Studies have shown that overexpression of gamma-tocopherol methyltransferase in Arabidopsis seeds shifted …

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The chloroplasts of higher plants produce numerous compounds that not only perform vital functions but also are important from agricultural and nutritional perspectives. Tocopherols, the lipid-soluble antioxidants known collectively as vitamin E, are one such group of compounds. The four naturally occurring tocopherols, -, -, - and -tocopherol, differ only in the number and position of methyl substituents on the aromatic ring (1). In addition to their role as antioxidants (1), tocopherols stabilize polyunsaturated fatty acids within lipid bilayers by protecting them from lipoxygenase attack (2).

Of tocopherol species present in foods, -tocopherol is the most important to human health, has the highest vitamin E activity (3), and occurs as a single (R,R,R)--tocopherol isomer (4). Although all tocopherols are absorbed equally during digestion, only (R,R,R)--tocopherol is preferentially retained and distributed throughout the body (5).

The most recent U.S. recommended daily allowance (RDA) suggests that 10 to 13.4 international units (IU) of vitamin E [equal to 7 to 9 mg of (R,R,R)--tocopherol] be consumed daily (6). Because of the abundance of plant-derived components in most diets, this RDA is often met in the average diet. However, daily intake of vitamin E in excess of the RDA (100 to 1000 IU) is associated with decreased risk of cardiovascular disease and some cancers, improved immune function, and slowing of the progression of a number of degenerative human conditions (5). Obtaining these therapeutic levels of vitamin E from the average diet is nearly impossible unless a concerted effort is made to ingest large quantities of specific foods enriched in vitamin E.

In the United States, approximately 60% of dietary vitamin E intake is from vegetable oils (7). In soybean oil, which accounts for 80 and 25% of the edible oil consumed in the United States and the world, respectively (8), -tocopherol and its immediate biosynthetic precursor -tocopherol account for 7 and 70%, respectively, of the total tocopherol pool (9). The other major oilseed crops--corn, canola, cottonseed, and palm oils--have similarly low - to -tocopherol ratios (4).

Substantial increases in the -tocopherol content of major food crops are needed to provide the public with dietary sources of vitamin E that can approach the desired therapeutic levels. The observation that many oilseeds contain relatively high levels of tocopherol, the biosynthetic precursor to tocopherol, suggests that the final step of the -tocopherol biosynthetic pathway, catalyzed by -tocopherol methyltransferase (-TMT), is limiting in these tissues. Therefore, it may be possible to convert the large pool of tocopherol present in seeds such as soybeans to -tocopherol by targeted overexpression of the -TMT gene.

The -tocopherol biosynthetic pathway is known (10), and genes encoding one enzyme, p-hydroxyphenylpyruvate dioxygenase (HPPDase) have been cloned from carrot (11) and Arabidopsis (12). It has proven difficult to obtain gene probes for additional pathway enzymes because the remaining enzymes are membrane-bound. We used a genomics-based approach to clone the -TMT gene that exploits the complementary model photosynthetic organisms Arabidopsis thaliana and Synechocystis PCC6803. Both organisms synthesize and accumulate (R,R,R)--tocopherol (13) to greater than 95% of their tocopherol pools, which suggests that -TMT activity is not limiting in these photosynthetic cells. We used the gene encoding the Arabidopsis HPPDase (14) to search the Synechocystis PCC6803 genomic database (15) and identified a single open reading frame (ORF) that shared 35% amino acid sequence identity with the Arabidopsis HPPDase. The Synechocystis HPPDase gene was located within a predicted 10-gene operon encompassing bases 2,893,184 to 2,905,235 of the Synechocystis genome (Fig. 1A). Because related biosynthetic genes in bacteria are often organized into operons, we hypothesized that this operon might contain other genes involved in tocopherol biosynthesis, including -TMT. 

