Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production

Authors

  • Chunyu Zhang,

    1. National Key Laboratory of Crop Genetic Improvement and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
    2. Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA
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  • Rebecca E. Cahoon,

    1. Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA
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  • Sarah C. Hunter,

    1. Plant Genetics Research Unit, Donald Danforth Plant Science Center, United States Department of Agriculture-Agricultural Research Service, Saint Louis, MO, USA
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  • Ming Chen,

    1. Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA
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  • Jixiang Han,

    1. Donald Danforth Plant Science Center, Saint Louis, MO, USA
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  • Edgar B. Cahoon

    Corresponding author
    1. Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA
    • National Key Laboratory of Crop Genetic Improvement and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
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For correspondence (e-mail ecahoon2@unl.edu).

Summary

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2–1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis.

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