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First published online November 20, 2003; 10.1105/tpc.015875 American Society of Plant Biologists Engineering Vitamin E Content: From Arabidopsis Mutant to Soy Oil
a Monsanto Company, Calgene Campus, Davis, California 95616 5 To whom correspondence should be addressed. E-mail henry.e.valentin{at}monsanto.com; fax 636-737-6429
We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of  -tocopherol and decreased  -tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed -tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (-tocopherol methyltransferase) in soybean, the seed accumulated to >95%  -tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of -tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.
The nutritional value of vitamin E in the human diet was recognized >75 years ago (Evans and Bishop, 1922  ). Vitamin E is made up of a group of structurally related compounds (vitamers), eight major forms of which are known to occur in nature: -,  -, -, and -tocopherol and four corresponding unsaturated derivatives, -, -, -, and -tocotrienol (Figure 1A). The biological activities of these eight naturally occurring tocol isoforms vary according to the number and position of methyl groups on the chroman ring and by the configuration of the asymmetric carbons in the side chain (Table 1) (Sheppard et al., 1993; Bramley et al., 2000). The highest vitamin E activity in animals is seen with trimethylated RRR--tocopherol, which may be attributable in part to the fact that -tocopherol is more readily retained by humans (Traber and Sies, 1996). During the past 20 years, a large body of medical evidence has indicated that daily vitamin E supplementation of 400 IU (250 mg of RRR--tocopherol) results in decreased risk for cardiovascular disease and cancer, aids in immune function, and prevents or slows a number of degenerative diseases (Buring and Hennekens, 1997; Tangney, 1997; Bramley et al., 2000). Tocotrienols also have been found to have hypocholesterolemic properties (Qureshi and Qureshi, 1993; Qureshi et al., 1995), leading to the suggestion that tocotrienols should be considered nutrients independent of the tocopherols (Hendrich et al., 1994).
Tocopherols and tocotrienols are thought to have a number of vital functions in plants, including the protection of chloroplasts from photooxidative damage (Munné-Bosch and Alegre, 2002 ). Two recent findings underscore the fact that we do not understand all of the functions of tocopherols in plants. First, the altered plasmodesmata mutant sucrose export defective1 of maize appears to be defective in a tocopherol cyclase ortholog (Porfirova et al., 2002). This mutant has dramatic morphological alterations, presumably caused by altered photoassimilate export (Provencher et al., 2001). By contrast, the Arabidopsis loss-of-function cyclase mutant vitamin E pathway gene1 (vte1) is normal under standard growth conditions and shows only modest changes in photosynthesis and pigmentation under growth conditions that cause photooxidative stress (Porfirova et al., 2002).
Tocopherols are synthesized in photosynthetic microorganisms and plants, with the highest concentrations being found in seeds. The seeds of most plants have significant amounts of
Tocopherols are synthesized from precursors derived from two pathways. The methylerythritol phosphate pathway contributes to the side chain of tocopherol through the synthesis of phytyldiphosphate (Figure 1B) (Bramley et al., 2000
Tocopherol biosynthesis occurs in the plastids of higher plants, and the biosynthetic enzymes are associated with the chloroplast envelope. It has proven difficult to isolate and purify the membrane-bound proteins of the tocopherol biosynthetic pathway using protein purification techniques (Soll et al., 1980
A screen was used to identify mutants with altered seed tocopherols in Arabidopsis. We hypothesized that mutants with increased
Isolation of High -Tocopherol Arabidopsis Lines in a Mutant ScreenNormally, the tocopherol composition in Arabidopsis seed is predominately -tocopherol, with small amounts of -tocopherol, as shown in Figure 2A. A screen was designed to detect mutants with altered tocopherol seed composition. Tocopherols were extracted and analyzed from the M3 seed of each of the  8000 plant lines. Individual plant lines with altered tocopherol composition were selected as putative mutants. To both confirm the mutant phenotype and attempt to isolate true breeding lines, tocopherol levels were analyzed in M4 seeds. We confirmed 18 high (hd) tocopherol mutants. An HPLC trace of the representative mutant hd2 is shown in Figure 2B, and relative seed - and -tocopherol levels for five of the hd mutants are shown in Figure 2C. The total tocopherol accumulation in these mutants was not substantially different from wild-type levels; rather, the ratio of -tocopherol to -tocopherol was shifted. As a result of their heavy mutational load, any nonseed composition phenotypes noted could not be attributed definitively to the mutation responsible for the tocopherol phenotype. However, it was noted that there were no consistent phenotypes other than increased seed -tocopherol observed among the 18 hd mutants isolated.
Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase–Deficient High -Tocopherol MutantsThe recessive hd2 mutant was chosen for map-based cloning based on its strong high -tocopherol phenotype. This M4 line, Landsberg erecta ecotype, was outcrossed to the Columbia-0 wild type, segregating F2 populations were generated, F3 seeds were harvested from 384 individual F2 lines, tocopherol levels were measured in the F3 seeds, and 101 lines with a mutant phenotype were identified. Eighty-eight homozygous mutant lines were selected for genotyping with 25 molecular markers throughout the genome (Jander et al., 2002).
The mutation responsible for the hd2 phenotype showed linkage to a marker on BAC F28P10 at position 21,630 kb on chromosome III (Figure 3, marker d). The location of the mutation was narrowed further to between a marker on BAC T12C14 (Figure 3B, marker h) and the end of chromosome III, defining a region of five sequenced BACs (Figure 3B). Review of the existing GenBank annotations for this region revealed no likely candidate genes. By contrast, a search of Pfam Hidden Markov models (Bateman et al., 2002
Transgenic Restoration of Normal -Tocopherol Levels in hd2To confirm that mutation of the wild-type At-VTE3 was responsible for the hd2 phenotype, we transformed the mutant with the wild-type gene under the control of the seed-specific napin promoter, as shown in Figure 4A. As a control, transgenic plants also were produced with the Anabaena-VTE3 (Anab-VTE3) gene fused to the N-terminal CTP1 chloroplast-targeting sequence and placed under the control of the same napin promoter. The results of tocopherol analysis of T2 segregating seeds are shown in Figure 4B. There was a substantial decrease in -tocopherol and an accompanying increase in -tocopherol in the seeds from these transgenic plants, as expected if At-VTE3 complemented the mutation in hd2. The chloroplast targeted Anab-VTE3 gave a similar phenotype. Together, the genetic mapping, sequencing of five independently isolated mutant alleles, and genetic complementation provide extremely strong evidence that mutation of At-VTE3 causes a high -tocopherol phenotype.
Demonstration That At-VTE3 Has 2-Methyl-6-Phytylbenzoquinol Methyltransferase Activity in Escherichia coli Because of the sequence similarity of At-VTE3 to ubiquinone methyltransferase and the accumulation of -tocopherol in the loss-of-function mutants, we hypothesized that At-VTE3 encodes a plant MPBQ methyltransferase (Figure 1B). This hypothesis was tested by expressing the mature protein from both wild-type Arabidopsis and the hd2 mutant in E. coli (Figure 5A) and determining whether the recombinant proteins could methylate MPBQ to produce 2,3-dimethyl-5-phytylbenzoquinol (Figure 5B). Consistent with our hypothesis, activity was seen in both the Anab-VTE3 and At-VTE3 E. coli extracts, in addition to a positive control extract from pea chloroplast (Figure 5). By contrast, extracts from E. coli expressing At-vte3-1 mutant protein had greatly decreased activity compared with that of the wild-type At-VTE3 protein. As expected, no activity was seen in E. coli extracts transformed with the empty vector control. These results support the hypothesis that At-VTE3 protein has MPBQ methylation activity in planta. Based on the mutant phenotype, gene sequence, and enzymatic activity, we conclude that At-VTE3 encodes a MPBQ methyltransferase, and we have renamed the hd2, hd6, hd9, hd10, and hd16 alleles At-vte3-1 through At-vte3-5 to reflect this fact.
