids and several secondary compounds derived from these amino acids. Non-target plants can be exposed to low doses of glyphosate by herbicide drift of spray droplets and contact with treated weeds. Previous studies have reported that low doses of glyphosate stimulate growth, although these data are very limited. The objective of this study was to determine the effects of low glyphosate doses on growth of a range of plant species. RESULTS: Growth of maize, conventional soybean, Eucalyptus grandis Hill ex Maiden, Pinus caribea L. and Commelia benghalensis L. was enhanced by 1.8-36 g glyphosate ha(-1). Growth of glyphosate-resistant soybean was unaffected by any glyphosate dose from 1.8 to 720 g AE ha(-1). The optimum doses for growth stimulation were distinct for plant species and tissue evaluated. The greatest stimulation of growth was observed for C. benghalensis and P. caribea. Shikimic acid levels in tissues of glyphosate-treated soybean and maize were measured and found to be elevated at growth-stimulating doses. CONCLUSION: Subtoxic doses of glyphosate stimulate the growth of a range of plant species, as measured in several plant organs. This hormesis effect is likely to be related to the molecular target of glyphosate, since the effect was not seen in glyphosate-resistant plants, and shikimate levels were enhanced in plants with stimulated growth.     

Changes in free amino acid pools can predict the mode of action of herbicides

Studies were conducted to determine the short-term changes in free amino acid levels in the meristematic zone of maize after treatment with various herbicides with different modes of action. These herbicides included inhibitors of various amino acid biosynthetic pathways, photosynthesis, and fatty acids biosynthesis. Inhibitors of various amino acid biosynthetic pathways caused specific reduction in the pools of amino acids being produced by the particular pathway. Inhibitors of other metabolic pathways also caused significant changes in pools of various amino acids. Very similar changes in the pools of amino acids were seen in plants treated with different chemical classes of inhibitors affecting a certain metabolic pathway. However, the changes were quite different between inhibitors of different metabolic processes. The data generated from these studies could be used as a diagnostic tool to determine the mode of action of novel herbicides.     

Effects of SC-0224 and glyphosate on inflated duckweed (Lemna gibba) growth and EPSP-synthase activity from Klebsiella pneumoniae

The effects of the herbicides SC-0224 (trimethylsulfonium carboxymethylaminomethylphosphonate) and glyphosate [N-(phosphonomethyl)glycine] (PMG) on the inhibition of inflated duckweed (Lemna gibba L.) growth and on the conversion of shikimate to anthranilate in a cell-free extract of Klebsiella pneumoniae (ATCC 25306) were compared. SC-0224 is the trimethylsulfonium (TMS) salt of N-(phosphonomethyl)-glycine (PMG), whereas glyphosate is commercially formulated as the isopropylamine (IPA) salt of PMG. Both formulated and technical grade forms of SC-0224 were found to be much more phytotoxic to duckweed than either formulated or technical grade forms of glyphosate. The growth inhibition caused by glyphosate was partially prevented by different combinations of the aromatic amino acids Phe, Tyr, and Trp; whereas, the duckweed growth inhibition caused by SC-0224 could not be reduced by the same amino acid combinations. Trimethylsulfonium ion (TMS), the cationic constituent of the SC-0224 salt,and SC-0224 were found to be equally phytotoxic to duckweek indicating that the phytotoxic effects of TMS may be responsible for differences in the action of glyphosate and SC-0224 on duckweed. SC-0224 and glyphosate equally inhibited the production of anthranilate in the cell-free extract of K. pneumoniae, whereas TMS caused no inhibition. These results indicate that both constituents of the SC-0224 salt, TMS and PMG, are phytotoxic and may act independently.     

