Aryloxyalkanoate Dioxygenase-12 Research Articles

Computational Study of the Fe(II) and α-Ketoglutarate-Dependent Aryloxyalkanoate Dioxygenase (AAD-1) in the Degradation of the Herbicide 2,4-Dichlorophenoxyacetic Acid.

The AAD-1 enzyme belongs to the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent nonheme aryloxyalkanoate dioxygenase family (AADs), which catalyzes the breakdown of 2,4-dichlorophenoxyacetic acid (2,4-D, an active ingredient of thousands of commercial herbicides) by using the highly active Fe(IV)═O complex. Multiple species of bacteria degrade 2,4-D via a pathway initiated by AADs; however, the detail of how they promote the cleavage of the ether C-O bond to generate 2,4-dichlorophenol (2,4-DCP) and glyoxylate is still unclear, which is the prerequisite for the further degradation of these halogenated aromatics. In this work, based on the crystal structure of AAD-1, the computational models were constructed, and a series of QM/MM and QM-only calculations were performed to explore the cleavage of the ether bond in 2,4-D with the catalysis of AAD-1. Our calculations reveal that AAD-1 may be only responsible for the hydroxylation of the substrate to generate the intermediate hemiacetal, which corresponds to an overall energy barrier of 14.2 kcal/mol on the quintet state surface, and the decomposition of the hemiacetal in the active site center of AAD-1 was calculated to be rather slow, corresponding to an energy barrier of 24.5 kcal/mol. In contrast, the decomposition of the free hemiacetal molecule in a solvent was calculated to be quite easy. Whether the decomposition of the hemiacetal occurs inside or outside the activation site is still worthy of experimental verification.

Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12

Soybeans (Glycine max (L.) Merr.) genetically modified to express aryloxyalkanoate dioxygenase-12 (AAD-12), an enzyme that confers resistance to the herbicide 2,4-D, can sometimes exhibit a darker seed coat coloration than equivalent unmodified soybeans. The biochemical basis for this coloration was investigated in a non-commercial transgenic event, DAS-411Ø4-7 that exhibited more pronounced AAD-12-associated seed coat coloration than the commercial event, DAS-444Ø6-6. Analysis of color-enriched seed coat fractions from DAS-411Ø4-7 showed that the color was due to localized accumulation of iron-chelating phenolics, particularly the isoflavone genistin, that are associated with seed coat pectic polysaccharide and produce a brown chromophore. The association between genistin, iron, and pectic polysaccharide was characterized using a variety of analytical methods. Darker seeds from commercial soybean event DAS-444Ø6-6 also show higher genistin content localized to the darker colored portions of the seed coat (with no increase in whole seed genistin levels).

Safety evaluation of genetically modified DAS-40278-9 maize in a subchronic rodent feeding study

Genetically modified (GM) maize, DAS-40278-9, expresses the aryloxyalkanoate dioxygenase-1 (AAD-1) protein, which confers tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. The aad-1 gene, which expresses the AAD-1 protein, was derived from Gram-negative soil bacterium, Sphingobium herbicidovorans. A 90-day sub-chronic toxicity study was conducted on rats as a component of the safety evaluation of DAS-40278-9 maize. Rats were given formulated diets containing maize grain from DAS-40278-9 or a non-GM near isogenic control comparator at an incorporation rate of 12.5%, 25%, or 50% (w/w), respectively for 90 days. In addition, another group of rats was fed a basic rodent diet. Animals were evaluated by cage-side and hand-held detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, serum chemistry, selected organ weights, and gross and histopathological examinations. Under the condition of this study, DAS-40278-9 maize did not cause any treatment-related effects in rats compared with rats fed diets containing non-GM maize.

