Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.

The EPSPS Pro106Ser substitution solely accounts for glyphosate resistance in a goosegrass(Eleusine indica) population from Tennessee, United States

Previous studies have documented the occurrence of glyphosate-resistant （GR） goosegrass （Eleusine indica （L.） Gaertn.） and, in at least some cases, resistance is due to an altered target site. Research was performed to determine if an altered target site was responsible for GR in a Tennessee, United States goosegrass population （TennGR）. DNA sequencing revealed a mutation in TennGR plants conferring the Prol06Ser 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） substitution previously identified in other GR populations. F2 populations were derived from TennGR plants crossed with plants from a glyphosate-susceptible population （TennGS） and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F2 populations segregated 1：2：1 sensitive：intermediate：resistant in response to a selec- tive dose of glyphosate, and these responses co-segregated with the EPSPS genotypes （PP106, PS106, and SS106）. To separately investigate the effect of the Prol06Ser substitution on GR, glyphosate dose-response curves and 50% effective dose （EDso） values were compared among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Prol06Ser EPSPS mutation is solely responsible for GR in the TennGR goosegrass population.

Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene,which confers glyphosate resistance of goosegrass (Eleusine indica)

Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.

Varying Tolerance to Glyphosate in a Population of Palmer Amaranth with Low EPSPS Gene Copy Number

A Palmer amaranth population (seeds collected in the year 2000;Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha?1) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD50 of 0.75 kg·ha?1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.

Glyphosate as a direct or indirect activator of pro-inflammatory signaling and cognitive impairment

Glyphosate-based herbicides are widely used around the world, making it likely that most humans have significant exposure. Because of habitual exposure, there are concerns about toxicity including neurotoxicity that could result in neurological, psychiatric, or cognitive impairment. We recently found that a single injection of glyphosate inhibits long-term potentiation, a cellular model of learning and memory, in rat hippocampal slices dissected 1 day after injection, indicating that glyphosate-based herbicides can alter cognitive function. Glyphosate-based herbicides could adversely affect cognitive function either indirectly and/or directly. Indirectly, glyphosate could affect gut microbiota, and if dysbiosis results in endotoxemia(leaky gut), infiltrated bacterial by-products such as lipopolysaccharides could activate pro-inflammatory cascades. Glyphosate can also directly trigger pro-inflammatory cascades. Indeed, we observed that acute glyphosate exposure inhibits long-term potentiation in rat hippocampal slices. Interestingly, direct inhibition of long-term potentiation by glyphosate appears to be similar to that of lipopolysaccharides. There are several possible measures to control dysbiosis and neuroinflammation caused by glyphosate. Dietary intake of polyphenols, such as quercetin, which overcome the inhibitory effect of glyphosate on long-term potentiation, could be one effective strategy. The aim of this narrative review is to discuss possible mechanisms underlying neurotoxicity following glyphosate exposure as a means to identify potential treatments.

Glyphosate Exposure Associated with Human Neurodegenerative Disorders: A Scoping Review

Chemically engineered agricultural products such as pesticides, insecticides, and herbicides, although used considerably for both industrialized and personal agricultural use, have recently been associated with a number of serious human health disorders. This rapid literature review aims to accumulate and analyze research from the last ten years, focusing specifically on the effects of exposure to glyphosate-based herbicide products such as Roundup as associated with the formation of various neurological disorders. Specifically, this review focuses on laboratory research using animal models or human cell cultures as well as human population-based epidemiological studies. It associates exposure to glyphosate or glyphosate-based products with the formation or exacerbation of neurological disorders such as Parkinson’s disease, Alzheimer’s disease, seizures, and autism spectrum disorder. In addition, it examines the correlation between the gut-brain axis, exposure to glyphosate, and neurodegeneration.

Study on Antioxidant Enzyme Activity and Physiology and Biochemistry of Solanum nigrum L. under Glyphosate Stress

[Objectives] This study was conducted to investigate the scientific prevention and control of Solanum nigrum L. [Methods] Through experiments on S. nigrum from different sources, it was found that glyphosate stress had significant effects on antioxidant enzyme activity and oxidative damage of sensitive S. nigrum plants. [Results] Sensitive S. nigrum showed oxidative damage under glyphosate stress, while resistant S. nigrum responded to adversity damage by improving its antioxidant enzyme activity. The experimental results showed that the antioxidant enzymes and reduced glutathione of S. nigrum had certain metabolic detoxification effects under glyphosate stress. [Conclusions] This study provides a theoretical basis for scientific prevention and control of S. nigrum , and has a certain reference value for revealing the glyphosate resistance mechanism of S. nigrum .