Resistance to glyphosate in Lolium rigidum

Annual ryegrass (Lolium rigidum) is a widespread and important weed of Australia and populations of this weed have developed resistance to most major herbicides, including glyphosate. The possible mechanisms of resistance have been examined in one glyphosate-resistant Lolium population. No major differences were observed between resistant and susceptible biotypes in respect of (i) the target enzyme (EPSP synthase), (ii) DAHP synthase, the first enzyme of the target (shikimate) pathway, (iii) absorption of glyphosate, or (iv) translocation. Following treatment with glyphosate, there was greater accumulation of shikimate (derived from shikimate-3-Pi) in susceptible than in resistant plants. In addition, the resistant population exhibited cross-resistance to 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate, a herbicide which, although structurally similar to glyphosate, acts at an unrelated target site. On the basis of these observations we speculate that movement of glyphosate to its site of action in the plastid is involved in the resistance mechanism. © 1999 Society of Chemical Industry     

Natural tolerance of Cuscuta campestris to herbicides inhibiting amino acid biosynthesis

The response of Cuscuta campestris Yuncker, a non‐specific above‐ground holoparasite, to amino acid biosynthesis inhibitor (AABI) herbicides, was compared with other resistant and sensitive plants in dose–response assays carried out in Petri dishes. Cuscuta campestris was found to be much more resistant to all AABI herbicides tested. The I₅₀ value of C. campestris growth inhibition by glyphosate was eightfold higher than that of transgenic, glyphosate‐resistant cotton (RR‐cotton). The I₅₀ value for C. campestris shoot growth inhibition by sulfometuron was above 500 μM, whereas that of sorghum roots was only 0.004 μM. Cuscuta campestris exposed to glyphosate gradually accumulated shikimate, confirming herbicide penetration into the parasite and interaction with an active form of the target enzyme of the herbicide, 5‐enolpyruvylshikimate‐3‐phosphate synthase. More than half of the C. campestris plants associated with transgenic, glyphosate‐resistant sugarbeet (RR‐sugarbeet) treated with glyphosate or with transgenic, sulfometuron‐resistant tomato (SuR‐tomato) treated with sulfometuron recovered and resumed regular growth 20–30 days after treatment. New healthy stems developed, followed by normal flowering and seed setting. The results of the current study demonstrate the unique capacity of C. campestris to tolerate high rates of AABI. The mechanism of this phenomenon is yet to be elucidated.     

Phytotoxic sites of action for molecular design of modern herbicides (Part 2): Amino acid, lipid and cell wall biosynthesis, and other targets for future herbicides

A bird's eye review was tried in Part 2 of this series, 'Phytotoxic sites of action for molecular design of modern herbicides', in order to select the best selection of known and some novel plant-specific targets for molecular design of modern herbicides, which affect amino acid, lipid and cell wall biosynthesis. Although amino acid biosynthesis pathways, particularly those for aromatic amino acids, ammonia assimilation and branched amino acids, have been confirmed as reasonable herbicidal target domains, the other targets affecting plant growth more markedly than inhibition of 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase and acetolactate synthase are discussed. In three essential enzymes involved in fatty acid biosynthesis in or in the vicinity of chloroplasts, acetyl-CoA carboxylase (ACCase), elongase(s) for very long chain fatty acids (VLCFA) and linolate monogalactosyldiacylglycerol desaturase, ACCase and elongase are more important targets for new herbicides. Although the effect of cellulose biosynthesis inhibitors is restricted to cell wall formation in growing plant cells only, there is a good chance to design the low-use rate herbicides also in this class of inhibitors. Other possible targets for new herbicides are also discussed.     

草甘膦防治菟丝子的机理研究

作者以菟丝子幼苗为材料研究了草甘膦防治菟丝子的机理。结果表明:草甘膦处理后,幼苗内色氨酸和花色素的含量均减少了,且其蛋白质合成也受到抑制。外源苯丙氨酸(Phe)、酪氨酸(Tyr)和色氨酸(Trp)的供给既可保证幼苗内蛋白质合成不受草甘膦的影响,又可保证其在这一除草剂存在的介质中能够正常生长。所有结果表明3种芳香族氨基酸的生物合成是草甘膦防治菟丝子的唯一作用点。     

Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to glyphosate

Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to sublethal concentrations of glyphosate were examined in order to evaluate the possibilities of using a response pattern in plants as a measure of exposure to glyphosate through the growth media, more sensitive than the well-known biomarker shikimate. Rapeseed seedlings were grown in hydroponic nutrient solutions containing varying sublethal concentrations of glyphosate (1-50 μM). After 9 days of glyphosate exposure, the shoots of the seedlings were analysed with respect to the effects on selected metabolites downstream from the primary affected metabolite shikimate, which accumulated linearly in response to glyphosate exposure (from 0 to ∼126 μmol/g DW). The selected metabolites analysed, comprising the free amino acids, and the glucosinolates derived therefrom, showed complex patterns in response to glyphosate exposure. Most noteworthy was though that they responded at the lowest concentrations of exposure to glyphosate (1 μM), where no visual effects, decrease in shoot DW or shikimate could be detected, indicating that a biomarker response more sensitive than that of shikimate can be established for plants exposed to glyphosate.     

