HPPD Inhibitors Research Articles

Mechanism of atrazine resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth (Amaranthus palmeri S. Wats.) from Nebraska

Palmer amaranth (Amaranthus palmeri S. Wats.) is one of the most problematic weed species in agronomic crops in the United States. A Palmer amaranth biotype multiple-resistant to atrazine and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors was reported in a seed corn production field in Nebraska. Rapid detoxification mediated by cytochrome P450 monooxygenases and increased HPPD gene expression were reported as the mechanisms of mesotrione resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth biotype from Nebraska; however, the mechanism of atrazine resistance is unknown. The objectives of this study were to investigate target site or non-target site based mechanisms conferring atrazine resistance in Palmer amaranth from Nebraska. 14C-atrazine absorption and translocation studies revealed that reduced atrazine absorption or translocation were not involved as one of the mechanisms of atrazine resistance. Instead, greater 14C-atrazine absorption and recovery in treated leaves were observed in resistant compared with susceptible Palmer amaranth. No known mutations including Ser264Gly substitution in the psbA gene causing target site based atrazine resistance were observed. However, the parent 14C-atrazine was metabolized rapidly <4 h after treatment in resistant plants, conferring enhanced atrazine metabolism as the mechanism of resistance.

Basis of Atrazine and Mesotrione Synergism for Controlling Atrazine‐ and HPPD Inhibitor‐Resistant Palmer Amaranth

Palmer amaranth (Amaranthus palmeri S. Watson) resistant to atrazine [6‐chloro‐N‐ethyl‐N’‐(1‐methylethyl)‐1,3,5‐triazine2,4‐diamine] and 4‐hydroxyphenylpyruvate dioxygenase (HPPD)‐inhibiting herbicides was confirmed in a seed corn (Zea mays L.) production field in Nebraska, in 2014. Neither atrazine nor HPPD inhibitors (mesotrione [2‐(4‐mesyl2‐nitrobenzoyl)‐3‐hydroxycylohex‐2‐enone], tembotrione {2‐[2‐chloro‐4‐(methylsulfonyl)‐3‐[(2,2,2‐trifluoroethoxy) methyl]benzoyl]‐1,3‐cyclohexanedione}, or topramezone {[3‐(4,5‐dihydro‐3‐isoxazolyl)‐2‐methyl‐4‐(methylsulfonyl)phenyl](5‐hydroxy‐1‐methyl‐1H‐pyrazol‐4‐yl)methanone}) applied post‐emergence were able to control resistant Palmer amaranth even at greater than label rates. However, their tank mixtures even at lower than the label rate provided more than 90% control under greenhouse and field conditions. The objectives of this study were to investigate the effect of atrazine on mesotrione absorption and translocation when tank mixed or vice versa in atrazine‐ and HPPD inhibitor‐resistant Palmer amaranth from Nebraska. Tank mixing commercial formulation of atrazine at 560 g ha−1 increased 14C‐mesotrione absorption to 51% compared to 39% with 14C‐mesotrione alone. However, 14C‐atrazine absorption or translocation was not affected by mesotrione at 26 g ha−1 in the tank mixture. Similarly, mesotrione did not affect the metabolism of 14C‐atrazine in resistant or susceptible plants when tank mixed compared to 14C‐atrazine applied alone. Increased absorption of mesotrione when tank mixed with atrazine could be one of the reasons of atrazine and mesotrione synergism besides their biochemical interaction in the atrazine‐ and HPPD inhibitor‐resistant Palmer amaranth biotype from Nebraska.Core Ideas Atrazine applied in tank‐mixture increased mesotrione absorption. Mesotrione applied in tank mixture did not affect atrazine absorption and translocation. Atrazine metabolism was not affected by mesotrione applied in tank mixture.

Tissue-specific FAH deficiency alters sleep–wake patterns and results in chronic tyrosinemia in mice

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyrosine catabolism, and mutations in the FAH gene are associated with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. In a behavioral screen of N-ethyl-N-nitrosourea mutagenized mice we identified a mutant line which we named "swingshift" (swst, MGI:3611216) with a nonsynonymous point mutation (N68S) in Fah that caused age-dependent disruption of sleep-wake patterns. Mice homozygous for the mutation had an earlier onset of activity (several hours before lights off) and a reduction in total activity and body weight when compared with wild-type or heterozygous mice. Despite abnormal behavioral entrainment to light-dark cycles, there were no differences in the period or phase of the central clock in mutant mice, indicating a defect downstream of the suprachiasmatic nucleus. Interestingly, these behavioral phenotypes became milder as the mice grew older and were completely rescued by the administration of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. Mechanistically, the swst mutation had no effect on the enzymatic activity of FAH, but rather promoted the degradation of the mutant protein. This led to reduced FAH protein levels and enzymatic activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine-but not methionine, phenylalanine, or succinylacetone-increased in homozygous mice, suggesting that swst mutants provide a model of mild, chronic HT1.