The operon included one candidate for a Synechocystis -TMT gene, ORF SLR0089. This ORF predicted a protein that shared a high degree of amino acid sequence similarity with known plant -(24)-sterol-C-methyltransferases. SLR0089 also contains several structural features of a -TMT (Fig. 2) including S-adenosylmethionine (SAM)-binding domains (16) and a putative NH₂-terminal bacterial signal sequence (17), which is consistent with the presumed site of cyanobacterial tocopherol biosynthesis. The predicted 32,766-dalton mature SLR0089 protein is similar in size to the 33,000 daltons reported for the only -TMT purified from plants (18). We therefore hypothesized that the SLR0089 ORF encoded the Synechocystis -TMT. 

A null mutant for the SLR0089 gene was created by replacing the wild-type gene with a nonfunctional SLR0089 gene (19) through homologous recombination (20). The SLR0089 mutant was then analyzed by high-performance liquid chromatography (HPLC) (21) for alterations in the normal Synechocystis tocopherol profile. Both the amount and type of tocopherol were altered. The mutant was devoid of -tocopherol, accumulated the biosynthetic precursor -tocopherol, and produced less total tocopherol (Fig. 1C). The change in the tocopherol composition of the SLR0089 mutant is consistent with the phenotype expected for a mutant lacking -TMT activity. To confirm its identity, the SLR0089 ORF was expressed in Escherichia coli (22), and the recombinant protein was assayed for -TMT (18). The results showed that the recombinant SLR0089 protein was able to catalyze the methylation of -tocopherol to form -tocopherol (Table 1). Thus, the SLR0089 ORF encoded the Synechocystis -TMT (Syn.-TMT).

To identify a -TMT gene from higher plants, we used the Syn.-TMT protein sequence as a query in a search of the Arabidopsis expressed sequence tag (EST) database (23). One cDNA clone, Arabidopsis EST 165H5T7, showed 66% amino acid sequence similarity with Syn.-TMT (Fig. 2). The 165H5T7 protein contained structural features common to -TMTs (Fig. 2), including SAM binding domains and a predicted 47-amino acid chloroplast transit peptide (24), which is consistent with the known intracellular locale of tocopherol synthesis in plants (25). The mature 33,843-dalton 165H5T7 protein is similar in size to the deduced mature Syn.-TMT protein. To confirm that 165H5T7 encoded an Arabidopsis -TMT ortholog, the DNA sequence corresponding to the predicted mature 165H5T7 protein was expressed in E. coli (26). The recombinant 165H5T7 protein indeed possessed -TMT activity (Table 1) (18). Thus, the protein encoded by EST clone 165H5T7 was the Arabidopsis -TMT (A.t.-TMT).

The substrate specificities of the Synechocystis and Arabidopsis -TMTs (18, 27) were similar. When different methyl-substituted tocopherols were used as substrates, only -tocopherol and -tocopherol were methylated by the two -TMTs to yield -tocopherol and -tocopherol, respectively (Table 1). Thus, the 5 position of the tocopherol aromatic head group is the specific site of methylation by -TMT.

To determine whether -TMT is a key enzymatic activity involved in determining the tocopherol composition in seeds, Arabidopsis was transformed (28) with pDC3-A.t.-TMT (29), a plant expression construct containing the full-length Arabidopsis -TMT cDNA driven by the seed-specific carrot DC3 promoter (30). Arabidopsis was used because, like major oilseed crops, its seed tocopherol profile (>95% -tocopherol) suggests that -TMT activity is limiting. Primary transformants were selected by antibiotic resistance and grown to maturity. Pooled, segregating T2 seeds from individuals were analyzed for changes in tocopherol content and composition.