Expression characterization of the herbicide tolerance gene Aryloxyalkanoate Dioxygenase (aad-1) controlled by seven combinations of regulatory elements

BackgroundAvailability of well characterized maize regulatory elements for gene expression in a variety of tissues and developmental stages provides effective alternatives for single and multigene transgenic concepts. We studied the expression of the herbicide tolerance gene aryloxyalkanoate dioxygenase (aad-1) driven by seven different regulatory element construct designs including the ubiquitin promoters of maize and rice, the actin promoters of melon and rice, three different versions of the Sugarcane Bacilliform Badnavirus promoters in association with other regulatory elements of gene expression.ResultsGene expression of aad-1 was characterized at the transcript and protein levels in a collection of maize tissues and developmental stages. Protein activity against its target herbicide was characterized by herbicide dosage response. Although differences in transcript and protein accumulation were observed among the different constructs tested, all events were tolerant to commercially relevant rates of quizalafop-P-ethyl compared to non-traited maize under greenhouse conditions.DiscussionThe data reported demonstrate how different regulatory elements affect transcript and protein accumulation and how these molecular characteristics translate into the level of herbicide tolerance. The level of transcript detected did not reflect the amount of protein quantified in a particular tissue since protein accumulation may be influenced not only by levels of transcript produced but also by translation rate, post-translational regulation mechanisms and protein stability. The amount of AAD-1 enzyme produced with all constructs tested showed sufficient enzymatic activity to detoxify the herbicide and prevent most herbicidal damage at field-relevant levels without having a negative effect on plant health.ConclusionsDistinctive profiles of aad-1 transcript and protein accumulation were observed when different regulatory elements were utilized in the constructs under study. The ZmUbi and the SCBV constructs showed the most consistent robust tolerance, while the melon actin construct provided the lowest level of tolerance compared to the other regulatory elements used in this study. These data provide insights into the effects of differing levels of gene expression and how these molecular characteristics translate into the level of herbicide tolerance. Furthermore, these data provide valuable information to optimize future designs of single and multiple gene constructs for maize research and crop improvement.

No treatment-related effects with aryloxyalkanoate dioxygenase-12 in three 28-day mouse toxicity studies

The aryloxyalkanoate dioxygenase-12 (AAD-12) protein is expressed in genetically modified soybean events DAS-68416-4 and DAS-444Ø6-6. Expression of the AAD-12 protein in soybeans confers tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) providing an additional herbicide choice to farmers. This enzyme acts by catalyzing the degradation of 2,4-D into herbicidally inactive metabolites. To meet evolving interpretation of regulations in the European Union, three separate 28-day repeat-dose oral mouse studies were conducted at increasing doses of up to 1100 mg AAD-12 protein/kg bw/day. No treatment-related effects were seen in any of these three studies.

The aryloxyalkanoate dioxygenase-12 (AAD-12) protein is not acutely toxic in mice

Typically, when a protein is orally toxic, it acts via acute mechanisms, especially at high doses. Therefore, an acute oral toxicity study is considered appropriate for evaluating the safety of transgenic proteins. Soybean plants (events DAS-68416-4 and DAS-444Ø6-6) have been genetically modified to express the aryloxyalkanoate dioxygenase-12 (AAD-12) protein. The AAD-12 protein provides tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). This paper summarizes the study designs of two acute oral toxicity studies evaluating the AAD-12 protein and reports the results of these studies. No mortalities or adverse effects were observed in mice when AAD-12 was tested up to a limit dose of 5000 mg/kg body weight. Based on the results of these studies, it can be concluded that AAD-12 protein, as expressed in genetically modified DAS-68416-4 and DAS-444Ø6-6 soybeans, lacks acute toxicity via the oral route.

Comparative Analysis of 2,4-D Uptake, Translocation, and Metabolism in Non–AAD-1 Transformed and 2,4-D–Resistant Corn

The Enlist™ traits provide 2,4-D resistance in several crops. Though corn is naturally tolerant to 2,4-D, the engineered trait conferred by the aryloxyalkanoate dioxygenase-1 (AAD-1) enzyme provides enhanced 2,4-D tolerance and confers resistance to the graminicide herbicide family, the aryloxyphenoxypropionates. The objectives of this research were 2-fold: (1) measure and compare uptake, translocation, and metabolism of 2,4-D in Enlist™ (E, +AAD1) and non–AAD-1 transformed (NT, −AAD1) isogenic corn hybrids; and (2) and investigate the effect of glyphosate and/or the Enlist™ adjuvant system (ADJ) on these factors and corn injury. Uptake of radiolabeled 2,4-D acid applied alone in corn was not altered by the addition of ADJ when tank mixed at 24 h after application (HAA). By contrast, uptake of radiolabeled 2,4-D was significantly lower (69%) compared with 2,4-D plus ADJ (89%) at 24 HAA with a premixed formulation of 2,4-D choline plus glyphosate-dimethylamine (Enlist Duo™ herbicide [EDH]). Translocation of 2,4-D between the two corn hybrids was not different. E corn metabolized more 2,4-D (100% of absorbed) than NT corn (84%), and glyphosate did not alter 2,4-D metabolism. Furthermore, the metabolism of 2,4-D to nonphytotoxic dichlorophenol (DCP) and subsequent DCP-derived metabolites formed in E corn was examined. Injury to E corn is not typically observed in the field; however, injury symptoms were clearly evident in E corn (within 24 HAA) when formulated acetochlor was tank mixed with EDH, which correlated with an increase in 2,4-D uptake during this time period. In summary, the lack of injury in E corn following EDH applied alone may be attributed to a relatively low amount of 2,4-D uptake and the combination of natural and engineered 2,4-D metabolic pathways.

