Acetohydroxyacid Synthase Enzyme Research Articles

Isolation and characterization of a new imidazolinone‐tolerant mutant in cotton

AbstractCotton (Gossypium hirsutum L.) cultivars resistant to different herbicides have generated important advances in crop management. There is a wide range of cotton resistance to herbicides, however, commercial imidazolinone (IMI)‐tolerant cultivars are not available. In this research, a new mutation that confers tolerance to IMI in cotton was isolated in the M2 generation after seed mutagenic treatments with sodium azide water solutions. The mode of inheritance of the trait was evaluated in the F2 generation by the application of imazethapyr. The inheritance corresponds to a semidominant type with a 1:2:1 distribution of tolerant/intermediate tolerance/not tolerant, in agreement with that observed in most IMI‐tolerant crops. The IMI‐tolerant line behavior was evaluated in the field nursery after the application of the herbicide in the form of foliar and pre‐emergence sprays. The tolerance level was high in pre‐emergence applications and intermediate in foliar applications compared with the nontolerant genotype, which showed a decrease in fiber yield and quality with both methods of application. After sequencing the acetohydroxyacid synthase (AHAS) gene, a point mutation corresponding to Ala205Val in the AHAS enzyme was found, which suggests it could be responsible for the increased IMI tolerance observed in cotton line SP 4172‐32. Based on results from field assays, it can be concluded that this mutant is a promising experimental line that can be used in obtaining IMI‐ tolerant commercial cotton cultivars.

Computer-Assisted Improvement of Sulfonylureas with Antifungal Properties and Limited Herbicidal Activity: Potential Application in Forage Conservation.

This work reports studies at the molecular level of a series of modified sulfonylureas to determine the chemophoric sites responsible for their antifungal and herbicidal activities. For forage conservation, high antifungal potency and low phytotoxicity are required. A molecular modeling study based on multivariate image analysis applied to quantitative structure-activity relationship (MIA-QSAR) was performed to model these properties, as well as to guide the design of new agrochemical candidates. As a result, the MIA-QSAR models were reliable, robust, and predictive; for antifungal activity, the averages of the main validation parameters were r2 = 0.936, q2 = 0.741, and r2pred = 0.720, and for herbicidal activity, the model was very predictive (r2pred = 0.981 and r2m = 0.944). From the interpretation of the MIA-plots, 46 novel sulfonylureas with likely improved performance were proposed, from which 9 presented promising calculated selectivity indexes. Docking studies were performed to validate the QSAR predictions and to understand the interaction mode of the proposed ligands with the acetohydroxyacid synthase enzyme.

Pro197Thr Substitution in Ahas Gene Causing Resistance to Pyroxsulam Herbicide in Rigid Ryegrass (Lolium Rigidum Gaud.)

Lolium rigidum Gaud. is a cross-pollinated species characterized by high genetic diversity and it was detected as one of the most herbicide resistance-prone weeds, globally. Acetohydroxyacid synthase (AHAS) resistant populations cause significant problems in cereal production; therefore, monitoring the development of AHAS resistance is widely recommended. Using next-generation sequencing (NGS), a de novo transcriptome sequencing dataset was presented to identify the complete open reading frame (ORF) of AHAS enzyme in L. rigidum and design markers to amplify fragments consisting of all of the eight resistance-conferring amino acid mutation sites. Pro197Thr, Pro197Ala, Pro197Ser, Pro197Gln, and Trp574Leu amino acid substitutions have been observed in samples. Although the Pro197Thr amino acid substitution was already described in SU and IMI resistant populations, this is the first report to reveal that the Pro197Thr in AHAS enzyme confers a high level of resistance (ED50 3.569) to pyroxsulam herbicide (Triazolopyrimidine).

Development and molecular analysis of a novel acetohydroxyacid synthase rapeseed mutant with high resistance to sulfonylurea herbicides

With the increasing promotion of simplified rapeseed cultivation in recent years, the development of cultivars with high resistance to herbicides is urgently needed. We previously developed M342, which shows sulfonylurea herbicide resistance, by targeting acetohydroxyacid synthase (AHAS), a key enzyme in branched-chain amino acid synthesis. In the present study, we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis, yielding the novel mutant DS3, which harbored two mutations in AHAS genes and showed high sulfonylurea resistance. One mutation was the substitution Trp574Leu, as in M342, according to Arabidopsis protein sequencing. The other site was a newly recognized substitution, Pro197Leu. A KASP marker targeting Pro197Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population. The combination of Trp574Leu and Pro197Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance. Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance, AHAS enzyme activity, and AHAS gene expression.