Response of wild Brassica juncea populations to glyphosate

BACKGROUND: Wild Brassica juncea (L.) is a troublesome arable land weed and ruderal. It is critical to understand the responses of this weed to herbicides, because the assessment of its susceptibility profile has important ecological and evolutionary consequences for future cultivation of herbicide-tolerant oilseed rape. The response of 31 wild populations from different geographic origins in China to glyphosate was evaluated with two bioassay methods, and variable responses were found in initial studies. Dose-response assays were conducted to characterize the extreme populations further, and shikimate accumulation in vivo was determined using a spectrophotometric method. RESULTS: On the basis of ID(50) values, the resistance ratios (R/S) were 5.85 and 4.19 for two glyphosate-resistant B. juncea populations in germination tests, whereas they were 4 times more resistant to glyphosate in spray tests. There were differences in shikimate accumulation patterns between the two biotypes. Shikimate concentrations in resistant populations began to decline from 6 days after treatment (DAT), while they increased continually in susceptible populations. CONCLUSION: The results obtained suggest that the populations responded differentially to glyphosate, and this variability may provide the genetic basis for evolution of individuals with increased resistance to glyphosate, with important implications for herbicide resistance management, especially in the context of risk assessment of glyphosate-tolerant crops.     

Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to glyphosate

Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to sublethal concentrations of glyphosate were examined in order to evaluate the possibilities of using a response pattern in plants as a measure of exposure to glyphosate through the growth media, more sensitive than the well-known biomarker shikimate. Rapeseed seedlings were grown in hydroponic nutrient solutions containing varying sublethal concentrations of glyphosate (1–50 μM). After 9 days of glyphosate exposure, the shoots of the seedlings were analysed with respect to the effects on selected metabolites downstream from the primary affected metabolite shikimate, which accumulated linearly in response to glyphosate exposure (from 0 to ∼126 μmol/g DW). The selected metabolites analysed, comprising the free amino acids, and the glucosinolates derived therefrom, showed complex patterns in response to glyphosate exposure. Most noteworthy was though that they responded at the lowest concentrations of exposure to glyphosate (1 μM), where no visual effects, decrease in shoot DW or shikimate could be detected, indicating that a biomarker response more sensitive than that of shikimate can be established for plants exposed to glyphosate.     

Environmental fate of herbicides trifluralin, metazachlor, metamitron and sulcotrione compared with that of glyphosate, a substitute broad spectrum herbicide for different glyphosate-resistant crops

The introduction of crops resistant to the broad spectrum herbicide glyphosate, N-(phosphonomethyl)glycine, may constitute an answer to increased contamination of the environment by herbicides, since it should reduce the total amount of herbicide needed and the number of active ingredients. However, there are few published data comparing the fate of glyphosate in the environment, particularly in soil, with that of substitute herbicides. The objective of this study is to compare the fate of glyphosate in three soils with that of four herbicides frequently used on crops that might be glyphosate resistant: trifluralin, alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine, and metazachlor, 2-chloro-N-(pyrazol-1-ylmethyl)acet-2',6'-xylidide for oilseed rape, metamitron, 4-amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazin-5-one for sugarbeet and sulcotrione, 2-(2-chloro-4-mesylbenzoyl)cyclohexane-1,3-dione for maize. The distribution of herbicides between the volatilized, mineralized, extractable and non-extractable fractions was studied, along with the formation of their metabolites in laboratory experiments using 14C-labelled herbicides, over a period of 140 days. The main dissipation pathways were mineralization for glyphosate and sulcotrione, volatilization for trifluralin and non-extractable residues formation for metazachlor and metamitron. The five herbicides had low persistence. Glyphosate had the shortest half-life, which varied with soil type, whereas trifluralin had the longest. The half-lives of metazachlor and sulcotrione were comparable, whereas that of metamitron was highly variable. Glyphosate, metazachlor and sulcotrione were degraded into persistent metabolites. Low amounts of trifluralin and metamitron metabolites were observed. At 140 days after herbicide applications, the amounts of glyphosate and its metabolite residues in soils were the lowest in two soils, but not in the third soil, a loamy sand with low pH. The environmental advantage in using glyphosate due to its rapid degradation is counterbalanced by accumulation of aminomethylphosphonic acid specifically in the context of extensive use of glyphosate.     