HPLC (21) analysis showed that seed-specific overexpression of the Arabidopsis -TMT gene increased seed -tocopherol levels >80-fold (Table 2). Seeds of the lines overexpressing the largest amounts of -TMT contained >95% of their total tocopherol pool as -tocopherol (Table 2). In addition, the small -tocopherol pool normally present in wild-type Arabidopsis seed was completely converted to -tocopherol, confirming the in vitro finding that both - and -tocopherols are substrates for Arabidopsis -TMT in vivo. The observed changes in the seed tocopherol composition of -TMT overexpressers correlated with increased levels of -TMT enzyme activity in mature seed protein (Table 2) (18). The accumulation of tocopherol and -tocopherol in the seeds of plants overexpressing -TMT indicates that their low levels in wild-type Arabidopsis seed were due to limiting seed -TMT activity.

Overexpression of the -TMT gene altered the tocopherol composition of Arabidopsis seed but not the total tocopherol content (Table 2). Thus, the increased -tocopherol content of pDC3-A.t.-TMT seeds was due to the conversion of the large pool of -tocopherol already present in wild-type seeds. Therefore, it appears that -TMT plays an important role in determining the composition but not the total content of the seed tocopherol pool.

The vitamin E activity of seeds from Arabidopsis lines overexpressing -TMT was ninefold that of wild-type seeds. Given the differing vitamin E potencies of -, -, - and -tocopherols [100, 50, 10, and 3% relative vitamin E activity, respectively (3)], 50 gm of wild-type Arabidopsis seed oil would contain 9 IU of vitamin E, whereas 50 g of seed oil from the best transgenic lines would contain 84 IU. We may assume that -TMT activity is also limiting in commercially important oilseeds crops that have low - to -tocopherol ratios, such as soybean, corn, and canola. Overexpressing the -TMT gene in these crops should similarly elevate tocopherol levels and thereby increase the nutritional value of these important dietary sources of vitamin E.