Corrigendum to “Mechanistic study of the biosynthesis of R-phenylacetylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations” [Arch. Biochem. Biophys. 701 (2021) 108807

Corrigendum to “Mechanistic study of the biosynthesis of R-phenylacetylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations” [Arch. Biochem. Biophys. 701 (2021) 108807

Synthesis, herbicidal activity, enzyme activity, and molecular docking of novel aniline thiourea

A series of new acyl thiourea derivatives containing aniline moiety were designed and synthesized. The preliminary herbicidal activity tests showed that some compounds had good herbicidal activity against Digitaria adscendens, Amaranthus retroflexus, especially for compound 4j (N-((4-chloro-3-(trifluoromethyl)phenyl)carbamothioyl)-4-(methylsulfonyl)-2-nitrobenzamide) and 4 l (N-((2-bromo-5-fluorophenyl)carbamothioyl)-4-(methylsulfonyl)-2-nitrobenzamide) . The results showed that compound 4j and 4l had an inhibition rate of 98% and 96% on the root growth of Brassica napus L. at the concentration of 100 mg L−1. Compounds 4j and 4 l had higher comparative activity on Echinochloa crusgalli than the commercial herbicide bensulfuron-methyl. The preliminary structure–activity relationship (SAR) was also summarized. We also tested the in vivo AHAS enzyme activity inhibition experiment of 17 compounds at 100 mg L−1, and the results showed that they all have inhibitory activity on the enzyme, with the highest inhibition rate reaching 43.66% (compound 4l). Based on the results of molecular docking to yeast acetohydroxyacid synthase (AHAS), the possible herbicidal activity mechanism of these compounds was evaluated.

Mechanistic study of the biosynthesis of R-phenylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cβ atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4′-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4kcal mol−1 at 27 °C. From this point, the reaction continues with the abstraction of Hβ atom of the HEThDP intermediate by the Oβ atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9kcal mol−1 at 27 °C.

Synthesis, herbicidal activity study and molecular docking of novel pyrimidine thiourea

According to the pharmacophore binding strategy and principle of bioelectronic isobaric, used the sulfonylurea bridge as the parent structure, a series of novel thiourea compounds containing aromatic-substituted pyrimidines were designed and synthesized. The preliminary herbicidal activity tests showed that some compounds had good herbicidal activity against Digitaria adscendens, Amaranthus retroflexus, especially for compound 4d and 4f. The results showed that compound 4d had an inhibition rate of 81.5% on the root growth of Brassica napus L. at the concentration of 100 mg L−1, and compound 4f had an inhibition rate of 81% on the root growth of Digitaria adscendens at the concentration of 100 mg L−1. Compounds 4d and 4f had higher comparative activity on Echinochloa crus-galli than the commercial herbicide bensulfuron-methyl. The preliminary structure-activity relationship (SAR) was also summarized. We also tested the in vivo AHAS enzyme activity inhibition experiment of 14 compounds at 100 mg L−1, and the results showed that they all have inhibitory activity on the enzyme, with the highest inhibition rate reaching 44.4% (compound 4d). Based on the results of molecular docking to yeast acetohydroxyacid synthase (AHAS), the possible herbicidal activity mechanism of these compounds was evaluated.

Ser-653-Asn substitution in the acetohydroxyacid synthase gene confers resistance in weedy rice to imidazolinone herbicides in Malaysia.

The continuous and sole dependence on imidazolinone (IMI) herbicides for weedy rice control has led to the evolution of herbicide resistance in weedy rice populations across various countries growing IMI herbicide-resistant rice (IMI-rice), including Malaysia. A comprehensive study was conducted to elucidate occurrence, level, and mechanisms endowing resistance to IMI herbicides in putative resistant (R) weedy rice populations collected from three local Malaysian IMI-rice fields. Seed bioassay and whole-plant dose-response experiments were conducted using commercial IMI herbicides. Based on the resistance index (RI) quantification in both experiments, the cross-resistance pattern of R and susceptible (S) weedy rice populations and control rice varieties (IMI-rice variety MR220CL2 and non-IMI-rice variety MR219) to imazapic and imazapyr was determined. A molecular investigation was carried out by comparing the acetohydroxyacid synthase (AHAS) gene sequences of the R and S populations and the MR220CL2 and MR219 varieties. The AHAS gene sequences of R weedy rice were identical to those of MR220CL2, exhibiting a Ser-653-Asn substitution, which was absent in MR219 and S plants. In vitro assays were conducted using analytical grade IMI herbicides of imazapic (99.3%) and imazapyr (99.6%) at seven different concentrations. The results demonstrated that the AHAS enzyme extracted from the R populations and MR220CL2 was less sensitive to IMI herbicides than that from S and MR219, further supporting that IMI herbicide resistance was conferred by target-site mutation. In conclusion, IMI resistance in the selected populations of Malaysian weedy rice could be attributed to a Ser-653-Asn mutation that reduced the sensitivity of the target site to IMI herbicides. To our knowledge, this study is the first to show the resistance mechanism in weedy rice from Malaysian rice fields.

