Progress 10/01/05 to 09/30/06
Outputs
In the previous year, we demonstrated that homogentisate geranylgeranyl transferase (HGGT), which catalyzes the first step in the tocotrienol biosynthetic pathway in cereal grains, is five-fold more active with the substrate geranylgeranyl diphosphate (GGDP) than with phytyl diphosphate (PDP). By contrast, homogentisate phytyltransferase (HPT), which catalyzes the first step in the biosynthesis of the tocopherol form of vitamin E, is 50- to 80-fold more active with PDP than with GGDP. Despite this marked difference in substrate preferences, HGGTs from cereal grains share approximately 40 to 60% amino acid sequence identity with known HPTs. During the current year, research was initiated to determine the structural basis for the differences in substrate preference displayed by HGGTs and HPTs. To address this question, domain swapping experiments were conducted using the barley HGGT (HvHGGT) and Arabidopsis HPT (AtHPT). Two regions of the barley enzyme were substituted
for the corresponding regions in the Arabidopsis enzyme. The regions replaced were the amino acid sequences AtHPT138-178 and AtHPT294-347 with HvHGGT152-192 and HvHGGT308-361 singly and in combination. Both these regions have been proposed to be putative active sites of the related Escherichia coli ubiA prenyltransferase. The chimeric genes were transformed into an Arabidopsis mutant that is devoid of vitamin E due to a missense mutation in HPT. Leaves and seeds from the transformed plants were analyzed by HPLC to determine whether the Arabidopsis enzyme had been converted from a tocopherol producing enzyme into a tocotrienol producing enzyme. Preliminary results from these experiments indicate that plants transformed with either AtHPT138-178 swapped with HvHGGT152-192 or AtHPT294-347 swapped with HvHGGT301-361 singly have tocopherol but no detectable tocotrienol. However, lines transformed with a chimeric enzyme containing both domain swaps were found to produce tocotrienols. This
finding suggests that the functional differences between HGGT and HPT can largely be attributed to two relatively small domains within their structures. In a continuation of research initiated in the previous year, transgenic soybean lines were generated that express the barley HGGT under control of a strong seed-specific promoter for the alpha'-subunit of beta-conglycinin. Fourteen trait positive soybean lines have been generated. At least three of these have vitamin E antioxidant content in excess of 2,000 mg/kg seed weight, which is nearly a six-fold increase in vitamin E antioxidants compared to seeds from non-transformed plants. This increase in total vitamin E antioxidant content is the largest that has been reported in soybean by the use of a single transgene. Transgenic seeds accumulate primarily the delta- and gamma forms of tocotrienol, and in lines producing the highest levels of vitamin E antioxidants, tocotrienols account for nearly 90% of the total vitamin E
antioxidants. These results demonstrate that expression of HGGT is a viable approach for the metabolic engineering of enhanced vitamin E antioxidant content in soybean seeds
Impacts
Results from these studies will provide basic knowledge of the biosynthesis of the tocotrienol form of vitamin E in plants and facilitate biotechnological efforts to enhance the lipid soluble antioxidant content of crop plants. This research will contribute to the development of crops with improved productivity, grains with enhanced nutritional value for human and livestock diets, and vegetable oils with increased oxidative stability for food processing and industrial applications.
Publications
- Hunter, S.C., Cahoon, E.B. (2007) Enhancing vitamin E in oilseeds: unraveling tocopherol and tocotrienol biosynthesis. Lipids (in press)
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Progress 10/01/04 to 09/30/05
Outputs
Research was initiated to determine the relative substrate specificity of the recently discovered enzyme homogentisate geranylgeranyl transferase (HGGT) for geranylgeranyl diphosphate (GGDP) and phytyl diphosphate (PDP). HGGT catalyzes the committed step in the synthesis of the tocotrienol form of vitamin E in the endosperm of cereal grains. HGGT from barley was expressed in insect cells, and an in vitro assay for the recombinant enzyme was established. For comparison, parallel experiments were conducted with the closely related Arabidopsis homogentisate phytyltransferase (HPT), which catalyzes the committed step in the biosynthesis of the tocopherol form of vitamin E. HGGT was approximately five times more active with GGDP than with PDP. By contrast, the Arabidopsis HPT displayed about 40-fold more activity with PDP than with GGDP. These results demonstrate that, despite their amino acid sequence relation, HGGT and HPT have distinct substrate specificities. The
activity of barley HGGT was also examined by constitutive expression of the corresponding cDNA in an Arabidopsis mutant devoid of vitamin E. Consistent with the in vitro data, expression of the barley HGGT was accompanied by the production of primarily tocotrienols and lesser amounts of tocopherols, and the relative amounts of the two forms of vitamin E produced were dependent on the tissues examined, which is a likely reflection of the relative pool sizes of GGDP and PDP found throughout the Arabidopsis plant. Overall, these results provide a biochemical explanation for the occurrence of tocotrienols, as well as tocopherols, in the endosperm of cereal grains such as barley, wheat, rice, and corn. Domain-swap experiments are currently underway to determine the structural basis for the different substrate specificities of HGGT and HPT. In addition, it has been previously reported that tocotrienol synthesis can be conferred to plant cells by strong, up-regulation of the shikimate branch
of the vitamin E biosynthetic pathway. To examine the basis for this metabolic phenomenon, wild-type Arabidopsis and a vitamin E null Arabidopsis mutant was engineered to express a plastid-targeted bifunctional chorismate mutase/prephenate dehydrogenase (TyrA) from E. coli. This enzyme by-passes feed-back regulated steps in the synthesis of the homogentisate substrate for HGGT and HPT. Expression of TyrA in the transgenic Arabidopsis lines was confirmed by western blot analysis. To generate additional enhancement of flux through the shikimate branch of the vitamin E biosynthetic pathway, these plants are currently being re-transformed with an overexpression construct for hydroxyphenylpyruvate dioxygenase. We have also succeeded in expressing barley HGGT in soybean under control of a strong seed-specific promoter. Tocotrienols accounted for >80% of the total vitamin E antioxidants in developing seeds from these plants. Measurements of the total increase in vitamin E antioxidant content
await collection of mature seeds. It is anticipated that vegetable oils obtained from these seeds will have improved properties for food processing, nutraceutical, and industrial applications.
Impacts
Results from these studies will provide basic knowledge of the biosynthesis of the tocotrienol form of vitamin E in plants and facilitate biotechnological efforts to enhance the lipid soluble antioxidant content of crop plants. This research will contribute to the development of crops with improved productivity, grains with enhanced nutritional value for human and livestock diets, and vegetable oils with increased oxidative stability for food processing and industrial applications.
Publications
- No publications reported this period
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