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.

Co-expression of GR79 EPSPS and GAT generates highglyphosate-resistant alfalfa with low glyphosate residues

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide,thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy toassist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms ofGR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able totolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten timeslower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicideresistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Variation in 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Coding Sequences and Glyphosate Response among <i>Cyperus rotundus</i>L. Populations

The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymorphisms (SNPs) only a few of which were predicted to cause an amino acid change in the EPSP synthase. None had the proline to serine substitution or other substitutions responsible for glyphosate resistance reported in other species. A dendrogram generated from the cluster analysis of the EPSPS gene sequences indicated similarities between accessions from Tanzania, Indonesia, California-2, Greece, Brazil, Argentina and Iran much like cluster analysis previously reported based on RAPD scores and morphological traits possibly indicating a common genetic background or origin. Considering the differences in EPSPS sequences, the response of these purple nutsedge accessions to 0.84 kg·ae·ha-1 of glyphosate was assessed to determine whether differential tolerance was present. At 7 days after the first application control ranged from 9% for the accession from Greece to 73% for the accession from Tanzania. Control of these accessions increased to 45% and 93% respectively by 14 days after the second application. The I50’s for glyphosate inhibition of growth for four accessions from geographically distant countries (Mississippi, Brazil, Indonesia and Tanzania) were 0.21, 0.10, 0.25 and 0.06 kg·ha-1, respectively, which represented a 4-fold difference. The difference in sensitivity to glyphosate may be a result of a non-target site mechanism such as differences in sequestration, translocation or cuticle thickness rather than alterations in EPSPS.

Size-controlled preparation of hollow silica spheres and glyphosate release

Different-sized hollow SiO2 spheres of 249–1348 nm in diameter were successfully prepared by using Na2SiO3 as the precursor and using polystyrene and polystyrene-methyl acrylic acid latexes as the templates. The diameter and shell thickness of the hollow SiO2 spheres increase with increasing the latex template diameter at a given mass ratio of SiO2 to latex template. The diameter and shell thickness of the hollow SiO2 spheres also increase with increasing the mass ratios of SiO2 to latex template. The presence of carboxylic acid groups on the surfaces of polystyrene-methyl acrylic acid latex templates favors the formation of dense and uniform SiO2 shells. The hollow SiO2 sphere is constructed by mesoporous shell with large specific surface area. When glyphosate is used as a release model chemical, glyphosate release rate is tuned by varying the shell thickness.

Screening for Glyphosate Resistant Wild Soybean(Glycine soja) and Study on Its Physiological Mechanisms of Resistance

[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistant soybean. [Method] First,a screening for glyphosate resistant varieties among sixty-seven wild soybean materials was done in a field trial; subsequently, physiological indexes of the screened resistant variety ZYD0685 and the sensitive variety ZYD0790 were studied. [Result]At the glyphosate dose of 1.23 kg a.i/hm2, glyphoaste resistance varied greatly among different wild soybean materials, with the highest survival rate of 87% and83% occurring in ZYD0685 and ZYD2405, respectively, and that of another seven accessions ranged from 2.7% to 38%, and all the remaining fifty-eight soybean materials died. After treatment with glyphoaste at different doses, there were no significant differences in chlorophyll content and shikimate content in the resistant ZYD0685, but there was an evident increase in the activity of gultathione-S-transferases（GSTs）; while in the sensitive ZYD0790, the content of shikimic acid increased significantly, and chlorophyll content decreased significantly, and GSTs activity revealed a slight change. [Conclusion] Therefore, lowering the amount of accumulated shikimic acid is the major physiological response to glyphosate in wild soybean.

Efficacy of POST glyphosate applications in combination with other POST herbicides in glyphosate-resistant corn (Zea mays L.)

The use of glyphosate-resistant corn has facilitated a shift from a reliance on preemergence residual herbicides to postemergence (POST) herbicides, and in some cases exclusively glyphosate. Glyphosate is a non-selective herbicide that is relatively slow-acting, which may allow weeds to continue to compete with corn after application and potentially decrease crop yield. The addition of several POST corn herbicides, with some residual control, to an early-season glyphosate application was examined to determine if the tankmix combination would improve the speed of weed control compared to glyphosate applied alone. Seven field trials were conducted over three years (2009, 2010 and 2011) near Ridgetown and Exeter, Ontario. The control of common ragweed was improved 3 days after application (DAA) with three POST glyphosate tankmixes compared to glyphosate alone. However control was still less than 55%. Depending on the weed species examined, at 28 DAA two of the glyphosate tankmix treatments tested provided better common ragweed, common lambsquarters, or green foxtail control than glyphosate alone. Treatments providing better weed control at 28 DAA also typically decreased weed density compared to glyphosate alone.

Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glypbosateresistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L^-1. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L^-1. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.

抗草甘膦水稻突变体osgr-1 EPSPS基因克隆及生物信息学分析

为深入研究抗草甘膦水稻突变体osgr-1中编码5-烯醇式丙酮酸莽草酸-3-磷酸合成酶基因EPSPS的结构与功能,利用生物信息学分析方法对EPSPS基因结构进行分析,对其编码产物功能进行预测。对克隆的EPSPS基因序列分析表明,osgr-1突变体EPSPS基因中CDS序列发生了3个位点的突变,分别为第226位核苷酸残基C变为G,引起编码蛋白226位脯氨酸(Pro)变成丙氨酸(Ala);第301位核苷酸残基G变为T,导致编码产物311位丙氨酸(Ala)变为丝氨酸(Ser);第311位核苷酸残基A变为C,导致编码蛋白中多少位的谷氨酸(Glu)变为丙氨酸(Ala)。该突变基因编码蛋白含511个aa,Mr为53,823kDa,分子式为C_(2373)H_(3867)N_(659)O_(721)S_(23),等电点为8.33,为疏水性蛋白,定位于叶绿体上且不存在信号肽;其二级结构中β-转角、α螺旋和无规则卷曲分别占31.6%、17.7%和0.7%;三级结构呈单聚体,由2个亚基组成,有4个氯离子、3个镁离子、1个磷酸根离子的结合位点和2个结构域。

Occurrence of Glyphosate-Resistant Horseweed (Conyza canadensis) Population in China

Horseweed （Conyza canadensis）, an invasive alien weed, is one of the main weeds in orchards in China. Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in China, a case of glyphosate-resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries. Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history. Potted seedlings with 11-13-leaf growth stage were treated with glyphosate at 0.035, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, 4.48, and 8.96 kg a.i. ha-1. The dosage dependence response curve of each population was constructed with Log-logistic dose response regression equations. The ED50 value of each population was calculated and compared with the susceptible population from China. Different populations had different relative glyphosate-resistant levels which increased with the number of years of glyphosate application. Two populations with the highest resistance levels, 8.28 and 7.95 times, were found in Ningbo, Zhejiang Province, China, where glyphosate was used for weed control in orchards twice each year for 15 yr. The two resistant populations accumulated approximately two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application.

Cadmium adsorption in montmorillonite as affected by glyphosate

Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO 2- 3 and -COOH groups, on cadmium adsorption in montmorillonite was studied in detail. The results showed that the cadmium adsorption quantity in montmorillonite increased with increasing soil solution pH and cadmium concentration as usual, but decreased with glyphosate, which is due to the formation of a low affinity complex of Cd and glyphosate and decreasing solution pH induced by glyphosate addition. When the equilibrium solution pH was below 6.7, glyphosate has little effect on cadmium adsorption, but when the equilibrium solution pH was above 6.7, glyphosate significantly decreased cadmium adsorption quantity in montmorillonite. In addition, the adding order of Cd and glyphosate also influenced Cd adsorption quantity in montmorillonite.

Effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils

Glyphosate （GPS） is a non-selective, post-mergence herbicide that is widely used throughout the world. Due to the similar molecular structures of glyphosate and phosphate, adsorption of glyphosate on soil is easily affected by coexisting phosphate, especially when phosphate is applied at a significant rate in farmland. This paper studied the effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils including two variable charge soils and one permanent charge soil. The results indicated that Freundlich equations used to simulate glyphosate adsorption isotherms gave high correlation coefficients（0.990-0.998） with K values of 2751, 2451 and 166 for the zhuanhong soil（ZH soil, Laterite）, red soil（ RS, Udic Ferrisol） and Wushan paddy soil（WS soil, Anthrosol）, respectively. The more the soil iron and aluminum oxides and clay contained, the more glyphosate adsorbed. The presence of phosphate significantly decreased the adsorption of glyphosate to the soils by competing with glyphosate for adsorption sites of soils. Meanwhile, the effects of phosphate on adsorption of glyphosate on the two variable charge soils were more significant than that on the permanent charge soil. When phosphate and glyphosate were added in the soils in different orders, the adsorption quantities of glyphosate on the soils were different, which followed GPS-soil 〉 GPS-P-soil = GPS-Soil-P 〉 P-soil-GPS, meaning a complex interaction occurred among glyphosate, phosphate and the soils.