Molecular modelling to understand AFM tip functionalisation and imazaquin-AHAS interactions in order to design a new nanobiosensor

ABSTRACTThe use of herbicides represents an important risk to environmental health, thus their monitoring is of fundamental importance to prevent and control contamination. The development of nanobiosensors is a powerful alternative because of the speed and accuracy of analysis. In this work, we aimed to present a further development and description of a theoretical model, at the atomic level, that represents an enzyme-herbicide interaction process, in order to apply in the construction of an optimised nanobiosensor based on Atomic Force Microscope (AFM), to detect the herbicide imazaquin. Here, structural data and molecular surface charge distribution were obtained from molecular dynamics (MD) simulations of the acetohydroxyacid synthase enzyme in complex and not with the herbicide molecule. The results were useful to predict the molecular orientation of the AHAS and the direction of unbinding pathway of herbicide from enzyme. Steered Molecular Dynamics (SMD) Simulations, along the previously determined direction of unbinding pathway of the imazaquin from the active site, were carried out to obtain a force value of 1.87 (± 0.20) nN per enzyme. These results are in accurate agreement with experimental data described in the literature.

Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass (Beckmannia syzigachne)

American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) is one of the most competitive and malignant weeds in rice-wheat rotation fields in China. American sloughgrass populations in the Jiangsu Province of China became less sensitive to acetohydroxyacid synthase (AHAS) inhibitors after repeated application for many years in these areas. Two suspected resistant American sloughgrass populations (R1 and R2) collected in the field were detected the resistance to inhibitors of AHAS in whole-plant dose-response assays, compared to the susceptible (S) population. These assays indicated that R1 showed low resistance to mesosulfuron-methyl (3.32-fold), imazapic (2.84-fold) and pyroxsulam (1.55-fold), moderate resistance to flazasulfuron (4.67-fold) and pyribenzoxim (7.41-fold), and high resistance to flucarbazone (11.73-fold). However, using a combination of the cytochrome P450 inhibitor, malathion, with mesosulfuron-methyl resulted in a reduction in R1 resistance relative to mesosulfuron-methyl alone. Furthermore, R2 was highly resistant to flazasulfuron (34.90-fold), imazapic (11.30-fold), flucarbazone (49.20-fold), pyribenzoxim (12.94-fold), moderately resistant to mesosulfuron-methyl (9.77-fold) and pyroxsulam (6.26-fold), and malathion had no effect on R2 resistance to mesosulfuron-methyl. The full-length of AHAS genes was sequenced and the AHAS enzymes were assayed in vitro in order to clarify the mechanism of resistance to AHAS inhibitors in R1 and R2 populations. The results demonstrated that R2 had a Pro-197-Ser mutation in the AHAS gene, and the sensitivity of R2 to the five AHAS inhibitors was decreased, which may result in R2 resistance to AHAS inhibitors. There was no mutation in the AHAS gene of R1, and there were no significant differences in enzyme sensitivity between susceptible (S) and resistant (R1) populations. An enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors.

Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene

BackgroundSomatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent.ResultsFollowing 5 days’ exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants.ConclusionsSomatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.