Discovery of N-Aroyl Diketone/Triketone Derivatives as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibiting-Based Herbicides.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an important target site for discovering new bleaching herbicides. To explore novel HPPD inhibitors with excellent herbicidal activity, a series of novel N-aroyl diketone/triketone derivatives were rationally designed by splicing active groups and bioisosterism. Bioassays revealed that most of these derivatives displayed preferable herbicidal activity against Echinochloa crus-galli (EC) at 0.045 mmol/m2 and Abutilon juncea (AJ) at 0.090 mmol/m2. In particular, compound I-f was more potent compared to the commercialized compound mesotrione. Molecular docking indicated that the corresponding active molecules of target compounds and mesotrione shared similar interplay with surrounding residues, which led to a perfect interaction with the active site of Arabidopsis thaliana HPPD.

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

Pyrazole–Isoindoline-1,3-dione Hybrid: A Promising Scaffold for 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors

The discovery of 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) inhibitors has been an active area of research due to their great potential as herbicides for weed control. Starting from the binding mode of known inhibitors of HPPD, a series of HPPD inhibitors with new molecular scaffolds were designed and synthesized by hybridizing 2-benzoylethen-1-ol and isoindoline-1,3-dione fragments. The results of the in vitro tests indicated that the newly synthesized compounds showed good HPPD inhibitory activity with IC50 values against the recombinant Arabidopsis thaliana HPPD (AtHPPD) ranging from 0.0039 μM to over 1 μM. Most promisingly, compound 4ae, 2-benzyl-5-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4- carbonyl)isoindoline-1,3-dione, showed the highest AtHPPD inhibitory activity with a Ki value of 3.92 nM, making it approximately 10 times more potent than pyrasulfotole (Ki = 44 nM) and slightly more potent than mesotrione (Ki = 4.56 nM). In addition, the cocrystal structure of the AtHPPD-4ae complex was successfully resolved at a resolution of 1.8 Å. The X-ray diffraction analysis indicated that the two carbonyl groups of 2-benzoylethen-1-ol formed a bidentate chelating interaction with the metal ion, while the isoindoline-1,3-dione moiety formed pronounced π-π stacking interactions with Phe381 and Phe424. Moreover, water-mediated hydrogen bonding interactions were observed between Asn282 and the nitrogen atoms of the pyrazole ring of 4ae. The above results showed that the pyrazole-isoindoline-1,3-dione hybrid is a promising scaffold for developing HPPD inhibitors.

Synthesis and herbicidal activity of novel pyrazole aromatic ketone analogs as HPPD inhibitor.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been a good target for herbicide discovery. In order to discover novel HPPD herbicides, a series of pyrazole aromatic ketone analogs were designed and synthesized. The 25 pyrazole aromatic ketone analogs synthesized were tested for herbicidal activity and compounds A1, A3, A4, A17, A20 and A25 displayed excellent herbicidal activity against Chenopodium serotinum, Stellaria media and Brassica juncea at 37.5g ha-1 . In addition, compounds A1, A5, A9, A10, A16, A17, A20 and A25 exhibited good crop selectivity for wheat, maize and rice at 150 g ha-1 . Inhibition activities against AtHPPD proved the compounds were HPPD inhibitors. The structure-activity relationship of these pyrazole aromatic ketone analogs was studied using molecular docking. These pyrazole aromatic ketone derivatives could be used as lead structures for development of HPPD herbicides against dicotyledonous weeds with further structure modification. © 2019 Society of Chemical Industry.