Setting of an import tolerance for 2,4‐D in maize

In accordance with Article 6 of Regulation (EC) No 396/2005, the evaluating Member State (EMS) Greece received an application from Dow AgroSciences to set an import tolerance for the active substance 2,4‐D in genetically modified (GM) maize imported from Canada and the USA. The modification confers tolerance to the herbicide 2,4‐D. Greece drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005 which was submitted to the European Commission and forwarded to EFSA. According to EFSA, the data submitted are sufficient to conclude that a change of the existing maximum residue level (MRL) set at the limit of quantification (LOQ) of 0.05 mg/kg in maize is not required. Analytical enforcement methods are available to control the residues of 2,4‐D in cereals and no risk for consumers was identified for the notified use on GM maize expressing the aryloxyalkanoate dioxygenase 1 (AAD‐1) protein imported from Canada and the USA.

Comparative performance of broilers fed diets containing DAS-44406-6 and non-transgenic soybean meal

Broiler chickens are fast growing monogastric animals considered a sensitive test species to evaluate the safety and nutritional equivalence between transgenic and non-transgenic grains as part of the human safety evaluation process. DAS-44406-6 soybean expresses three herbicide-tolerant proteins: the aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme which provides tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) herbicides, the double-mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) enzyme encoded by a modified version of the epsps gene from maize (Zea mays), which provides tolerance to glyphosate herbicides, and the phosphinothricin acetyltransferase (PAT) enzyme from Streptomyces viridochromogenes, which provides tolerance to glufosinate herbicides. A 42-day broiler study was conducted with diets containing toasted DAS-44406-6 soybean meal to evaluate nutritional wholesomeness and safety compared with non-transgenic, near-isoline soybean and conventional comparators. The study used a randomized complete block design with five dietary treatments assigned randomly within six blocks of 12 pens per treatment, with 10 birds per pen. Broiler performance and carcass parameters were measured over a 6-week period of exposure to diets containing different sources of toasted soybean meal and hulls. Among the 13 endpoints measured, only thigh weight was numerically different between birds fed DAS-44406-6 soybean meal and those fed isoline soybean meal. However, no significant differences to thigh weight were observed between birds fed DAS-44406-6 soybean and any of the non-transgenic reference varieties. Results indicate that DAS-44406-6 soybeans are nutritionally equivalent to conventional varieties.

Scientific opinion on an application by Dow AgroSciences LLC (EFSA-GMO-NL-2012-106) for the placing on the market of genetically modified herbicide-tolerant soybean DAS-44406-6 for food and feed uses, import and processing under Regulation (EC) No1829/2003.

Soybean DAS‐44406‐6 expresses 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase (2mEPSPS), conferring tolerance to glyphosate‐based herbicides, aryloxyalkanoate dioxygenase (AAD‐12), conferring tolerance to 2,4‐dichlorophenoxyacetic acid (2,4‐D) and other related phenoxy herbicides, and phosphinothricin acetyl transferase (PAT), conferring tolerance to glufosinate ammonium‐based herbicides. The molecular characterisation data and bioinformatics analyses did not identify issues requiring assessment for food/feed safety. The agronomic and phenotypic characteristics revealed no relevant differences between soybean DAS‐44406‐6 and its conventional counterpart, except for pod count, seed count and yield. The compositional analysis identified no differences requiring further assessment, except for an increase (up to 31%) in lectin activity in soybean DAS‐44406‐6. Such increase is unlikely to raise additional concerns for food/feed safety and nutrition of soybean DAS‐44406‐6 as compared to its conventional counterpart and non‐GM reference varieties. There were no concerns regarding the potential toxicity and allergenicity of the three newly expressed proteins, and no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS‐44406‐6. Soybean DAS‐44406‐6 is as nutritious as its conventional counterpart and the non‐GM soybean reference varieties tested. There are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS‐44406‐6 plants, unless exposed to the intended herbicides. The likelihood of environmental effects from the accidental release of viable seeds from soybean DAS‐44406‐6 into the environment is therefore very low. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DAS‐44406‐6. In conclusion, the GMO Panel considers that the information available for soybean DAS‐44406‐6 addresses the scientific comments raised by Member States and that soybean DAS‐44406‐6, as described in this application, is as safe as its conventional counterpart and non‐GM soybean reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application.