Acetohydroxyacid synthase (AHAS) amino acid substitution Asp376Glu in Lolium perenne: effect on herbicide efficacy and plant growth

Recently, the acetohydroxyacid synthase (AHAS) amino acid substitution Asp376Glu was detected in a Lolium perenne population originating in France. This is the first documented occurrence of the Asp376Glu single-nucleotide polymorphism in a Lolium species. The effects on herbicide efficacy and plant fitness are presented. Separation of the original population into different genetic subgroups allowed the comparison of different L. perenne AHAS genotypes according to their susceptibility to AHAS-inhibiting herbicides. Root and shoot biomass accumulation as well as tiller production was analysed for resistant and sensitive populations. Genotype-specific AHAS activity and inhibition were studied in vitro, using recombinant Arabidopsis thaliana DNA. Plants expressing the AHAS Asp376Glu genotype were resistant against mesosulfuron + iodosulfuron, pyroxsulam and propoxycarbazone. To imazamox, reduced susceptibility for dosages below 140 g a.i. ha−1 was observed. In the absence of herbicide, no significant impact of the Asp376Glu genotype on shoot biomass and tiller accumulation could be observed, whereas root biomass of the Asp376Glu genotype was significantly reduced by 68 % compared to the AHAS genetic wild type. The AHAS enzyme study revealed reduced AHAS enzyme rate of 48 % for the Asp376Glu genotype compared to the AHAS genetic wild type. This study highlights the role of the amino acid exchange on resistance profile against the most relevant AHAS-inhibiting herbicides. First indications were found that fitness of the Asp376Glu genotype is hampered.

Nicosulfuron application in agricultural soils drives the selection towards NS-tolerant microorganisms harboring various levels of sensitivity to nicosulfuron.

The action mode of sulfonylurea herbicides is the inhibition of the acetohydroxyacid synthase (AHAS) required for the biosynthesis of amino acids valine and isoleucine in plants. However, this enzyme is also present in a range of non-targeted organisms, among which soil microorganisms are known for their pivotal role in ecosystem functioning. In order to assess microbial toxicity of sulfonylurea herbicide nicosulfuron (NS), a tiered microcosm (Tier I) to field (Tier II) experiment was designed. Soil bacteria harboring AHAS enzyme tolerant to the herbicide nicosulfuron were enumerated, isolated, taxonomically identified, and physiologically characterized. Results suggested that application of nicosulfuron drives the selection towards NS-tolerant bacteria, with increasing levels of exposure inducing an increase in their abundance and diversity in soil. Tolerance to nicosulfuron was shown to be widespread among the microbial community with various bacteria belonging to Firmicutes (Bacillus) and Actinobacteria (Arthrobacter) phyla representing most abundant and diverse clusters. While Arthrobacter bacterial population dominated community evolved under lower (Tier II) nicosulfuron selection pressure, it turns out that Bacillus dominated community evolved under higher (Tier I) nicosulfuron selection pressure. Different NS-tolerant bacteria likewise showed different levels of sensitivity to the nicosulfuron estimated by growth kinetics on nicosulfuron. As evident, Tier I exposure allowed selection of populations able to better cope with nicosulfuron. One could propose that sulfonylureas-tolerant bacterial community could constitute a useful bioindicator of exposure to these herbicides for assessing their ecotoxicity towards soil microorganisms.

Target-Site Resistance to Nicosulfuron in Johnsongrass (Sorghum halepense) from Chilean Corn Fields

Johnsongrass is a common weed of corn in Chile, which is most often controlled by nicosulfuron, an acetohydroxyacid synthase (AHAS)-inhibiting herbicide. Recurrent nicosulfuron use has resulted in selection for resistant johnsongrass biotypes. We conducted studies to determine nicosulfuron resistance levels in two johnsongrass biotypes from Chile and to investigate if this resistance was target-site mediated. Whole-plant resistance to nicosulfuron was 33 and 46 times higher in resistant (R) than in susceptible (S) plants grown from seed and rhizomes, respectively. The nicosulfuron concentrations for 50% inhibition of AHAS enzyme activity in vitro were more than 11 times higher in R than in S plants. Sequencing analysis of theAHAScoding sequence revealed a Trp-574-Leu substitution in both R biotypes. This study shows that resistance to nicosulfuron in the two R biotypes is conferred by an altered target site. We also report the first consensus sequence of the johnsongrassAHASgene corresponding to the known mutation sites conferring resistance to AHAS-inhibiting herbicides.