Method validation and dissipation kinetics of the new HPPD inhibitor QYR301 in rice, paddy water and paddy soil using a QuEChERS-based method and LC-MS/MS

A rapid and simple method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of sample preparation using QuEChERS was developed for detecting residues of QYR301, a new HPPD-inhibiting herbicide, in rice plant (straw), water, soil, rice hull and brown rice (BR). To eliminate matrix interference, matrix-matched calibrations with satisfactory linearity (R2 > 0.99) were used for accurate quantification. The method showed recoveries of 90.3–108.1% and relative standard deviations (RSDs) < 11%. The limits of quantification (LOQ) for QYR301 were 0.005 mg kg−1 in all five matrixes. Furthermore, the dissipation kinetics and terminal residues of QYR301 were determined at two sites in 2018. The days for 50% dissipation (DT50) of QYR301 in rice plants, water and soil were 3.6–4.4, 0.7–3.0 and 4.3–8.0 d, respectively, which indicated that QYR301 is a short-persistence herbicide. Moreover, no QYR301 residues were detected in BR, rice hull and straw collected at harvest following its application at 1.0 or 1.5 × of the recommended high rate. These results will help organizations and governments establish related principles/laws regarding the use of QYR301 in terms of environmental protection, food safety and other potential aspects.

Effect of hybrid varieties, application timing, and herbicide rate on field corn tolerance to tolpyralate plus atrazine

AbstractA wide margin of crop safety is a desirable trait of POST herbicides, and investigation of crop tolerance is a key step in evaluation of new herbicides. Six field experiments were conducted in Ontario, Canada, from 2017 to 2018 to examine the influence of corn (Zea mays L.) hybrid (DKC42-60RIB, DKC43-47RIB, P0094AM, and P9840AM), application rate (1X and 2X), and application timing (PRE, V1, V3, and V5) on the tolerance of field corn to tolpyralate, a new 4-hydroxyphenyl pyruvate dioxygenase inhibitor, co-applied with atrazine. Two corn hybrids (DKC42-60RIB and DKC43-47RIB) exhibited slightly greater visible injury from tolpyralate + atrazine, applied POST, than P0094AM and P9840AM at 1 to 2 wk after application (WAA); hybrids responded similarly with respect to height, grain moisture, and yield. Applications of tolpyralate + atrazine at a 2X rate (80 + 2,000 g ai ha−1) induced greater injury (≤31.6%) than the field rate (40 + 1,000 g ha−1) (≤11.6%); the 2X rate applied at V1 or V3 decreased corn height and slightly increased grain moisture at harvest. On average, field rates resulted in marginally higher grain yields than 2X rates. Based on mixed-model multiple stepwise regression analysis, the air temperature at application, time of day, temperature range in the 24 h before application, and precipitation following application were useful predictor variables in estimating crop injury with tolpyralate + atrazine; however, additional environmental variables also affected crop injury. These results demonstrate the margin of corn tolerance with tolpyralate + atrazine, which provides a basis for optimization of application timing, rate, and corn hybrid selection to mitigate the risk of crop injury with this herbicide tank mixture.

Quantitative Structure Activity Relationship Studies and Molecular Dynamics Simulations of 2-(Aryloxyacetyl)cyclohexane-1,3-Diones Derivatives as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a significant enzyme in the biosynthesis of plastoquinone and tocopherol. Moreover, it is also a potential target to develop new herbicide. The technology of computer-aided drug design (CADD) is a useful tool in the efficient discovery of new HPPD inhibitors. Forty-three compounds with known activities were used to generate comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models based on common framework and molecular docking. The structural contribution to the activity was determined, which provided further information for the design of novel inhibitors. Molecular docking was used to explain the changes in activity caused by the binding mode between ligand and protein. The molecular dynamics (MD) results indicated that the electrostatic energy was the major driving force for ligand–protein interaction and the Phe403 made the greatest contribution to the binding. The present work has provided useful information for the rational design of novel HPPD inhibitors with improved activity.

Identification of novel inhibitors of p-hydroxyphenylpyruvate dioxygenase using receptor-based virtual screening

Abstract p-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potential target for herbicide discovery, which catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisate. Triketone HPPD inhibitors have been widely applied to weed control for many years. To discover potential HPPD inhibitors with novel scaffold, a virtual screening procedure combining pharmacophore model, molecular docking and molecular dynamics (MD) simulation was carried out. The crystal complex (PDB: 6ISD ) with sulcotrione was selected to establish the receptor-ligand pharmacophore model (CBP). The verified CBP model with key features and interactions was employed to seek structures with potential inhibitory from ZINC database. The best-fit compounds from pharmacophore screening (2050 compounds) were subsequently subjected to molecular docking with CDOCKER method to identify the binding affinity of inhibitors with amino acid residues in active pocket. Six compounds were obtained as the final hits with the Fe(Ш) coordination and π-π interaction with Phe381 and Phe424. MD results demonstrated that Phe424 made great contributions to binding affinities of all the systems. This work provided powerful insights into the design and development of novel HPPD inhibitor in silico techniques. ZINC000255329531 was identified as the most promising candidate due to its high HPPD inhibition effect (ΔGbind = 25.66 kcal mol−1), in comparison with the commercial triketone HPPD herbicide sulcotrione (ΔGbind = 18.94 kcal mol−1).