Demonstration That At-VTE3 Expression Significantly Alters the Tocopherol Composition in Transgenic Soybean To determine if a multigenic approach could convert the majority of tocopherols to -tocopherol in soybean seed, At-VTE3 and At-VTE4 were expressed either independently or simultaneously in transgenic soy seed. Numerous plants were generated, and plants that tested positive for the presence of the CP4 selectable marker were analyzed for tocopherol composition. A representative analysis of pools of ten seed from the first generation is shown for eight representative lines (Table 2). Because this is the first generation, the sample represents a mixture of transgenic and null seeds. Seed populations from plants transformed with the P7S'-At-VTE3 construct showed a dramatic decrease in - and -tocopherol levels in all lines, with a proportionate increase in - and -tocopherol levels. To examine this phenotype further, single seed analysis was performed on the progeny of several transgenic insertion events. The results for event 27930 are shown in Figure 6A. Because this analysis was performed on a population of R1 seeds segregating for the transgene, each individual seed may be a null or may carry one or more copies of the gene. For the eight single seeds tested, one seed was a null and the other seven seed showed the trait from At-VTE3 expression. In the transgenic seeds, the majority of the tocopherol accumulated as -tocopherol (75 to 85%) with increased -tocopherol as well. By contrast, these seeds had very low levels of - and -tocopherol, with these representing only 0.5 to 1.5% of total tocopherols. These data demonstrate that the At-VTE3 MPBQ methyltransferase enzyme converts - to -tocopherol in soybean. Soybean lines transformed with a P7S'-VTE4 expression construct had substantially decreased levels of - and -tocopherol, with a proportional increase in - and -tocopherol, respectively (Table 2). This finding is consistent with results reported previously in Arabidopsis (Shintani and DellaPenna, 1998).
Simultaneous expression of At-VTE3 and At-VTE4 in soybean seeds resulted in a dramatic increase in -tocopherol and decrease in -tocopherol levels (Table 2). This finding is consistent with the hypothesis that the At-VTE4 enzyme efficiently converts -tocopherol produced by At-VTE3 to -tocopherol. One line, 27936, did not fit this pattern and showed the phenotype seen if only At-VTE4 enzyme were produced: an increase in - and -tocopherol with a decrease in - and -tocopherol. Single seed analysis was performed on seeds from several lines transformed with the double gene construct. Analysis of seeds from a representative line (A28096) revealed the presence of two null seeds and six seeds showing the At-VTE3 plus At-VTE4 expression phenotype (Figure 6B). These seeds contained substantial levels of -tocopherol (96 to 98%) and -tocopherol (2 to 4%), whereas - and -tocopherol levels were below the detection limit (<0.2%). Similar results were seen in single seed analysis of other lines (data not shown). Seed-specific expression of At-VTE3 and At-VTE4 as single genes or in combination did not cause a significant effect on total tocopherol levels in the seed (Table 2).
We report the use of Arabidopsis "forward" genetics to identify a gene that encodes a plant MPBQ methyltransferase, At-VTE3, and demonstrate its efficacy in the conversion of - and -tocopherol to - and -tocopherol in transgenic soybean seed. Eighteen mutants with increased levels of seed -tocopherol were identified in a screen of 8000 mutagenized Arabidopsis lines. The range of increase in -tocopherol was 4- to 25-fold (Figure 2C). This change in -tocopherol was accompanied by a reduction of -tocopherol in the seed, resulting in no net change in total tocopherol levels. It seemed likely that this change in composition could result from a mutation affecting the activity of the enzyme responsible for the methylation of MPBQ (Figure 1). A reduction in activity in this enzyme would cause more MPBQ to be converted to -tocopherol via the tocopherol cyclase enzyme, VTE1. Because VTE4 activity is limiting in seeds (Shintani and DellaPenna, 1998), efficient conversion of - to -tocopherol is not expected, and this would result in the accumulation of abnormally large amounts of -tocopherol. We hypothesized three different molecular mechanisms that could lead to this recessive phenotype: a mutation in the methyltransferase enzyme structural gene, reduced synthesis of a cofactor required for the activity of this enzyme, or production of a defective regulator of the enzyme.
