Abstract
Tocopherol cyclase (VTE1) plays a key role in promoting the production of γ-tocopherol and improving total tocopherol content in photosynthetic organisms. Walnut is an important source of tocopherols in the human diet, and γ-tocopherol is the major tocopherol compound in walnut kernels. In this study, a full-length cDNA of the VTE1 gene was isolated from walnut using RT-PCR and RACE, and designated as JrVTE1. The full-length cDNA of the JrVTE1 gene contained a 1353-bp open-reading frame encoding a 451-amino-acid protein with a calculated molecular weight of 49.5 kDa. The deduced JrVTE1 protein had a considerable homology with other plant VTE1s and belonged to the tocopherol cyclase family. Functional characterization of JrVTE1 by heterologous expression was carried out in E. coli BL21 (DE3) and microshoot lines of the fruit trees jujube (Zizyphus jujuba var. spinosa) and pear (Pyrus communis) cultivar ‘Old Home’. JrVTE1 in E. coli expressed as a 50 kDa protein, as expected. One or two copies of the transferred JrVTE1 gene were detected in the genomes of representative transgenic lines (from the initial transgenic plants) of jujube and pear by gel blots analysis. Over-expression of JrVTE1 in jujube and pear resulted in an accumulation of tocopherol and a shift in tocopherol composition in leaf, root and stem tissues. In the transgenic jujube, the total tocopherol content increased by 29.8 μg/g in the stems of line J3, 43.7 and 22.5 μg/g in the roots and leaves of line J1, respectively, whereas in the transgenic pear it increased by 47.3 μg/g in the leaf of line P3, and 16.7 and 10.4 μg/g in roots and stems of line P9, respectively. In the examined tissues of transgenic plants, the highest accumulation rate was the γ-tocopherol. These results indicate that JrVTE1 is one of the rate-limiting enzymes for tocopherol production and could be used to improve the tocopherol content of tree crops through genetic engineering. Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0414-2) contains supplementary material, which is available to authorized users.
Highlights
Vitamin E, a group of minor but ubiquitous lipidsoluble compounds, consists of four forms of tocopherol (a, b, c, d-tocopherol) and tocotrienol (a, b, c, d-tocotrienol)
In the examined tissues of transgenic plants, the highest accumulation rate was the ctocopherol. These results indicate that JrVTE1 is one of the rate-limiting enzymes for tocopherol production and could be used to improve the tocopherol content of tree crops through genetic engineering
Total tocopherol content was approximately 32, 96 and 108 lg/g in walnut embryos at 60, 90, and 120 days after flowering (DAF), respectively, and c-tocopherol was the main form at each time period
Summary
Vitamin E, a group of minor but ubiquitous lipidsoluble compounds, consists of four forms of tocopherol (a-, b-, c-, d-tocopherol) and tocotrienol (a-, b-, c-, d-tocotrienol). A-tocopherol (aT) was considered the most biologically active form and had captured much attention (Jiang et al 2004; Amaral et al 2005). The tocopherol cyclase (TC, VTE1) has been reported to be the key enzyme that catalyzes conversion of 2,3-dimethyl6-phytyl-1,4-benzoquinone (DMPBQ) to c-tocopherol and promotes the production of c-tocopherol and the total vitamin E content (Porfirova et al 2002; Cheng et al 2003; Kanwischer et al 2005; Vidi et al 2006). Much effort has been expended in overexpressing VTE1 to increase c-tocopherol production and vitamin E content in plants such as Arabidopsis (Kanwischer et al 2005), transgenic rapeseed (Kumar et al 2005), transgenic lettuce (Lee et al 2007) and transgenic tobacco (Yabuta et al 2013)
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