Molecular characterization and detection of a spontaneous mutation conferring imidazolinone resistance in rapeseed and its application in hybrid rapeseed production

Imidazolinone herbicide (IMI)-resistant varieties of many crops, induced by mutations to acetohydroxyacid synthase (AHAS) genes, are planted worldwide. However, in the case of rapeseed, which is a common source of edible oil for nearly one-half of China’s population, no IMI resistance has been reported for any of the varieties currently cultivated. We have developed an imazethapyr-resistant rapeseed (M9) derived from a naturally occurring mutant plant. The goals of this study were to determine the biochemical and molecular bases of herbicide resistance in M9, to develop molecular markers for the detection of herbicide-resistant genes, and to utilize herbicide-resistant traits to enhance seed purity in hybrid rapeseed production. An in vitro AHAS activity assay indicated that the AHAS enzyme from M9 conferred a specific resistance to IMIs. Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 653 (AGT) to asparagine (AAT) at the herbicide-binding site of the rapeseed BnAHAS1 gene. This substitution mutation (Ser653Asp) did not change the transcription levels of BnAHAS1 in M9 compared with the wild type. An allele-specific PCR marker for the BnAHAS1 mutant sequence was developed and cosegregated with IMI resistance in the F2, BC1, and BC2 populations. Finally, the CMS restorer line 10M169 was developed to show the resistance of M9, and F1 seeds of different purity were generated from 10M169 and Ning A7 under different pollination conditions. The increases in seed purity under natural hybridization and hybridization in tents reached 13.41 and 16.41 %, respectively, after IMI treatment, suggesting that the herbicide-resistant trait can be utilized for the efficient elimination of false hybrids in hybrid rapeseed production, leading to increases in yield of up to 322 and 394 kg ha−1, respectively. The molecular mechanism and molecular marker of herbicide resistance described here provide the basis for the release of IMI-resistant rapeseed cultivars.

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for de novo synthesis of branched-chain amino acids. The genome sequence of Mycobacterium tuberculosis revealed genes encoding four catalytic subunits, ilvB1 (Rv3003c), ilvB2 (Rv3470c), ilvG (Rv1820) and ilvX (Rv3509c), and one regulatory subunit, ilvN (Rv3002c), of AHAS. All these genes were found to be expressed in M. tuberculosis growing in vitro. Each AHAS subunit gene was cloned and expressed in Escherichia coli. AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology of M. tuberculosis, expression of AHAS genes was analysed in different physiological conditions. ilvB1, ilvB2 and ilvG were differentially expressed. The role of ilvB1 in persistence is known, but the upregulation of ilvB2 and ilvG in extended stationary phase, ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of M. tuberculosis by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.

The identification of enzyme targets for the optimization of a valine producing Corynebacterium glutamicum strain using a kinetic model

The enzyme targets for the rational optimization of a Corynebacterium glutamicum strain constructed for valine production are identified by analyzing the control of flux in the valine/leucine pathway. The control analysis is based on measurements of the intracellular metabolite concentrations and on a kinetic model of the reactions in the investigated pathway. Data-driven and model-based methods are used and evaluated against each other. The approach taken gives a quantitative evaluation of the flux control and it is demonstrated how the understanding of flux control is used to reach specific recommendations for strain optimization. The flux control coefficients (FCCs) with respect to the valine excretion rate were calculated, and it was found that the control is distributed mainly between the acetohydroxyacid synthase enzyme (FCC = 0.32), the branched chain amino acid transaminase (FCC = 0.27), and the exporting translocase (FCC = 0.43). The availability of the precursor pyruvate has substantial influence on the valine flux, whereas the cometabolites are less important as demonstrated by the calculation of the respective response coefficients. The model is further used to make in-silico predictions of the change in valine flux following a change in enzyme level. A doubling of the enzyme level of valine translocase will result in an increase in valine flux of 31%. By optimizing the enzyme levels with respect to valine flux it was found that the valine flux can be increased by a factor 2.5 when the optimal enzyme levels are implemented.

Genetics of Resistance to Acetohydroxyacid Synthase Inhibitors in Populations of Eastern Black Nightshade (Solanum Ptychanthum) from Ontario

Abstract Resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides in populations of eastern black nightshade from Ontario has been linked to an Ala205Val substitution in the AHAS enzyme. The aim of this study was to determine the mechanism of inheritance of AHAS inhibitor resistance and the genetic relationships among resistant (R) and susceptible (S) eastern black nightshade populations from Ontario. Homozygous R and S parental populations were crossed and the inheritance was analyzed in F1 (S × R), reciprocal F1 (R × S), F2, and backcross (S × F1) progenies after application of imazethapyr at 150 g ai ha−1. Compared to parental lines, the progenies were rated as R, intermediate (I), and S phenotypes. All the F1 progenies were of the I phenotype. The backcross progenies segregated in a 1:1 (S:I) ratio, and the F2 families segregated in a 1:2:1 (R:I:S) ratio. These results indicate that a single nuclear gene, with incomplete dominance, controls resistance to AHAS-inhibiting herbicides in R pop...