Constraints on the evolution of tolerance to herbicide in the common morning glory: resistance and tolerance are mutually exclusive

Evolutionary biologists explain the maintenance of intermediate levels of defense in plant populations as being due to trade-offs, or negative genetic covariances among ecologically important traits. Attempts at detecting trade-offs as constraints on the evolution of defense have not always been successful, leading some to conclude that such trade-offs rarely explain current levels of defense in the population. Using the agricultural pest Ipomoea purpurea, we measured correlations between traits involved in defense to glyphosate, the active ingredient in Roundup, a widely used herbicide. We found significant allocation costs of tolerance, as well as trade-offs between resistance and two measures of tolerance to glyphosate. Selection on resistance and tolerance exhibited differing patterns: tolerance to leaf damage was under negative directional selection, whereas resistance was under positive directional selection. The joint pattern of selection on resistance and tolerance to leaf damage indicated the presence of alternate peaks in the fitness landscape such that a combination of either high tolerance and low resistance, or high resistance and low tolerance was favored. The widespread use of this herbicide suggests that it is likely an important selective agent on weed populations. Understanding the evolutionary dynamics of herbicide defense traits is thus of increasing importance in the context of human-mediated evolution.     

核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶的酶学性质

核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶（EPSP合酶）是AROM多功能酶的活性之一.该酶催化莽草酸磷酸（S3P）和磷酸烯醇式丙酮酸（PEP）产生5-烯醇丙酮酰莽草酸-3-磷酸和无机磷酸的可逆反应,受除草剂草甘膦（N-（膦羧甲基）甘氨酸）抑制.纯化了核盘菌AROM蛋白并对EPSP合酶进行了酶学特征研究.结果显示,该酶反应的最适pH值为7.2,最适温度为30℃.热失活反应活化能是69.62 kJ/mol.底物S3P和PEP浓度分别高于1 mmol/L和2 mmol/L时,对EPSP合酶反应产生抑制作用.用双底物反应恒态动力学Dalziel方程求得的Km(PEP)为140.98 μmol/L,K _m(S3P)为139.58 μmol/L.酶动力学模型遵循顺序反应机制.草甘膦是该酶反应底物PEP的竞争性抑制剂（Ki为0.32 μmol/L）和S3P的非竞争性抑制剂.正向反应受K⁺激活.当［K⁺］增加时,K _m(PEP)随之降低,Km(S3P)不规律变化,而K _i(PEP)随［K⁺］增加而提高.     

Genetic analysis of glyphosate tolerance in Halomonas variabilis strain HTG7

Summary A highly glyphosate-tolerant bacterium strain HTG₇ was isolated from glyphosate-polluted soil in north China, and identified as Halomonas variabilis. It was a Gram-negative motile rod giving convex colony. The strain HTG₇ could tolerate up to 900 mM glyphosate in minimal medium. The 16S rDNA sequence was amplified by PCR using universal primers. The region essential for glyphosate tolerance was localized to a 3.5-kb fragment from a cosmid library of HTG₇. The DNA fragment consisted of one complete open reading frame (ORF) and one partial ORF. The partial ORF was homologous to prephenate dehydrogenase of Pseudomonas aeruginosa PA01. The complete ORF contained the tyrA and aroA genes. Only the 1.35-kb aroA encoding EPSP synthase conferred glyphosate tolerance, and complemented with E. coli aroA mutant ER2799. E. coli JM109 harboring aroA grew well in Mops supplemented with 80 mM glyphosate.     

Enzyme Responses of <Emphasis Type="Italic">Abutilon Theophrasti</Emphasis> in an Enhanced Biocontrol System

Weak virulence has limited the development of Colletorichum coccodes (Wallr.) Hughes (DAOM 183088) as a bioherbicide for Abutilon theophrasti Medik. (velvetleaf) control. This study examines the role of chemical synergy to suppress host defense mechanisms. Dry weight of A. theophrasti was reduced by treatment with C. coccodes in a tank-mix with 0.25 kg a.i. ha⁻¹ bentazon more than by the treatment with C. coccodes or bentazon alone, while the effect of a sub-lethal rate of glyphosate was not significant. C. coccodes did not affect phenylalanine ammonia lyase (PAL) activity in infected leaves of A. theophrasti, whereas PAL activity was significantly inhibited by the presence of bentazon 5 days after treatment. The inhibition was stronger when bentazon was applied alone than when bentazon was applied with C. coccodes. Treatment with glyphosate or a mixture of glyphosate and C. coccodes did not affect PAL activity. Peroxidase activity was strongly induced by C. coccodes treatment and increased over time. Peroxidase activity was not induced by 0.25 kg a.i. ha⁻¹ bentazon alone. However, when bentazon was applied in combination with C. coccodes, it prevented the activation of peroxidase by C. coccodes infection. Treatment with 0.2 kg a.i. ha⁻¹ glyphosate did not affect peroxidase activity. These results suggest that these enzymes are involved in the resistance mechanism of A. theophrasti, and the synergistic effect of bentazon on C. coccodes efficacy as a bioherbicide may result from suppressing the activation of these defense-related enzymes.     