A double-edged rice paddy herbicide

Plant Science A useful herbicide kills weeds but spares the crop of interest. For many rice paddies, the herbicide benzobicyclon (BBC) serves this purpose. But some rice strains are susceptible to BBC, which diminishes its value in weed control. Maeda et al. uncovered the genetic cause controlling the response to the herbicide: resistant rice cultivars have an oxidase that detoxifies BBC herbicides. Susceptible rice varieties carried genetic mutations that disabled the oxidase. The gene HPPD INHIBITOR SENSITIVE 1 , which encodes the oxidase, may be useful for developing BBC-resistant crops. Science , this issue p. [393][1] [1]: /lookup/doi/10.1126/science.aax0379

Alternative Options to Glyphosate for Control of Large Echinochloa colona and Chloris virgata Plants in Cropping Fallows.

The over-reliance on the herbicide glyphosate for knockdown weed control in fallows under minimum and zero-till cropping systems has led to an increase in populations of glyphosate-resistant weeds. Echinochloa colona and Chloris virgata are two major grass weeds in the cropping regions of northern New South Wales and southern Queensland, Australia, that have become harder to kill due to a steady rise in the occurrence of glyphosate-resistant weed populations. Therefore, to help growers contain these hard to kill fallow weeds, an alternate approach to glyphosate application is needed. With this purpose in mind, a pot study was carried out during the summer seasons of 2015 and 2016 at the Tamworth Agricultural Institute, Tamworth, NSW, Australia, to evaluate the efficacy of tank mixtures and sequential applications of Group H (4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor), Group C (inhibitors of photosynthesis at photosystem II), Group A (ACCase inhibitors) and Group L (photosystem I inhibitor) herbicides on late tillering E. colona and C. virgata plants. These herbicide groups are a global classification by the Herbicide Resistance Action Committee. Highly effective results were achieved in this study using combinations of Groups H, C, A and L herbicides applied as tank mixtures for controlling large E. colona plants. Additionally, sequential applications of Group H, C and A herbicides followed by (fb) paraquat were shown to be very effective on large E. colona plants. Late tillering C. virgata plants were generally well controlled by tank mixtures, and sequential applications proved to be highly effective on this grass weed as well. Haloxyfop in combination with paraquat as a tank mixture, via sequential application or as a stand-alone treatment, was highly effective for C. virgata control; however, using combinations of herbicide groups is the preferred choice when combating herbicide resistant weed populations. There was a clear synergy shown using Group H, Group C and Group A herbicides in combination with the Group L herbicide paraquat in this study for controlling advanced E. colona and C. virgata plants. These combinations were shown to be successful on plants grown under glasshouse conditions; however; these treatments would need to be tested on plants grown in a field situation to show whether they will be a useful solution for farmers who are trying to control these weeds in fallow.

Cross-resistance to acetolactate synthase (ALS) inhibitors associated with different mutations in Japanese foxtail (Alopecurus japonicus)

AbstractJapanese foxtail (Alopecurus japonicus Steud.) is an invasive grass weed that severely threatens the production of wheat (Triticum aestivum L.) and canola (Brassica napus L.) crops in eastern Asia. Mesosulfuron-methyl is a highly efficient acetolactate synthase (ALS)-inhibiting herbicide widely used for control of this species in China. However, in recent years, some A. japonicus populations have evolved resistance to mesosulfuron-methyl by different amino acid substitutions (AASs) within the ALS gene. In the current study, 11 populations of A. japonicus were collected from Anhui Province, China, where the wheat fields were severely infested with this weed. Based on single-dose screening, eight of these populations evolved resistance to mesosulfuron-methyl, and gene sequencing revealed three AASs located in codon 197 or 574 of the ALS gene in the different resistant populations. Subsequently, three typical populations, AH-1, AH-4, and AH-10 with Trp-574-Leu, Pro-197-Thr, and Pro-197-Ser mutations, respectively, in ALS genes were selected to characterize their cross-resistance patterns to ALS inhibitors. Compared with the susceptible population AH-S, AH-1 showed broad-spectrum cross-resistance to sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), and sulfonyl-aminocarbonyl-triazolinones (SCTs); whereas AH-4 and AH-10 were resistant to SUs, TPs, and SCTs but sensitive to IMIs. Moreover, all three resistant populations were sensitive to both photosystem II inhibitor isoproturon and 4-hydroxyphenylpyruvate dioxygenase inhibitor QYM201 (1-(2-chloro-3-(3-cyclopropyl-5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-6-(trifluoromethyl)phenyl)piperidin-2-one). Based on the current state of knowledge, this study is the first report of A. japonicus evolving cross-resistance to ALS-inhibiting herbicides due to a Pro-197-Ser mutation in the ALS gene.

