Article pubs.acs.org/JAFC

Vitamin E Levels in Soybean (Glycine max (L.) Merr.) Expressing a p‑Hydroxyphenylpyruvate Gene from Oat (Avena sativa L.)† Catherine M. Kramer* Syngenta Crop Protection, LLC, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States

Karen L. Launis Syngenta Crop Protection, LLC, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States

Maret G. Traber Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, Oregon 97331, United States

Dennis P. Ward Syngenta Seeds, Inc., 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States ABSTRACT: The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) is ubiquitous in plants and functions in the tyrosine catabolic pathway, resulting in the formation of homogentisate. Homogentisate is the aromatic precursor of all plastoquinones and tocochromanols, including tocopherols and tocotrienols. Soybean (Glycine max (L.) Merr.) has been genetically modified to express the gene avhppd-03 that encodes the protein AvHPPD-03 derived from oat (Avena sativa L.). The AvHPPD-03 isozyme has an inherent reduced binding affinity for mesotrione, a herbicide that inhibits the wild-type soybean HPPD enzyme. Expression of avhppd-03 in soybean plants confers a mesotrione-tolerant phenotype. Seeds from three different avhppd-03expressing soybean events were quantitatively assessed for content of eight vitamin E isoforms. Although increased levels of two tocopherol isoforms were identified for each of the three soybean events, they were within, or not substantially different from, the ranges of these isoforms found in nontransgenic soybean varieties. The increases of these tocopherols in the avhppd-03expressing soybean events may have a slight benefit with regard to vitamin E nutrition but, given the commercial processing of soybeans, are unlikely to have a material impact on human nutrition with regard to vitamin E concentrations in soybean oil. KEYWORDS: vitamin E, tocochromanol, tocopherol, HPPD, soybean, Glycine max, Avena sativa

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INTRODUCTION

is important in both photosynthesis and cellular metabolism via the citric acid cycle. The tocochromanols, collectively known as vitamin E, consist of four tocopherol isoforms and four tocotrienol isoforms. They are lipophilic antioxidants that are synthesized exclusively in photosynthetic organisms and are an essential part of human and animal diets. Tocochromanol content and composition vary considerably among plant species. In higher order plants, αtocopherol is the predominant isoform in green leaf tissue, and γtocopherol is the predominant isoform in seeds, comprising 70% of the total tocopherols in soybean oil and as high as 1200 μg/g oil in one variety of soybean.5 Oilseeds are particularly rich in tocochromanols.6 In soybean, the natural range is broad due to the influence of genetics and environmental effects7−10 such as temperature during plant development and seed filling,11−15

Eukaryotic organisms catabolize tyrosine to homogentisate (HGA) as a central intermediate in the tyrosine catabolic pathway. The conversion of p-hydroxyphenylpyruvate to HGA is catalyzed by the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD)1−4 (Figure 1). The primary biosynthetic products of the tyrosine catabolic pathway are the eight tocochromanols (tocopherols and tocotrienols), plastoquinone, acetoacetate, and fumarate (Figure 2). This biochemical pathway is found in nearly all aerobic organisms, including plants, animals, and bacteria, and

Received: Revised: Accepted: Published:

Figure 1. Conversion of p-hydroxyphenylpyruvate to homogentisate. © 2014 American Chemical Society

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October 30, 2013 March 19, 2014 March 31, 2014 March 31, 2014 dx.doi.org/10.1021/jf4048995 | J. Agric. Food Chem. 2014, 62, 3453−3457

Journal of Agricultural and Food Chemistry

Article

Figure 2. Tyrosine catabolic pathway. DMGGBQ, 2,3-dimethyl-5-geranylgeranyl-1,4-benzoquinone; DMPBQ, 2,3-dimethyl-5-phytyl-1,4benzoquinone; HGGT, homogentisate geranylgeranyl transferase; HmgA, homogentisate dioxygenase; HmgB, fumarylacetoacetate hydrolase; HmgC, maleylacetoacetate isomerase; HPP, 4-hydroxyphenylpyruvate; HPPD, p-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; HST, homogentisate solanyltransferase; MGGBQ, 2-methyl-6-geranylgeranyl-1,4-benzoquinone; MPBQ, 2-methyl-6-phytyl-1,4benzoquinone; MPBQ MT, MPBQ methyltransferase; MSBQ, 2-methyl-6-solanyl-1,4-benzoquinone; PDP, phytyldiphosphate; TAM, L-tyrosine aminotransferase; TC, tocopherol cyclase; TMT, tocopherol methyltransferase; TYRA, bifunctional chorismate mutase-prephenate.