The vte3-1 (hd2) mutant was chosen for map-based cloning because it had a 25-fold increase in
Sequencing of the At-VTE3 open reading frame from five independently isolated hd mutants showed unique single base pair changes, all predicted to cause amino acid substitutions. Interestingly, four of the five alleles have mutations in the first exon (Figure 3D), suggesting that this protein region may be crucial for enzyme activity or that mutations in this area are less detrimental. It is noteworthy that none of the five mutations was predicted to create a null allele, such as a splice site change or protein truncation. This finding, coupled with the observation that significant amounts of
Mutations in multiple genes can lead to the high
Enzyme assays and transgenic Arabidopsis and soybean data support the hypothesis that At-VTE3 encodes a tocopherol methyltransferase responsible for methylating MPBQ. The transgenic Arabidopsis data indicate that the expression of At-VTE3 under the control of the napin seed-specific promoter substantially restores the wild-type phenotype to vte3-1 seed (Figure 4B). Interestingly, the napin–At-VTE3 construct further decreased the already low levels of
The inferred At-VTE3 protein-coding region has structural features that are consistent with its identity as an S-adenosylmethionine–dependent methyltransferase, as shown in Figure 7. Three regions of sequence similarity—called motifs I, II, and III—are seen in a broad group of methyltransferases (Kagan and Clarke, 1994
At-VTE3 has a surprisingly low level of sequence similarity ( 35% amino acid identity over an area of 68 amino acids) with the cyanobacterial VTE3 genes (Figure 7), which is why homology searching of full Arabidopsis genomic and EST sequences did not reveal its presence. Furthermore, a transmembrane domain identified by Motohashi et al. (2003) at the C-terminal end of At-VTE3 is located in a C-terminal extension of the Arabidopsis sequence and cannot be defined clearly in the Syn-VTE3 sequence (Figure 7). One possible reason for this difference in sequence homology could relate to substrate specificity. Syn-VTE3 was found to be active on both MPBQ and 2-methylsolanylbenzoquinol (Shintani et al., 2002), suggesting that prenyl side chain length is not a factor in substrate preference for this enzyme. However, characterization of the Arabidopsis apg1 mutant suggests that At-VTE3 is involved in the methylation of 2-methylsolanylbenzoquinol as well (Motohashi et al., 2003). It would be interesting to test the activity of At-VTE3 on 2-methylsolanylbenzoquinol to determine whether the sequence differences between the plant and cyanobacterial VTE3 proteins can be explained partially by a divergence in substrate specificity. It also would be interesting to examine whether either VTE3 can use the geranylgeranyl-substituted quinols that produce tocotrienol, because biochemical assays found that geranylgeranyl-substituted quinols were methylated to a greater extent than phytyl-substituted quinols in spinach chloroplasts (Soll and Schultz, 1979).
The transgenic soy seed data clearly demonstrate that At-VTE3 will be useful for modifying the composition and thus the quality of the tocopherols in plants. The expression of At-VTE3 protein caused a significant decrease in seed
Soybean is not the only crop in which increased VTE3 activity could have a beneficial impact. Several other important oilseed crop species have a relatively high percentage of seed
Plants and Growth Conditions For the mutant screen, Arabidopsis thaliana was grown under continuous illumination at 120 µmol·m-2·s-1 and 21°C in Conviron MTPC432 chambers (Conviron, Winnipeg, Canada). The plants were grown in Metro Mix 200 soil (Scotts, Marysville, OH) in two three-eighth-inch pots at a density of 32 plants per flat, three flats to a group of 96. The three flats of a group were grown side by side at all times. A collection of Columbia and Landsberg erecta ethyl methanesulfonate–treated mutagenized plants was from two sources, Lehle Seeds (Red Rock, TX) and those generated at Cereon Genomics by the method described previously (Barczak et al., 1995). For Agrobacterium tumefaciens–mediated germline transformation, Arabidopsis plants were grown in a 21°C chamber under light at 200 µmol·m-2·s-1 for a 16-h photoperiod. After transformation, T0 plants recovering from transformation were grown at 19°C and T1 plants were grown at 21°C. Transgenic plants were selected by spraying with a 1:200 dilution of Finale (AgrEvo Environmental Health, Montvale, NJ) at both 7 and 14 days after seeding. Transformed hemizygous plants were grown to maturity, and the T2 segregating seeds were harvested for tocopherol analysis.
Tocopherol Analysis
Mutant Isolation and Gene Identification For sequencing of the candidate Arabidopsis gene, PCR amplification was performed with DNA isolated from the mutant or wild-type lines using primer pairs 1 to 11 listed in Table 3. The reaction mixture for PCR was preincubated for 10 min at 94°C. The product then was amplified for 45 cycles of 94°C for 15s, 56°C for 15s, and 72°C for 1.5 min, followed by a 10-min hold at 72°C.