Scientific Opinion on an application by Dow AgroSciences LLC (EFSA-GMO-NL-2011-91) for the placing on the market of genetically modified herbicide-tolerant soybean DAS-68416-4 for food and feed uses, import and processing under Regulation (EC) No1829/2003.

Soybean DAS‐68416‐4 was developed by Agrobacterium tumefaciens‐mediated transformation to express the aryloxyalkanoate dioxygenase‐12 (AAD‐12) protein, conferring tolerance to 2,4‐dichlorophenoxyacetic acid (2,4‐D) and other related phenoxy herbicides, and the phosphinothricin acetyltransferase (PAT) protein, conferring tolerance to glufosinate ammonium‐based herbicides. The molecular characterisation data and bioinformatics analyses did not identify issues requiring further assessment for food/feed safety. The agronomic and phenotypic characteristics tested revealed no relevant differences between soybean DAS‐68416‐4 and its conventional counterpart, except for ‘days to 50% flowering’. The compositional analysis identified no differences requiring further assessment, except for an increase (up to 36%) in lectin activity in soybean DAS‐68416‐4. Such increase is unlikely to raise additional concerns for food/feed safety and nutrition for soybean DAS‐68416‐4 as compared to its conventional counterpart and the non‐GM reference varieties. There were no concerns regarding the potential toxicity and allergenicity of the two newly expressed proteins, and no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS‐68416‐4. Soybean DAS‐68416‐4 is as nutritious as its conventional counterpart and the non‐GM reference varieties. There are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS‐68416‐4 plants, unless these are exposed to the intended herbicides. The likelihood of environmental effects resulting from the accidental release of viable seeds from soybean DAS‐68416‐4 into the environment is therefore very low. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DAS‐68416‐4. The GMO Panel concludes that the information available addresses the scientific comments of the Member States and that soybean DAS‐68416‐4, as described in this application, is as safe as its conventional counterpart and the tested non‐GM reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application.

Tolerance of Corn with Glyphosate Resistance and the Aryloxyalkanoate Dioxygenase Trait (AAD-1) to 2,4-D Choline and Glyphosate

In 2011, 14 Midwest trial locations evaluated tolerance of an AAD-1 and glyphosate-resistant corn hybrid to a 2,4-D choline+glyphosate premix formulation applied single and sequential POST at V4 and/or V7 corn with and without a PRE application of 2,4-D dimethylamine (DMA). Herbicides were applied at 1X and 2X maximum use rates with 1X rates of 1120 g ae ha−1 for glyphosate and 2,4-D DMA and 1065+1120 g ae ha−1 for the 2,4-D choline+glyphosate premix, respectively. Crop response was greatest 2 d after 2X rate applications, resulting in 4 to 10% visible injury to corn across application timings. No brace root injury or effect on corn grain yield were observed.

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression

Key messageThe choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize.Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

Scientific Opinion on an application by DOW AgroSciences LLC (EFSA‐GMO‐NL‐2010‐89) for placing on the market the genetically modified herbicide‐tolerant maize DAS‐40278‐9 for food and feed uses, import and processing under Regulation (EC) No 1829/2003