Joint action of amino acid biosynthesis-inhibiting herbicides

The joint action of binary mixtures of the amino acid biosynthesis‐inhibiting herbicides glyphosate, glufosinate‐ammonium, metsulfuron‐methyl and imazapyr was assessed in pot experiments applying the Additive Dose Model (ADM). Plants of Sinapis arvensis or S. alba were sprayed with seven doses of the herbicides alone and binary fixed‐ratio mixtures of the four herbicides. In total, 73 binary mixtures were studied in six separate experiments. Mixtures of glyphosate and glufosinate‐ammonium were less phytotoxic than predicted by ADM whether commercial formulations or technical grade products were applied. In contrast, mixtures of glyphosate and metsulfuron‐methyl, glyphosate and imazapyr, glufosinate‐ammonium and metsulfuron‐methyl, glufosinate‐ammonium and imazapyr, and metsulfuron‐methyl and imazapyr either followed ADM or were synergistic. Synergism was observed most frequently for mixtures of glyphosate or glufosinate‐ammonium with metsulfuron‐methyl. Synergism was also more pronounced for commercial formulations of glyphosate and glufosinate‐ammonium than for the corresponding technical grade formulations, implying that synergism was caused by the presence of the formulation constituents of the commercial glyphosate and glufosinate‐ammonium formulations in the spray solution.     

Auxin herbicides: current status of mechanism and mode of action

Synthetic compounds that act like phytohormonal ‘superauxins’ have been among the most successful herbicides used in agriculture for more than 60 years. These so‐called auxin herbicides are more stable in planta than the main natural auxin, indole‐3‐acetic acid (IAA), and show systemic mobility and selective action, preferentially against dicot weeds in cereal crops. They belong to different chemical classes, which include phenoxycarboxylic acids, benzoic acids, pyridinecarboxylic acids, aromatic carboxymethyl derivatives and quinolinecarboxylic acids. The recent identification of receptors for auxin perception and the discovery of a new hormone interaction in signalling between auxin, ethylene and the upregulation of abscisic acid biosynthesis account for a large part of the repertoire of auxin‐herbicide‐mediated responses, which include growth inhibition, senescence and tissue decay in sensitive dicots. An additional phenomenon is caused by the quinolinecarboxylic acid quinclorac, which also controls grass weeds. Here, the accumulation of phytotoxic levels of tissue cyanide, derived ultimately from quinclorac‐stimulated ethylene biosynthesis, plays a key role in eliciting the herbicidal symptoms in sensitive grasses. Copyright © 2009 Society of Chemical Industry     

Low glyphosate rates do not affect Citrus limonia (L.) Osbeck seedlings

BACKGROUND: Glyphosate is used to control weeds in citrus orchards, and accidental spraying or wind drift onto the seedlings may cause growth arrest owing to metabolism disturbance. Two experiments were carried out to investigate the effect of non‐lethal rates (0, 180, 360 and 720 g AI ha^?1) of glyphosate on four‐month‐old ‘Cravo’ lime, Citrus limonia (L.) Osbeck, seedlings. Photosynthesis and the concentrations of shikimic acid, total free amino acids and phenolic acids were evaluated. RESULTS: Only transitory effects were observed in the contents of shikimate and total free amino acids. No visual effects were observed. CONCLUSION: The present study showed that glyphosate at non‐lethal rates, which is very usual when accidental spraying or wind drift occurs in citrus orchard, did not cause severe metabolic damage in ‘Cravo’ lime seedlings. Copyright © 2009 Society of Chemical Industry     