Design, synthesis, SAR and molecular docking of novel green niacin-triketone HPPD inhibitor

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a very important target for the discovery of novel bleaching herbicides. In order to discover novel triketone green HPPD inhibitors with excellent herbicidal activity and crop selectivity, the natural product leptospermone was selected as the template compound, and niacin was employed as bioisostere to design and synthesize a series of novel substituted niacin-triketone derivatives. Some of the target compounds showed potent Arabidopsis thaliana HPPD (AtHPPD) inhibitory activity in vitro. Especially compound II-1 displayed the best herbicidal activity (IC50 = 0.262 μM, AtHPPD), and was safe for Zea mays at concentration of 300 g ai/ha. Molecular docking results indicated that the triketone moiety of compound II-1 could form a bidentate complex with Fe(II), and the pyridine subunit interacted with Phe430 and Phe360 via π−π packing. The present work indicated that 3-hydroxy-2-nicotinoylcyclohex-2-en-1-one could be a potential lead structure for developing novel green HPPD inhibitors.

Influence of application timing and herbicide rate on the efficacy of tolpyralate plus atrazine

AbstractEffective POST herbicides and herbicide mixtures are key components of integrated weed management in corn; however, herbicides vary in their efficacy based on application timing. Six field experiments were conducted over 2 yr (2017–2018) in southwestern Ontario, Canada, to determine the effects of herbicide application timing and rate on the efficacy of tolpyralate, a new 4-hydroxyphenyl pyruvate dioxygenase inhibitor. Tolpyralate at 15, 30, or 40 g ai ha−1in combination with atrazine at 500 or 1,000 g ai ha−1was applied PRE, early POST, mid-POST, or late POST. Tolpyralate + atrazine at rates ≥30 + 1,000 g ha−1provided equivalent control of common lambsquarters and Powell amaranth applied PRE or POST, whereas no rate applied PRE controlled common ragweed, velvetleaf, barnyardgrass, or green foxtail. Common ragweed, common lambsquarters, velvetleaf, and Powell amaranth were controlled equally regardless of POST timing. In contrast, control of barnyardgrass and green foxtail declined when herbicide application was delayed to the late-POST timing, irrespective of herbicide rate. Similarly, corn grain yield declined within each tolpyralate + atrazine rate when herbicide applications were delayed to late-POST timing. Overall, the results of this study indicate that several monocot and dicot weed species can be controlled with tolpyralate + atrazine with an early to mid-POST herbicide application timing, before weeds reach 30 cm in height, and Powell amaranth and common lambsquarters can also be controlled PRE. Additionally, this study provides further evidence highlighting the importance of effective, early-season weed control in corn.

Characterization of a waterhemp (Amaranthus tuberculatus) population from Illinois resistant to herbicides from five site-of-action groups

AbstractExperiments were initiated to characterize a waterhemp population (CHR) discovered in a central Illinois corn field after it was not controlled by the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor topramezone. Field experiments conducted during 2014–2015 indicated that acetolactate synthase (ALS)-, protoporphyrinogen oxidase (PPO)-, photosystem II (PSII)-, and HPPD-inhibiting herbicides and the synthetic auxin 2,4-D did not control the CHR population. Laboratory experiments confirmed target site–based resistance mechanisms to ALS- and PPO-inhibiting herbicides. Herbicide doses required to reduce dry biomass 50% (GR50) were determined in greenhouse dose–response experiments, and indicated 16-fold resistance to the HPPD inhibitor mesotrione, 9.5-fold resistance to the synthetic auxin 2,4-D, and 252-fold resistance to the PSII inhibitor atrazine. Complementary results from field, laboratory, and greenhouse investigations indicate that the CHR population has evolved resistance to herbicides from five sites of action (SOAs): ALS-, PPO-, PSII-, and HPPD-inhibiting herbicides and 2,4-D. Herbicide use history for the field in which CHR was discovered indicates no previous use of 2,4-D.

