Progress 05/01/07 to 04/30/08
Outputs
OUTPUTS: Objective 1. Biochemical characterization of HGGT. The monocot homogentisate geranylgeranyl transferase (HGGT), the initial enzyme in the biosynthesis of the tocotrienol form of vitamin E, was determined to be biochemically distinct from the closely related homogentisate phytyltransferase (HPT), the initial enzyme in the biosynthesis of the tocopherol form of vitamin E. Recombinant barley HGGT produced by expression in insect cells was 12-times more active with the substrate geranylgeranyl diphosphate (GGDP) than with phytyl diphosphate (PDP). Conversely, recombinant Arabidopsis PDP was 20-times more active with PDP than with GGDP. The structural basis for the substrate specificity differences of HGGTs and HPTs was determined by domain swapping experiments in which an HPT was functionally converted into a HGGT. Expression of barley HGGT in an Arabidopsis vitamin E null HPT-mutant revealed that the relative amounts of tocotrienols and tocopherols generated by this enzyme in planta is organ- and age-dependent, which is a likely reflection of the spatial and temporal pool sizes of GGDP and PDP in Arabidopsis. Objective 2. Identification of dicot-type HGGTs. In contrast to most dicots, seeds of Apiaceae species are rich sources of the tocotrienol form of vitamin E. An HGGT-related enzyme was identified in Coriandrum sativum (coriander) and several other Apiaceae. This enzyme shares <50% amino acid identity to monocot HGGTs, but contains many of the amino acid residues that distinguish monocot HGGTs from HPTs. Expression of this enzyme in an Arabidopsis vitamin E null mutant was accompanied by tocotrienol production, confirming the functional identity of this enzyme as an HGGT. Tocotrienols were identified as the primary form of vitamin E in roots and young leaves of the dicot Manihot esculenta (cassava). A cDNA for an HPT-like enzyme was identified from this plant and shown to generate tocotrienols during the early stages of seedling growth in a HPT knockout mutant of Arabidopsis. Objective 3. Biochemical basis for tocotrienol production in response to enhanced shikimate pathway flux. Tocotrienol production was detected in wild type Arabidopsis engineered for enhanced shikimate pathway flux by co-expression of the bacterial tyrA and Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) genes. No tocotrienol production was detected in an HPT-knockout Arabidopsis mutant engineered to co-express tyrA and HPPD, indicating the essential role of HPT in generating tocotrienols under these altered metabolic conditions. Objective 4. Biotechnological enhancement of tocotrienols in crops. Soybeans engineered to express the barley HGGT in a seed-specific manner accumulated vitamin E antioxidants, principally as tocotrienols, in excess of 3,000 mg/kg seed weight, a nearly 10-fold enhancement over wild type levels. Co-expression of HGGT and gamma-tocopherol methyltransferase resulted in a shift in tocotrienol production from the delta and gamma forms to the beta and alpha species. Constitutive expression of tyrA and HPPD along with HGGT yielded increased levels of tocotrienols in Arabidopsis leaves, above that achieved with HGGT expression alone. PARTICIPANTS: Dr. Edgar Cahoon (PD), Donald Danforth Plant Science Center, directed the project, provided technical guidance to the post-doctoral associate, and planned experimental approaches in coordination with the postdoctoral associate. Dr. Cahoon critiqued research conducted by the post-doctoral associate and toke lead responsibility for the writing and submission of manuscripts arising from the proposed studies. In addition, Dr. Cahoon coordinated and provided technical input, as needed. Dr. Sarah Hunter, USDA-ARS, served as a post-doctoral scientist and reported directly to the PD. Dr. Hunter conducted biochemical studies on barley homogentisate geranylgeranyl transfrerase. Dr. Wenyu Yang, Donald Danforth Plant Science Center, served as a post-doctoral scientist and reported directly to the PD. He conducted recombinant expression and biochemical analyses of putative homogentisate geranylgeranyl transferases from dicots. Dr. Chunyu Zhang, Visiting Scientist, Donald Danforth Plant Science Center, conducted research on the characterization of transgenic Arabidopsis lines that have been engineered to co-express the E. coli bifunctional chorismate mutase/prephenate dehydrogenase and hydroxyphenylpyruvate dioxygenase. To extend the results to a practical outcome, the PD conducted research sponsored by the Gate Foundation's Grand Challenges in Global Health program. This research involved efforts to metabolically engineer cassava storage roots to produce higher levels of vitamin E antioxidants TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project explored the biochemistry underlying the biosynthesis of tocotrienols, the primary form of vitamin E in seeds of cereal crops, including rice, wheat, and barley as well as seeds of some dicots, including members of the Apiaceae family. Tocotrienols have important nutritional value and are superior chemical antioxidants for the stabilization of vegetable oils used in frying and processing of foods as well as in industrial lubricants. Results from this research demonstrated that the homogentisate geranylgeranyl transferase (HGGT)-catalyzed production of tocotrienols is dependent not only on the marked substrate preference of HGGT for geranylgeranyl diphosphate (GGDP) but is also dependent on the organ in which HGGT is expressed, which is a likely reflection of the pool sizes of GGDP in a given plant organ. In Objective 2 of the grant, a new class of dicot-specific HGGT was identified in Apiaceae seeds that is structurally and biochemically distinct from homogentisate phytyltransferase (HPT), which catalyzes the first step in the synthesis of the tocopherol form of vitamin E. The identification of this HGGT and the corresponding gene expands the repertoire of transgenes that are available for the biotechnological enhancement of vitamin E antioxidant content in crops and also provides new information for deciphering the structural basis for the differing substrate preferences of HGGT and HPT. In Objective 3, the role of HPT in the synthesis of tocotrienols was demonstrated in transgenic plants with engineered enhancement of shikimate pathway flux. These findings provide basic information regarding an alternative route of tocotrienol production that can be used for biofortification of crop species. The findings from the first three objectives of the grant were used as the basis for the biotechnology-based enhancement of tocotrienol and total vitamin E content of soybean seeds in Objective 4. The increases in total vitamin E antioxidants achieved in soybean from these studies (6- to 10-fold increase over wild type seeds) are the largest reported to date through the use of a single transgene. Pending functionality studies, it is anticipated that vegetable oils extracted from these seeds will have improved oxidative stability for food processing and for bio-based lubricant applications.
Publications
- Hunter S.C., Cahoon E.B. (2007) Enhancing vitamin E in oilseeds: unraveling tocopherol and tocotrienol biosynthesis. Lipids 42: 97-108.
- Cahoon E.B. (2007) Metabolic redesign of vitamin E biosynthesis in soybean seeds for tocotrienol production and increased antioxidant content. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America Annual Meeting Abstract Book, New Orleans, LA (166-1).
- Cahoon E.B., Solomon F.S., Hunter S.C., Taylor N.J. (2007) Vitamin E and provitamin A biofortification of cassava. American Society of Plant Biologists meeting, Chicago, IL (Abstract # 2625).
- Cahoon, E.B. (2007) Understanding the biosynthesis of the tocotrienol form of vitamin E for the biofortification of crops. Phytochemical Society of North America Annual Meeting, Saint Louis, MO (Abstract # S1-3).
- Cahoon, E.B. (2006) Metabolic redesign of vitamin E biosynthesis in soybean seeds for increased antioxidant content. Soy2006: Molecular and Cellular Biology of the Soybean Conference, Lincoln, NE. (Conference Book, Page 55).
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