REFERENCES AND NOTES

1. M. J. Fryer, Plant Cell Environ. 15, 381 (1992).
2. A. N. Erin, V. V. Skrypin, V. E. Kagan, Biochim. Biophys. Acta 815, 209 (1985).
3. A. Kamal-Eldin and L.-A. Appelqvist, Lipids (Rev.) 31, 671 (1996) .
4. R. R. Eitenmiller, Food Technol. 51, 78 (1997) 
5. M. G. Traber and H. Sies, Annu. Rev. Nutr. 16, 321 (1996) .
6. National Research Council Food and Nutrition Board, NAS-NRC Recommended Dietary Allowances (National Academy Press, Washington, DC, ed. 10, 1989).
7. VERIS Information Service, Vitamin E Fact Book (LaGrange, IL, 1994).
8. These figures are from the American Soybean Association (available at www.ag.uiuc.edu/~stratsoy/97soystats).
9. P. Taylor and P. Barnes, Chem Ind. (17 October 1981), p. 722.
10. D. R. Trelfall and G. R. Whistance, in Aspects of Terpenoid Chemistry and Biochemistry, T. W. Goodwin, Ed. (Academic Press, London, 1971), pp. 357-404.
11. I. Garcia, et al., Biochem. J. 325, 761 (1997).
12. S. R. Norris, X. Shen, D. DellaPenna, Plant Physiol. 117, 1317 (1998).
13. G. R. Whistance and D. R. Threlfall, Biochem. J. 117, 593 (1970) .
14. S. R. Norris, T. R. Barrette, D. Dellapenna, Plant Cell 7, 2139 (1995).
15. T. Kaneko, et al., DNA Res. 3, 109 (1996) .
16. R. Kagan and S. Clarke, Arch. Biochem. Biophys. 310, 417 (1993) .
17. K. Nakai and M. Kanehisa, Proteins Struct. Funct. Genet. 11, 95 (1991)
18. A. D'Harlingue and B. Camara, J. Biol. Chem. 260, 15200 (1985) .
19. Synechocystis genomic DNA was purified as described (20). The SLR0089 gene was amplified from genomic DNA by polymerase chain reaction (PCR) using Taq polymerase (Gibco-BRL, Grand Island, NY) and primers SLR0089F (5'-ACGGATCCAAAAATGCCTATGGTTCATCATCGGGG-3') and SLR0089R (5'-GGGGATCCTGTGGACTTCAAACTAAAGGCTTTATC-3'). The SLR0089 PCR fragment was subcloned into pBluescript KS II (Stratagene), generating plasmid pSLR0089. The aminoglycoside 3 162-phosphotransferase gene from pUC4K [ L. A. Taylor and R. E. Rose, Nucleic Acids Res. 16, 358 (1988) was cloned into a unique Nco I site in pSLR0089, generating plasmid pSLR0089:Kan.
20. J. Williams, Methods Enzymol. 167, 766 (1988) .
21. Tocopherols from weighed Synechocystis cells grown on BG-11 agar medium [M. M. Allen, J. Phycol. 4, 1, (1968)] and Arabidopsis seeds were extracted [ E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959)] and analyzed on a LiChrosorb Si60A 4.6 × 250 mm HPLC column [ E. L. Syvaoja , et al., J. Am. Oil Chem. Soc. 63, 328 (1986)].
22. The amplified SLR0089 gene was subcloned into pET3D (Novagen, Madison, WI), generating plasmid pSyn.-TMT. Protein expression was induced as described [ F.W. Studier, et al., Methods Enzymol. 185, 60 (1990)], and extracts from induced pSyn.-TMT and pET3D empty vector control cells were prepared by homogenizing cell pellets in 10 mM Hepes (pH 7.8), 5 mM dithiothreitol, 0.24 M sorbitol, and 1 mM phenylmethylsulfonyl fluoride. Triton X-100 was added to a final concentration of 1%, and the homogenates were incubated on ice for 30 min and centrifuged at 30,000g for 30 min at 4[1/4]C, after which the protein concentration of each supernate was determined [ M. Bradford, Anal. Biochem. 72, 248 (1976)].
23. T. Newmann, et al., Plant Physiol. 106, 1241 (1994).
24. K. Nakai and M. Kanehisa, Genomics 14, 897 (1992).
25. J. Soll, G. Schultz, J. Joyard, R. Douce, M. A. Block, Arch. Biochem. Biophys. 238, 290 (1985) .
26. The mature 165H5T7 DNA sequence was amplified from the original 165H5T7 cDNA by PCR using Pfu polymerase (Stratagene), 165matF (5'-CCATGGCTGTGGCGGCTGCTGCTAC-3'), and 165matR (5'-GTCGACGCATGCACGCGTACGTAA-3'). The amplified sequence was subcloned into pET3D (Novagen, Madison, WI), generating plasmid pA.t.-TMT. Protein expression was induced and assayed as described (18, 22).
27. Substrate specificity assays were performed as described (18), except that 5 mM of the appropriate tocopherol substrate was substituted for -tocopherol. Radiolabeled reaction products were then collected after normal phase HPLC fractionation (21), and radiolabel incorporation was determined by scintillation counting.
28. N. Bechtold, J. Ellis, G. Pelletier, C. R. Acad. Sci. Paris 1144, 204 (1993) .
29. The seed-specific plant gene expression plasmid pDC3 was constructed from a derivative of the Agrobacterium tumefaciens plant transformation vector pBIB-Hyg [ D. Becker, Nucleic Acids Res. 18, 203 (1990) ] and the DC3 promoter (30). 165H5T7 cDNA was subcloned in the sense orientation into pDC3, generating pDC3-A.t.-TMT.
30. W. Seffens, C. Almoguera, H. Wilde, R. Vonder Haar, T. Thomas, Dev. Genet. 11, 65 (1990) .
31. We thank K. Osteryoung, D. Schooley, C. Condit, C. Pristos, C. Howard, and members of the DellaPenna lab for their comments on this manuscript; H. Wada and D. Vermas for their advice in culturing Synechocystsis PCC6803; T. L. Thomas for the DC3 promoter; and H. Banno at the Eisai Company for providing the tocol internal standard. This work was supported in part by U.S. Department of Agriculture NRICGP grant 98-01445.