Effects of alachlor on anthocyanin and lignin synthesis in etiolated sorghum (Sorghum bicolor (L.) Moench) mesocotyls

Alachlor, a preemergence herbicide used to control grasses and some broadleaf weeds, was found to inhibit anthocyanin and lignin accumulation in excised sections of mesocotyls from 6 day-old, etiolated sorghum (Sorghum bicolor (L.) Moench) seedlings. Alachlor inhibited anthocyanin and lignin synthesis by 50% at 20 and 28 μM, respectively, with anthocyanin synthesis being inhibited in 1 hr. Other chloroacetanilide herbicides also inhibited anthocyanin synthesis in this system, but all were less active than alachlor. Inhibition of anthocyanin synthesis could not be reversed by compounds from the shikimic acid or phenylpropanoic acid pathways. Anthocyanin synthesis could be restored by removal of alachlor from the incubation solution. Evidence is presented which suggests that alachlor inhibits anthocyanin formation at a point late in the phenylpropanoic acid pathway and not in the shikimic acid pathway.     

藜、铁苋菜和苘麻对草甘膦的耐受性及其与莽草酸含量的关系

采用整株生物测定法研究了藜、铁苋菜和苘麻对草甘膦的耐受性,药后14 d测定结果表明,草甘膦对上述3种杂草的ED50分别为215.27、954.34、1 522.54 g a.i./hm~2。通过比较杂草植株地上部莽草酸积累量的变化发现,草甘膦1 230 g a.i./hm~2处理后,藜、铁苋菜和苘麻莽草酸积累量最大值分别为1 400.65、1247.19、581.28μg/g,莽草酸积累量越少的杂草对草甘膦耐受性也越强。药后5 d,对杂草不同部位莽草酸积累量的比较显示,3种杂草顶部叶片莽草酸积累量明显大于根部,敏感种和耐受种相比,顶部叶片莽草酸积累量的差异更为明显,该部位可以准确地评价杂草对草甘膦的耐受程度。     

Molecular basis of multiple resistance to ACCase-inhibiting and ALS-inhibiting herbicides in Lolium rigidum

Herbicide resistance in Lolium rigidum is widespread across much of the agricultural land in Australia. As the incidence of herbicide resistance has increased, so has the incidence of multiple herbicide resistance. This reduces the herbicide options available for control of this weed. This study reports on the successful amplification and sequencing of the acetolactate synthase (ALS) gene of L. rigidum using primers designed from sequence information of related taxa. This enables, for the first time, the successful determination of a mutation in the ALS gene of this species that provides resistance to ALS‐inhibiting herbicides. This mutation causes amino acid substitution at Trp574 (numbering standardised to Arabidopsis thaliana) to Leu which had been reported to confer a high level of resistance against all classes of ALS inhibitor herbicides. In addition, multiple resistance to ALS‐inhibiting and acetyl‐coenzyme A carboxylase‐inhibiting herbicides is acquired through the independent accumulation of mutant alleles for the target sites. This may thus explain some of the irregular, mosaic resistance patterns that occur in this predominantly outcrossing species.     

The plasma membrane as a barrier to herbicide penetration and site for adjuvant action

Adjuvants are traditionally thought to exert their main effect on the cuticle or spray droplet to enhance foliar-applied herbicide penetration. However, considerable evidence exists indicating that the plasma membrane is a barrier to intracellular penetration of herbicides and a site of action for adjuvants. Surfactants may penetrate through the cuticle and into the region of the plasma membrane. Insertion into the membrane causes a general “ loosening” to provide greater penetration by highly polar herbicides such as glyphosate. Weak acid herbicides typically have a lipophilic moiety and, therefore, can move more easily through the membrane but the rate and accumulation is dependent on pH conditions across the membrane. Ammonium salts have been shown to affect the pH of the apoplast in a manner which allows faster penetration and greater accumulation of weak acid herbicides. Examination and understanding of the plasma membrane as a barrier to herbicide penetration will aid in defining the mechanisms of adjuvant action and improve the efficiency of agrochemical use.