water deficit,16,17 and crop management practices such as seeding rate, row spacing, and planting date.8 Syngenta has genetically modified soybean (Glycine max (L.) Merr.) to express an hppd gene derived from oat (Avena sativa L.). This gene, designated avhppd-03, encodes a p-hydroxyphenylpyruvate dioxygenase isozyme (AvHPPD-03) that has >99.7% sequence identity with the HPPD isozyme from A. sativa. In comparison with the endogenous soybean HPPD, the AvHPPD-03 isozyme from oat has reduced binding affinity for mesotrione, a herbicide that inhibits HPPD. The avhppd-03 gene is linked to a constitutive promoter that expresses AvHPPD-03 at a higher level than the wildtype soybean hppd. Expression of avhppd-03 in soybean plants confers a mesotrione tolerance phenotype. Syngenta has produced three mesotrione-tolerant soybean events (SYHT04R, SYHT06W, and SYHT0H2) and has evaluated the effect of expression of avhppd-03 on tocochromanol levels in seeds from these events grown under field conditions in two consecutive years.

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treated with mesotrione herbicide (0.1 kg active ingredient per hectare) at the V3−V5 growth stage.19 The other transgenic entry was not treated with mesotrione. All entries within a trial were treated with maintenance chemicals as needed for optimum plant health. Seed samples were obtained from 30 randomly selected plants from each plot after plants reached full maturity (R8 stage).19 Representative subsamples were finely ground on dry ice and maintained at approximately −20 °C until they were analyzed. Quantitation of Tocopherol and Tocotrienol Levels in Soybean Seed. The levels of tocopherols and tocotrienols in the seed were determined by Covance Laboratories, Inc. (Madison, WI, USA) using validated methods based on literature references. Representative aliquots from the homogeneous samples were analyzed in a random order. Following quantitation of the tocochromanols, all results were converted to equivalent units of dry weight based on the moisture content of the seed samples. Tocopherols. To release vitamin E, the aliquots (approximately 6 g) were saponified to break down any fat. The saponified mixture was extracted with diethyl ether, evaporated to near dryness, and diluted to a volume of 2 mL in hexane. The levels of α-, β-, γ-, and δ-tocopherols were quantitated by high-performance liquid chromatography (HPLC) using fluorescence detection (excitation, 292 nm; emission, 325 nm). HPLC was performed with the following parameters: a silica column (ES Ind. Chromegabond Diol 3 μm 60 Å, 15 cm × 4.0 mm), guard column (Diol, 12.5 mm × 4.6 mm, 4 μm packing), flow rate of 1.0 mL/ min, run time of 20 min, and injection volume of 15 μL, with mobile phase A [3% tetrahydrofuran (THF), 0.05% glacial acetic acid (HOAc) in iso-octane] and mobile phase B (30% THF, 0.05% HOAc in isooctane).20−22 Tocotrienols. Aliquots (approximately 2 g) were treated with sodium bicarbonate and sodium ascorbate to buffer the solution. Pyrogallol (4 mL) was added as an antioxidant. The samples were extracted with 8 mL of 2,2,4-trimethylpentane (iso-octane), and dimethyl sulfoxide (4 mL) was added to increase extraction efficiency. Methanolic sulfuric acid solution (3.33 N) was added to further break down the sample matrix. Additional iso-octane (8 mL) was added to aid in phase separation. The samples were centrifuged, and the α-, β-, γ-, and δ-tocotrienols in the supernatant were quantitated by HPLC and fluorescence detection (excitation, 292 nm; emission, 325 nm). The HPLC was performed with

MATERIALS AND METHODS

Production of Seed Samples. During 2009 and 2010, three soybean events expressing avhppd-03 and the corresponding nontransgenic, near-isogenic control variety ‘Jack’18 (maturity group 2.9) were grown according to local agronomic practices in the United States. The field trials were conducted at six locations (in Nebraska, Ohio, Michigan, Arkansas, Missouri, and Iowa) in 2009 and at eight locations [in Nebraska, Missouri, Iowa, Illinois (2), North Carolina, Pennsylvania, and Indiana] in 2010. One event (SYHT04R) was grown in both years, and the other two events (SYHT06W, SYHT0H2) were each grown in one year (2009 or 2010, respectively). To establish a range of natural variability, six nontransgenic precommercial Syngenta soybean reference varieties (maturity groups 2.3−3.5) were also grown at the same locations in the same years. At each location, two entries of each transgenic soybean event, the corresponding control soybean, and the reference varieties were grown in a randomized complete block design with four replicate plots for each entry. In each trial, one entry of each transgenic soybean event was 3454

dx.doi.org/10.1021/jf4048995 | J. Agric. Food Chem. 2014, 62, 3453−3457

Journal of Agricultural and Food Chemistry

Article

Table 1. Tocopherol Levelsa in Three Field-Grown avhppd-03-Expressing Soybean Events line

N

statistic

α-tocopherol

β-tocopherol

γ-tocopherol

δ-tocopherol

−