Maize DAS-40278-9 was developed by direct Whiskers-mediated transformation to express the aryloxyalkanoate dioxygenase-1 (AAD-1) protein, conferring tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. The molecular characterisation of maize DAS-40278-9 did not raise safety issues. The agronomic, phenotypic and compositional characteristics of maize DAS-40278-9 tested under field conditions revealed no differences between maize DAS-40278-9 and its non-genetically modified (GM) comparator that would give rise to food and feed or environmental safety concerns. There were no concerns regarding the potential toxicity and allergenicity of the newly expressed protein AAD-1, and no evidence that the genetic modification might significantly change the overall allergenicity of maize DAS-40278-9. The nutritional characteristics of maize DAS-40278-9 are not expected to differ from those of non-GM maize varieties and no post-market monitoring of food/feed is considered necessary. Maize DAS-40278-9 is as nutritious as its non-GM comparator and other non-GM commercial varieties. There are no indications of an increased likelihood of establishment and spread of occasional feral maize DAS-40278-9 plants, unless these plants are exposed to the intended herbicides. However, this will not result in different environmental impacts compared to conventional maize. Considering the scope of the application, interactions with the biotic and abiotic environment were not considered an issue. Risks associated with the unlikely but theoretically possible horizontal gene transfer from maize DAS-40278-9 to bacteria were not identified. The post-market environmental monitoring plan and reporting intervals are in line with the scope of the application. In conclusion, the EFSA GMO Panel considers that the information available for maize DAS-40278-9 addresses the scientific comments raised by the Member States and that maize DAS-40278-9, as described in this application, is as safe as the non-GM comparator and non-GM maize reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application.

Metabolism and Residues of 2,4-Dichlorophenoxyacetic Acid in DAS-40278-9 Maize (Zea mays) Transformed with Aryloxyalkanoate Dioxygenase-1 Gene.

DAS-40278-9 maize, which is developed by Dow AgroSciences, has been genetically modified to express the aryloxyalkanoate dioxygenase-1 (AAD-1) protein and is tolerant to phenoxy auxin herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D). To understand the metabolic route and residue distribution of 2,4-D in DAS-40278-9 maize, a metabolism study was conducted with 14C-radiolabeled 2,4-D applied at the maximum seasonal rate. Plants were grown in boxes outdoors. Forage and mature grain, cobs, and stover were collected for analysis. The metabolism study showed that 2,4-D was metabolized to 2,4-dichlorophenol (2,4-DCP), which was then rapidly conjugated with glucose. Field-scale residue studies with 2,4-D applied at the maximum seasonal rate were conducted at 25 sites in the U.S. and Canada to measure the residues of 2,4-D and free and conjugated 2,4-DCP in mature forage, grain, and stover. Residues of 2,4-D were not detectable in the majority of the grain samples and averaged <1.0 and <1.5 μg/g in forage and stover, respectively. Free plus conjugated 2,4-DCP was not observed in grain and averaged <1.0 μg/g in forage and stover.

Rapid simulated gastric fluid digestion of in-seed/grain proteins expressed in genetically engineered crops

The speed of simulated gastric digestion of proteins expressed in genetically engineered (GE) crops is commonly used to inform the allergenicity risk assessment. However, persistence of purified proteins in simulated gastric fluid (SGF) is poorly correlated with the allergenic status of proteins. It has been proposed that the plant or food matrix may affect the digestion of proteins and should be considered in interpreting digestion results. Here the SGF digestion of several GE proteins both as purified preparations and in soybean, corn, and cotton seed/grain extracts (in-matrix) are compared. Cry1F, Cry1Ac, phosphinothricin acetyltransferase (PAT), aryloxyalkanoate dioxygenase-1 (AAD-1), aryloxyalkanoate dioxygenase-12 (AAD-12), and double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) were all found to rapidly digest both as purified protein preparations and in seed/grain extracts from GE crops expressing these proteins. Based on these results, purified protein from microbial sources is a suitable surrogate for proteins in-matrix when conducting SGF digestion studies.

Control of Volunteer Corn with the AAD-1 (aryloxyalkanoate dioxygenase-1) Transgene in Soybean

