Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes 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 deregulation of homogentisate synthesis. In this report, the basis for this pathway and its impact on vitamin E ion when combined with HGGT were explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bifunctional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol ion 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-DP 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.