Using RNA-seq to characterize responses to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide resistance in waterhemp (Amaranthus tuberculatus)

BackgroundWaterhemp (Amaranthus tuberculatus (Moq.) J.D. Sauer) is a problem weed commonly found in the Midwestern United States that can cause crippling yield losses for both maize (Zea mays L.) and soybean (Glycine max L. Merr). In 2011, 4-hydroxyphenylpyruvate-dioxygenase (HPPD, EC 1.13.11.27) inhibitor herbicide resistance was first reported in two waterhemp populations. Since the discovery of HPPD-herbicide resistance, studies have identified the mechanism of resistance and described the inheritance of the herbicide resistance. However, no studies have examined genome-wide gene expression changes in response to herbicide treatment in herbicide resistant and susceptible waterhemp.ResultsWe conducted RNA-sequencing (RNA-seq) analyses of two waterhemp populations (HPPD-herbicide resistant and susceptible), from herbicide-treated and mock-treated leaf samples at three, six, twelve, and twenty-four hours after treatment (HAT). We performed a de novo transcriptome assembly using all sample sequences. Following assessments of our assembly, individual samples were mapped to the de novo transcriptome allowing us to identify transcripts specific to a genotype, herbicide treatment, or time point. Our results indicate that the response of HPPD-herbicide resistant and susceptible waterhemp genotypes to HPPD-inhibiting herbicide is rapid, established as soon as 3 hours after herbicide treatment. Further, there was little overlap in gene expression between resistant and susceptible genotypes, highlighting dynamic differences in response to herbicide treatment. In addition, we used stringent analytical methods to identify candidate single nucleotide polymorphisms (SNPs) that distinguish the resistant and susceptible genotypes.ConclusionsThe waterhemp transcriptome, herbicide-responsive genes, and SNPs generated in this study provide valuable tools for future studies by numerous plant science communities. This collection of resources is essential to study and understand herbicide effects on gene expression in resistant and susceptible weeds. Understanding how herbicides impact gene expression could allow us to develop novel approaches for future herbicide development. Additionally, an increased understanding of the prolific traits intrinsic in weed success could lead to crop improvement.

Free energy calculations elucidate substrate binding, gating mechanism, and tolerance-promoting mutations in herbicide target 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide-tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C-terminal helix H11 in HPPD. We compared gating equilibria in Arabidopsis thaliana (At) and Zea mays (Zm) wild-type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open-closed transition and discovered a ligand-mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in At mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new At mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.

Transcriptome responses to the natural phytotoxin t-chalcone in Arabidopsis thaliana L.

New modes of action are needed for herbicides. The flavonoid synthesis intermediate t-chalcone causes apoptosis-like symptoms in roots and bleaching of shoots of Arabidospsis, suggesting a unique mode of action as a phytotoxin. Using RNA-Seq, transcriptome changes were monitored in Arabidopsis seedlings during the first 24 h of exposure (at 1, 3, 6, 12 and 24 h) to 21 μmt-chalcone (I50 dose), examining effects on roots and shoots separately. Expression of 892 and 1000 genes was affected in roots and shoots, respectively. According to biological classification, many of the affected genes were transcription factors and genes associated with oxidative stress, heat shock proteins, xenobiotic detoxification, ABA and auxin biosynthesis, and primary metabolic processess. These are secondary effects found with most phytotoxins. Potent phytotoxins usually act by inhibiting enzymes of primary metabolism. KEGG pathway analysis of transcriptome results from the first 3 h of t-chalcone exposure indicated several potential primary metabolism target sites for t-chalcone. Of these, p-hydroxyphenylpyruvate dioxygenase (HPPD) and tyrosine amino transferase were consistent with the bleaching effect of the phytotoxin. Supplementation studies with Lemna paucicostata and Arabidiopsis supported HPPD as the target, although in vitro enzyme inhibition was not found. t-Chalcone is possibly a protoxin that is converted to a HPPD inhibitor in vivo. © 2019 Society of Chemical Industry.