Volunteer Enlist corn with the AAD-1 (aryloxyalkanoate dioxygenase-1) transgene can become a problem when glyphosate-resistant (GR) soybean follows Enlist corn in the rotation. Field trials were conducted at Ridgetown, Ontario in 2013 and 2014 to evaluate the control of volunteer Enlist corn in GR soybean. Glyphosate plus clethodim at 30 g ai ha−1provided 75 to 92% control of volunteer Enlist corn at 1, 2, 4, and 8 weeks after treatment application (WAT) and reduced volunteer Enlist corn density and dry weight 95 to 97%. Glyphosate plus clethodim at 60 g ai ha−1provided 84 to 98% control of volunteer Enlist corn at 1, 2, 4, and 8 WAT and reduced volunteer Enlist corn density and dry weight 97 to 99%. Glyphosate plus sethoxydim at 150 g ai ha−1provided 66 to 86% control of volunteer Enlist corn at 1, 2, 4, and 8 WAT and reduced volunteer Enlist corn density and dry weight 91 to 97%. Glyphosate plus sethoxydim at 300 g ha−1provided 84 to 96% control of volunteer Enlist corn at 1, 2, 4, and 8 WAT and reduced volunteer Enlist corn density and dry weight 96 to 98%. Glyphosate plus fenoxaprop-p-ethyl, fluazifop-p-butyl, and quizalofop-p-ethyl applied POST provided 0 to 9% control of volunteer Enlist corn at 1, 2, 4, and 8 WAT and reduced volunteer Enlist corn density and dry weight 18 to 44%. Soybean yields closely reflected the level of volunteer Enlist corn control. Based on these results, the cyclohexanedione herbicides, clethodim and sethoxydim, provide adequate control of volunteer Enlist corn in GR soybean. In contrast, the aryloxyphenoxypropionate herbicides, fenoxaprop-p-ethyl, fluazifop-p-butyl and quizalofop-p-ethyl do not provide control of volunteer Enlist corn in GR soybean.

Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

Response of Aryloxyalkanoate Dioxygenase-12 Transformed Soybean Yield Components to Postemergence 2,4-D

New trait technology incorporating 2,4-dichlorophenoxyacetic acid (2,4-D) resistance in soybean provides an alternative method to control weeds. However, the effect of postemergence treatments of 2,4-D on aryloxyalkanoate dioxygenase-12 (AAD-12) soybean on injury and yield components has not been reported. Our objectives were to characterize the effect of 2,4-D (dimethylamine salt) rates (0, 1,120, and 2,240 g ae ha−1) and soybean growth stage (V5, R2, or V5 followed by R2) on AAD-12 soybean injury and yield components. Less than 3% soybean injury was observed when 2,240 g ha−1of 2,4-D was applied to R2 soybean, and less than 1% soybean injury was caused by 1,120 g ha−1of 2,4-D. Seed yield, seed mass, pod number, seed number, seed per pod, reproductive node number, pods per reproductive node, node number, and percent reproductive nodes were not affected by 2,4-D treatments when applied at the V5, R2, or the V5 followed by R2 soybean growth stage. This research demonstrates that soybean transformed with AAD-12 can tolerate foliar applications of 2,4-D at rates up to 2,240 g ha−1with no effect on soybean grain yield components.

Aryloxyalkanoate Dioxygenase-12 Soybean Protein Expression

New trait technology incorporating 2,4-D resistance in soybean is dependent upon the ability of the plant to metabolize 2,4-D by the aryloxyalkanoate dioxygenase-12 protein (AAD-12). Our objectives were to determine AAD-12 expression during the daytime, throughout the leaf canopy, and before and after 2,4-D treatment for the events DAS-68416-4 and DAS-21606-3. Field experiments were conducted near Wanatah, IN in 2009 and Fowler, IN in 2009, 2010, and 2011. During the daytime, total AAD-12 expression was lowest between 12:30 and 15:30, averaging 161 ng cm−2, as compared to an average of 245 ng cm−2 in the morning and 243 ng cm−2 in the evening. The youngest fully emerged trifoliate in the DAS-68416-4 event had the highest AAD-12 expression, with means ranging from 369 to 390 ng cm−2, while the older leaves maintained a lower level of expression, 171 to 211 ng cm−2. The youngest leaves of event DAS-21606-3 had the highest level of AAD-12 expression (205 to 225 ng cm−2), while the level of AAD-12 was lower in older leaves (71 to 149 ng cm−2). In general, 2,4-D treatments did not reduce AAD-12 expression at 3, 7, 14, and 21 days after treatment; however, in a few instances AAD-12 expression was increased or decreased by 8 to 11% after 2,4-D treatment. Expression of AAD-12 was between 152 to 390 ng cm−2 for DAS-68416-4 and from 71 to 244 ng cm−2 for DAS-21606-3.