Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Characterization of Cytochrome P450s with Key Roles in Determining Herbicide Selectivity in Maize.

Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.

Fitness Cost Associated With Enhanced EPSPS Gene Copy Number and Glyphosate Resistance in an Amaranthus tuberculatus Population.

The evolution of resistance to pesticides in agricultural systems provides an opportunity to study the fitness costs and benefits of novel adaptive traits. Here, we studied a population of Amaranthus tuberculatus (common waterhemp), which has evolved resistance to glyphosate. The growth and fitness of seed families with contrasting levels of glyphosate resistance was assessed in the absence of glyphosate to determine their ability to compete for resources under intra- and interspecific competition. We identified a positive correlation between the level of glyphosate resistance and gene copy number for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) glyphosate target, thus identifying gene amplification as the mechanism of resistance within the population. Resistant A. tuberculatus plants were found to have a lower competitive response when compared to the susceptible phenotypes with 2.76 glyphosate resistant plants being required to have an equal competitive effect as a single susceptible plant. A growth trade-off was associated with the gene amplification mechanism under intra-phenotypic competition where 20 extra gene copies were associated with a 26.5 % reduction in dry biomass. Interestingly, this growth trade-off was mitigated when assessed under interspecific competition from maize.

Characterization of 4-hydroxyphenylpyruvate dioxygenases, inhibition by herbicides and engineering for herbicide tolerance in crops.

4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with kcat/KmHPP values ranging from 0.8 to 2.5 s-1 µM-1. Most were also highly sensitive to herbicides with, for example, Ki values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, Ki value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (CallistoTM) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of Ki to KmHPP, by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds.

Mechanism of resistance to mesotrione in an Amaranthus tuberculatus population from Nebraska, USA.

Amaranthus tuberculatus is a troublesome weed in corn and soybean production systems in Midwestern USA, due in part to its ability to evolve multiple resistance to key herbicides including 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we have investigated the mechanism of resistance to mesotrione, an important chemical for managing broadleaf weeds in corn, in a multiple herbicide resistant population (NEB) from Nebraska. NEB showed a 2.4-fold and 45-fold resistance increase to mesotrione compared to a standard sensitive population (SEN) in pre-emergence and post-emergence dose-response pot tests, respectively. Sequencing of the whole HPPD gene from 12 each of sensitive and resistant plants did not detect any target-site mutations that could be associated with post-emergence resistance to mesotrione in NEB. Resistance was not due to HPPD gene duplication or over-expression before or after herbicide treatment, as revealed by qPCR. Additionally, no difference in mesotrione uptake was detected between NEB and SEN. In contrast, higher levels of mesotrione metabolism via 4-hydroxylation of the dione ring were observed in NEB compared to the sensitive population. Overall, the NEB population was characterised by lower levels of parent mesotrione exported to other parts of the plant, either as a consequence of metabolism in the treated leaves and/or impaired translocation of the herbicide. This study demonstrates another case of non-target-site based resistance to an important class of herbicides in an A. tuberculatus population. The knowledge generated here will help design strategies for managing multiple herbicide resistance in this problematic weed species.

Synthesis and evaluation of hydroxyazolopyrimidines as herbicides; the generation of amitrole in planta.

BACKGROUND: Exploiting novel herbicidal modes of action is an important method to overcome the challenges faced by increasing resistance and regulatory pressure on existing commercial herbicides. Recent reports of inhibitors of enzymes in the non-mevalonate pathway of isoprenoid biosynthesis led to the design of a novel class of azolopyrimidines which were assessed for their herbicidal activity. Studies were also undertaken to determine the mode of action responsible for the observed herbicidal activity. RESULTS: In total, 30 novel azolopyrimidines were synthesised and their structures were unambiguously determined by 1 H NMR, mass spectroscopy and X-ray crystallographic analysis. The herbicidal activity of this new chemical class was assessed against six common weed species, with compounds from this series displaying bleaching symptomology in post-emergence tests. A structure-activity relationship for the novel compounds was determined, which showed that only those belonging to the hydroxytriazolopyrimidine subclass displayed significant herbicidal activity. Observed similarities between the bleaching symptomology displayed by these herbicides and amitrole suggested that hydroxytriazolopyrimidines could be acting as elaborate propesticides of amitrole, and this was subsequently demonstrated in plant metabolism studies using Amaranthus retroflexus. It was shown that selected hydroxytriazolopyrimidines that displayed promising herbicidal activity generated amitrole, with peak concentrations of amitrole generally being observed 1 day after application. Additionally, the herbicidal activity of selected compounds was profiled against tobacco plants engineered to overexpress 4-diphosphocytidyl-2C-methyl-d-erythritol synthase (IspD) or lycopene ß-cyclase, and the results suggested that, where significant herbicidal activity was observed, inhibition of IspD was not responsible for the activity. Tobacco plants overexpressing lycopene ß-cyclase showed tolerance to amitrole and the two most herbicidally active triazolopyrimidines. CONCLUSIONS: Inhibition of IspD leading to herbicidal activity has been ruled out as the mode of action for the hydroxytriazolopyrimidine class of herbicides. Additionally, tobacco plants overexpressing lycopene ß-cyclase showed tolerance to amitrole, which indicates that this is the main herbicidal mode of action for amitrole. Results from the metabolic fate study of selected hydroxytriazolopyrimidines suggested that the herbicidal activity displayed by these compounds is due to amitrole production, which was confirmed when tobacco plants overexpressing lycopene ß-cyclase also showed tolerance towards two triazolopyrimidines from this study. © 2016 Society of Chemical Industry.