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.     

Effects of methamidophos and glyphosate on copper sorption-desorption behavior in soils

A batch-equilibration technique was employed to study the impact of two organophosphorus pesticides methamidophos (MDP) and glyphosate (GPS) on copper (Cu2+) sorptiondesorption for phaeozem and burozem collected from Northeastern China. The addition of the two pesticides decreased Cu2+ sorption, increased Cu2+ desorption and prolonged the equilibrium time of Cu2+ sorption-desorption. But GPS appeared to exert a stronger influence on Cu2+ sorption-desorption due to its stronger complexion with Cu2+. When MDP was added, Cu2+ sorption-desorption was linearly correlated with MDP treatment concentrations. But in the presence of GPS, Cu2+ sorption first underwent a rapid decrease period, and then slowly tended towards a steady period. The reverse pattern could be found for Cu2+ desorption in the presence of GPS.Without pesticides and with the existence of MDP, Cu2+ sorption-desorption kinetics was well conformed to two-constant equation and Elovich equation. But that was not the case for Cu2+ desorption kinetics in the presence of GPS although its sorption could be also described by these two equations.     

Natural Fusarium Grain Infection Level in Wheat, Barley and Oat after Early Application of Fungicides and Herbicides

In Norway, early application of fungicides against cereal leaf diseases (before Zadoks 60) is common practice amongst farmers. Whether this procedure has any effect on Fusarium infection of the mature grain has been little investigated. To evaluate effects on Fusarium grain infection, cereal grains were sampled during 1996, 1997 and 1998 from 12 field trials where early spraying against fungal diseases in spring wheat, spring barley and oats was carried out. Percentage infected grains and frequency of different Fusarium species was analysed in every grain sample. The effect of fungicides, glyphosate and postemergence herbicides on Fusarium grain infection was studied. Significant increase in Fusarium infection was detected in fungicide‐treated plots compared with untreated plots. Fusarium avenaceum and F. tricinctum were the most frequent species detected. The internal ranking of Fusarium species remained the same after spraying. No significant effects were found on the level of Fusarium infection after glyphosate treatment in autumn or herbicide treatment during the growing season.     

Comparative study of two forms of <Emphasis Type="Italic">aro</Emphasis> A CP4 gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The enzyme CP4 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) from Agrobacterium tumefaciens CP4, encoded by the aroA gene, has been used for the construction of genetically modified crops resistant to total herbicide glyphosate. During the study of possible horizontal gene transfer of aroA CP4 gene from genetically modified food in gastrointestinal tract to bacterial community living in the animal gut, we have discovered and characterized truncated form of aroA CP4 within the cloning experiments in Escherichia coli. We have compared properties of the recombinant E. coli strains with both CP4 EPSPS enzyme forms.     

新型柱前衍生试剂分析草甘膦的高效液相色谱研究

以2,5-二甲氧基苯磺酰氯(DMOSC)为柱前衍生化试剂,建立了柱前衍生草甘膦的紫外检测反相高效液相色谱法,并优化了衍生化条件,得最佳条件:衍生温度35℃,时间15 min,pH 10.0,草甘膦与DMOSC的摩尔比为1∶6。HPLC分析条件:采用Kromasil C18柱,流速1.0 mL/min,柱温30℃,检测波长220 nm,流动相为甲醇-乙腈-磷酸盐缓冲溶液(0.02 mol/L、pH 5.5),三者的体积比为15∶5∶80。结果表明:草甘膦质量浓度在5~100μg/mL范围内线性关系良好,相关系数为0.996 2,检测限为0.067μg/mL。实验表明该方法反应条件温和,灵敏度高,衍生产物稳定。     

Aldo‐keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants

In recent years, concerns about the use of glyphosate‐resistant crops have increased because of glyphosate residual levels in plants and development of herbicide‐resistant weeds. In spite of identifying glyphosate‐detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo‐keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate‐mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1‐ or OsAKRI‐expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.