Plant Vector Construction and Transformation The At-VTE3 cDNA was amplified from an Arabidopsis accession Col-0 leaf cDNA library using the Expand High-Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, IN) and primer pair 12 (Table 3). The sequence of Syn-VTE3 was used to identify an Anab-VTE3 coding sequence (Kaneko et al., 2001 ). This Anab-VTE3 sequence was amplified from genomic DNA derived from 3-day-old Anabaena sp (ATCC 27893) cultures. Cultures were spun, and the pellet was washed with 1 mL of PBS to remove the medium. The suspension was centrifuged, and the supernatant was discarded. The pellet was resuspended in 1 mL of water and boiled for 10 min. Anabaena amplification reactions contained 10 µL of boiled Anabaena extract, the Expand High-Fidelity PCR System, and primer pair 13 (Table 3).
After amplification, PCR products were purified using a Qiagen PCR cleanup column and cloned into pDONR201 using the Gateway cloning system (Life Technologies, Rockville, MD). DNA sequences were confirmed and then recloned into a napin-driven cassette derived from pCGN3223 (Kridl et al., 1991
Three soy expression constructs were synthesized: two constructs containing either At-VTE3 or At-VTE4 under the control of the P7S
The At-VTE3 and At-VTE4 expression cassettes were cloned individually and jointly into a plant binary expression vector containing a Pe35S-CP4 gene as a selectable marker (Martinell et al., 2002
Expression of Wild-Type At-VTE3, Mutant At-vte3-1, and Anab-VTE3 Proteins in a Prokaryotic Expression System
The mature At-VTE3 and vte3-1 sequences were amplified by PCR from cDNA using the Expand High-Fidelity PCR System as described previously and primer pair 15 (Table 3). PCR products were cloned into pDONR201 and sequenced and then At-VTE3, vte3-1, and Anab-VTE3 (as described previously) sequences were recombined behind the T7 promoter in the prokaryotic expression vector pET-DEST42 (Life Technologies) using the Gateway cloning system (Figure 5A). Overnight starter cultures of E. coli BL21 (DE3) cells transformed previously with one of these Anab-VTE3, At-VTE3, or vte3-1 prokaryotic expression constructs were inoculated into 100 mL of Luria-Bertani medium with appropriate antibiotics, and the cultures were grown at 25°C to a density of OD600 = 0.6, at which time isopropylthio-
2-Methyl-6-Phytylbenzoquinol (MPBQ) Methyltransferase Enzyme Assay
The reaction mixture was extracted with 4 mL of 2:1 CHCl3/methanol with 1 mg/mL butylated hydroxy toluene and vortexed for 30 s. The organic phase (bottom) was transferred to a fresh 15-mL glass tube after a 5-min low-speed centrifugation. The CHCl3 was evaporated off under a stream of nitrogen gas at 37°C for Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Henry E. Valentin, henry.e.valentin{at}monsanto.com.
Accession Numbers
While this manuscript was in press, a paper appeared describing cloning of the Arabidopsis VTE3 gene and a role for this enzyme in tocopherol and plastoquinone synthesis. The citation is Cheng, Z., Sattler., S., Maeda, H., Sakuragi, Y., Bryant, D.A., and DellaPenna, D. (2003). Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes. Plant Cell 15, 2343–2356.
This work was a team effort between four Monsanto research sites: Davis, California; Madison, Wisconsin; St. Louis, Missouri; and the former Cereon site in Cambridge, Massachusetts. We thank our current and past colleagues at all of these sites, particularly those in plant transformation, the plant growth facilities, the greenhouses, and leadership roles for their expert assistance and support. We also thank our colleagues at Renessen (Bannockburn, IL) for their staunch support and advice. Any views, opinions, or conclusions expressed in this article are those of the author (R.L.L.) and do not represent the official views, opinions, or policy of the National Science Foundation.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015875.
1 These authors contributed equally to this report.
2 Current address: Department of Animal Science, One Shields Avenue, University of California, Davis, CA 95616.
3 Current address: Cambria Biosciences, 8A Henshaw Street, Woburn, MA 01801.
4 Current address: Department of Biochemistry, University of Missouri–Columbia, 117 Schweitzer Hall, Columbia, MO 65211.
6 Current address: Blugoose Consulting, Woodland, CA 95776.
7 Current address: Cantata Pharmaceuticals, 300 Technology Square, 5th Floor Cambridge, MA 02139.
8 Current address: Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, MO 63167.
9 Current address: National Science Foundation, Plant Genome Research Program, 4201 Wilson Boulevard, Room 615, Arlington, VA 22230. Received August 1, 2003; accepted September 22, 2003.
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Wang, Q., and Dubois, P. (2003). Seed specific 7S This article has been cited by other articles:
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