Children's perceptions of injuries: a qualitative study in Sweden.

To understand one of the major public health problems for children, it is important to consider the children's perspective. The purpose of this qualitative study was to explore, describe, and categorize children's perceptions of injury severity and children's explanations of the injuries they experience. A total of 29 students from six randomly selected schools were interviewed in age groups of 9, 13, and 17 years. Manifest content analysis according to Graneheim and Lundman (2004) was used to categorize children's own statements. Need of medical attention, long-term consequences, and familiarity with the injury risk situation were identified as important determinants of children's perception of injury severity. Three categories emerged from children's explanations of their injuries: "Because of Me" (beliefs, lack of concentration, health conditions, and lack of awareness of risk), "Because of the Situation" (rain, ice, wind, animals, inanimate objects, constructions, and the children's games), and "Just Inexplicable" to the children. Findings suggest that children have a wide perception of injury severity and that children's beliefs of injury causation, as well as children's familiarity with injury risk situations, need to be considered in future studies focusing on the development of childhood injury prevention strategies. Additionally, results suggest that sometimes children cannot or do not want to explain their injuries.

Role of a novel I1781T mutation and other mechanisms in conferring resistance to acetyl-CoA carboxylase inhibiting herbicides in a black-grass population.

BACKGROUND: Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2). METHODOLOGY/PRINCIPAL FINDINGS: Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. CONCLUSION/SIGNIFICANCE: This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances surpassed that of target site resistance, including the one endowed by the most commonly encountered I1781L mutation.

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

BACKGROUND: Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). METHODOLOGY/PRINCIPAL FINDINGS: The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. CONCLUSION/SIGNIFICANCE: This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.

Alcohol environment, gender and nonfatal injuries in young people. An ecological study of fourteen Swedish municipalities (2000-2005).

BACKGROUND: Sweden has had a restrictive alcohol policy, but there are gender and geographical differences in alcohol consumption and injury rates within the country. Whether and how the Swedish alcohol environment influences gender differences in injuries in young people is still unclear. Thus, the aim of this study was to analyse the associations between the local alcohol environment and age- and gender-specific nonfatal injury rates in people up to 24 years in Sweden. METHODS: The local alcohol environment from 14 municipalities was studied using indicators of alcohol access, alcohol consumption and alcohol-related crimes. A comprehensive health care register of nonfatal injuries was used to estimate mean annual rates of nonfatal injuries by gender and age group (2000-2005). Pearson's correlation coefficients were used to analyse linear associations. RESULTS: Associations were shown for both alcohol access and alcohol consumption with injury rates in boys aged 13-17 years; no other associations were observed between alcohol access or per capita alcohol consumption and nonfatal childhood injuries. The prevalence of crimes against alcohol laws was associated with injury rates in children of both genders aged 6-17 years. CONCLUSIONS: This study found no strong area-level associations between alcohol and age and gender specific nonfatal injuries in young people. Further, the strength of the area-level associations varied by age, gender and type of indicator used to study the local alcohol environment.

Broad resistance to ACCase inhibiting herbicides in a ryegrass population is due only to a cysteine to arginine mutation in the target enzyme.

BACKGROUND: The design of sustainable weed management strategies requires a good understanding of the mechanisms by which weeds evolve resistance to herbicides. Here we have conducted a study on the mechanism of resistance to ACCase inhibiting herbicides in a Lolium multiflorum population (RG3) from the UK. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of plant phenotypes and genotypes showed that all the RG3 plants (72%) that contained the cysteine to arginine mutation at ACCase codon position 2088 were resistant to ACCase inhibiting herbicides. Whole plant dose response tests on predetermined wild and mutant 2088 genotypes from RG3 and a standard sensitive population indicated that the C2088R mutation is the only factor conferring resistance to all ten ACCase herbicides tested. The associated resistance indices ranged from 13 for clethodim to over 358 for diclofop-methyl. Clethodim, the most potent herbicide was significantly affected even when applied on small mutant plants at the peri-emergence and one leaf stages. CONCLUSION/SIGNIFICANCE: This study establishes the clear and unambiguous importance of the C2088R target site mutation in conferring broad resistance to ten commonly used ACCase inhibiting herbicides. It also demonstrates that low levels "creeping", multigenic, non target site resistance, is not always selected before single gene target site resistance appears in grass weed populations subjected to herbicide selection pressure.

A novel P106L mutation in EPSPS and an unknown mechanism(s) act additively to confer resistance to glyphosate in a South African Lolium rigidum population.

Glyphosate resistance evolution in weeds is a growing problem in world agriculture. Here, we have investigated the mechanism(s) of glyphosate resistance in a Lolium rigidum population (DAG1) from South Africa. Nucleotide sequencing revealed the existence of at least three EPSPS homologues in the L. rigidum genome and identified a novel proline 106 to leucine substitution (P106L) in 52% DAG1 individuals. This mutation conferred a 1.7-fold resistance increase to glyphosate at the whole plant level. Additionally, a 3.1-fold resistance increase, not linked to metabolism or translocation, was estimated between wild-type P106-DAG1 and P106-STDS sensitive plants. Point accepted mutation analysis suggested that other amino acid substitutions at EPSPS position 106 are likely to be found in nature besides the P106/S/A/T/L point mutations reported to date. This study highlights the importance of minor mechanisms acting additively to confer significant levels of resistance to commercial field rates of glyphosate in weed populations subjected to high selection pressure.

D-glufosinate as a male sterility agent for hybrid seed production.

A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F1 hybrid seed production.