Genetic variation of g-tocopherol methyltransferase gene contributes to elevated a-tocopherol content in soybean seeds

Background: Improvement of a-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the a-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to a-tocopherol biosynthesis or through changes to crop management practices. Varieties with high a-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high a-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low a-tocopherol variety Ichihime. In this study, the genetic mechanism of the high a-tocopherol content trait of KAS was elucidated. Results: Through QTL analysis and fine mapping in populations from a cross between KAS and a Japanese variety Ichihime, we identified g-TMT3, which encodes g-tocopherol methyltransferase, as a candidate gene responsible for high a-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of g-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in a-tocopherol concentration. Therefore, we focused on transcriptional regulation of g-TMT3 in developing seeds and leaves. An F5 line that was heterozygous for the region containing g-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the g-TMT3 locus were chosen for further evaluation. The expression level of g-TMT3 was higher both in developing seeds and leaves of plants homozygous for the g-TMT3 allele from KAS. The higher expression level was closely correlated with high atocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of g-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of g-TMT3 promoter from KAS was higher than that of Ichihime. Conclusions: The genetic variation in g-TMT3, which plays a major role in determining a-tocopherol concentration, provides significant information about the regulation of tocopherol biosynthesis in soybean seeds. This knowledge will help breeding programs to develop new soybean varieties with high a-tocopherol content. Background The vitamin E family comprises tocopherols (a, b, g, and δ forms) and tocotrienols (a, b, g, and δ forms). All isoforms possess lipid antioxidant activity, and a-tocopherol possesses the highest vitamin E activity in mammals [1,2]. Vitamin E is widely used as an antioxidant in foods and oils, as a nutrient additive in poultry and cattle feeds to improve meat quality, and as a supplement in the human diet to help prevent diseases such as cancer and cardiovascular diseases. The market size is expected to grow because of the increasing interest in functional food and increasing demand for meat products. About 85% of commercial vitamin E is synthesized by chemical reaction [3]. This vitamin E usually includes the naturally occurring RRR-a-tocopherol and 7-stereoisomers as secondary products, whose biological activity is only 50%-74% of that of the natural a-tocopherol [4]. Thus, it is very important to increase natural vitamin E production in crops and vegetables [2]. Soybean (Glycine max (L.) Merr.) is one of the major crops for food, oil, and animal feed. In seed processing, * Correspondence: [email protected] Laboratory of Plant Genetics and Evolution, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9 Sapporo 060-8589, Hokkaido, Japan Dwiyanti et al. BMC Plant Biology 2011, 11:152 http://www.biomedcentral.com/1471-2229/11/152 © 2011 Dwiyanti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tocopherols are extracted together with the oil fraction. The tocopherol content is only about 1.5% of the oil; nevertheless, tocopherols are critical for oxidative stability [5]. Since tocopherols contribute to both the nutritional value of seeds and the oxidative stability of soybean oil, enhancing tocopherol content in soybean will improve its market value. In common soybean cultivars, the main forms of seed tocopherols are g-tocopherol and δ-tocopherol, which account for 60% to 70% and 20% to 25% of the total tocopherol, respectively. The proportion of a-tocopherol is usually less than 10% of total tocopherol in soybean seeds [1,6,7]. There have been some efforts to improve soybean vitamin E through genetic engineering. The Arabidopsis VTE4 gene encodes g-tocopherol methyltransferase (g-TMT), which catalyzes the last step of a-tocopherol biosynthesis (Figure 1); overexpression of VTE4 in soybean seeds resulted in a-tocopherol elevation to 75% of total tocopherol. When VTE4 was coexpressed with VTE3, which encodes methyl-6-phytyl-1,4-benzoquinol (MPBQ)methyltransferase (Figure 1), a-tocopherol increased to more than 95% of total tocopherol, and vitamin E activity increased to up to five times the level in nontransgenic soybean [6]. Meanwhile, overexpression of Perilla frutescens g-TMT alone increased a-tocopherol to more than 90% of total tocopherol [8]. Several studies have suggested the importance of other tocopherol forms. For example, g-tocopherol may prevent inflammation or improve kidney function, which are distinct from its antioxidant activity [9,10]. These studies triggered us to look for natural tocopherol variants, which may have unique characteristics. Such variants may make it possible to breed soybean cultivars with a wide range of atocopherol (from 10% to 90% of total tocopherol), and to develop soybean cultivars tailor-made for certain purposes. Tocopherols are present in leaves, stems, flower petals, and seeds of higher plants and green algae [1,11]. While a-tocopherol is usually the predominant form in leaves, there are diverse variations of tocopherol composition in seeds [1]. For example, in soybean, rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana), most of the tocopherols are g-tocopherol or δ-tocopherol; in sunflower (Helianthus annuus) and safflower (Carthamus tinctorius) seeds, the content of a-tocopherol is more than 95% of the total tocopherol content [12,13]. Variations in a-tocopherol content (a-tocopherol weight [μg] per 100 mg seed powder) and concentration (atocopherol as a percentage of total tocopherol) have been reported in crops such as maize (from 0.9 to 6.5 μg 100 mg), sunflower (>95% in wild type and <10% in mutants), safflower (>85% in wild type and <15% in mutants), rapeseed (a/g-tocopherol ratio ranged from 0.54 to 1.70) and in the model plant Arabidopsis [12-16]. Previous studies have shown that variation is also present in soybean. Three soybean varieties with atocopherol concentration of 20% to 30%, Keszthelyi Aproszemu Sarga (KAS), Dobrogeance, and Dobrudza 14 Pancevo, were identified through analysis of more than 1,000 cultivars and varieties from soybean germplasms collections [7]. These varieties showed higher atocopherol content compared to typical cultivars over two planting years, indicating that high a-tocopherol content was a stable trait [7]. QTL analysis using Chinese (Hefeng 25) and Canadian (OAC Bayfield) soybean varieties revealed four QTLs for tocopherol content in linkage groups B2, C2, D1b, and I, which correspond to chromosome 14, 6, 2, and 20, respectively. However, the causal genes involved in these QTLs are yet to be identified [17]. In our previous study, the genetic characteristics of the high a-tocopherol concentration trait were evaluated in an F2 population derived from a cross between KAS and a typical variety, Ichihime [18]. a-Tocopherol Homogentisic acid PP