Soybean Power!

1. Soybean Power!

2. Engineering Vitamin E Content: From Arabidopsis Mutant to Soy Oil Van Eenennaam, et al Presented by: Judy, Grant and Jenny March 3, 2005

3. Vitamin E Vitamin E belong to a group called tocopherols Eight major forms occur in nature: • α,β,δ,and γ-tocopherols • α,β,δ,and γ-tocotrienols (unsaturated derivatives) The most potent antioxidant and most useful for humans is the α-tocopherol

4. Vitamin E Difference between α,β,δ,and γ-tocopherols are the location of methylated groups on the R positions Me H H Me H Me Me

5. Vitamin E The main function of Vitamin E is to maintain the integrity of the body’s intracellular membrane by protecting its physical stability and providing a defense line against tissue damage caused by oxidation. Hence antioxidant properties Antioxidative properties of vitamin E health benefits gaining both humans and plants

6. Health benefits Humans: Decreased risk for : • Cardio vascular disease • Cancer • Alzheimer’s disease • Diabetes Plants: Protecting chloroplasts from photooxidative damage However, do not understand all functions of tocopherols in plants

7. Where to Find Vitamin E Tocopherols are synthesized in photosynthetic microorganisms and plants Highest concentration of tocopherols found in seeds. The leafs of plants contain the α-tocopherol The seeds contain mostly γ-tocopherol • One-tenth the vitamin E activity to α-tocopherol

8. Vitamin E intake • 20-30% of Vitamin E intake in US comes from oil based products: • Margarines • Dressings • Mayonnaise

9. Vitamin E intake • 20-30% of Vitamin E intake in US comes from oil based products: • Margarines • Dressings • Mayonnaise • Soybean oil accounts for 70% of the edible oil in US and 30% in the rest of the world. • 100 grams of oil contain 100mg tocopherols • Mostly γ-tocopherols (60-65%) and δ-tocopherols (20-26%).

10. Purpose To manipulate the tocopherol biosynthetic pathway in seeds to convert the less active tocopherols to the beneficial α-tocopherol

11. Tocopherol biosynthesis • Synthesized from precursors of two pathways • Methylerythritol phosphate pathway • Shikimate pathway VTE 1- tocopherolcyclase VTE2 – prenyltransferase VTE3 – methyltransferase VTE4- methyltransferase VTE 3 VTE 1 MPBQ VTE 1 VTE 4 VTE 4

12. Tocopherol biosynthesis • Occurs in plastids of higher plants and the biosynthetic enzymes associated with the chloroplast envelope. • Difficult to isolate and purify the membrane bound proteins using protein purification techniques.

13. Tocopherol biosynthesis • Homology based genomic database searching techniques has lead to identification of several tocopherol biosynthetic genes in cyanobacteria • No apparent orthologs to these genes in higher plants

14. Hypothesis • Mutants with increased accumulation of δ-tocopherol might be defective in the MPBQ methyl transferase

15. Overview Screening and isolation of Arabidopsis mutants containing high δ-tocopherols Identification of MPBQ gene in high δ-tocopherol mutant using map-based cloning Transformed mutant with wild type to confirm that the gene of interest (At-VTE3) was responsible for the mutant phenotype Demonstrated that At-VTE3 has MPBQ activity in E-coli Demonstrated that expression of At-VTE3 alters the tocopherol composition in transgenic soybean

16. Isolation of High δ-Tocopherol Arabidopsis Lines in a Mutant Screen Recall... Seeds normally have high levels of γ-tocopherol ? Need to isolate VTE3 mutant A VTE3 mutant should have abnormal tocopherol composition

17. Isolation of High δ-Tocopherol Arabidopsis Lines in a Mutant Screen The Mutant Screen 1. Extract and analyzed tocopherols from M3 seeds of ~8000 plants using HPLC 2. Plant lines with abnormal tocopherol levels were selected as potential VTE3 mutants 3. Confirm mutant phenotype and isolate true breeding lines by analyzing the tocopherol levels of the M4 seeds

18. Isolation of High δ-Tocopherol Arabidopsis Lines in a Mutant Screen 18 high δ (hd) tocopherol mutants were isolated, including hd2 WT hd2 However, the level of δ-tocopherol is greater so that the total amount of tocopherol in the wild type and mutant plants are the same γ-tocopherol level is high in the WT arabdopsis but lower in the hd2 mutant

19. Isolation of High δ-Tocopherol Arabidopsis Lines in a Mutant Screen 18 high δ (hd) tocopherol mutants were isolated, including hd2 WT hd2 However, the level of δ-tocopherol is greater so that the total amount of tocopherol in the wild type and mutant plants are the same γ-tocopherol level is high in the WT arabdopsis but lower in the hd2 mutant Many mutants had abnormal tocopherol composition, however, hd2 had a significantly increased percentage of δ-tocopherol so it was used for further study

20. Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase-Deficient High δ-Tocopherol Mutants Next, locate At-VTE3 using map-based cloning... Crossed hd2 to Columbia-0 wild type strain and mutant phenotypes in F3 seeds were isolated Homozygous mutant lines were selected for genotyping with 25 molecular markers throughout the genome The mutation was showed linkage to a marker near the end of chromosome III

21. Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase-Deficient High δ-Tocopherol Mutants Next, locate At-VTE3 using map-based cloning... Using markers near the end of chromosome III, the location of the genewas narrowed down further to a region containing five BAC’s

22. Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase-Deficient High δ-Tocopherol Mutants Next, locate At-VTE3 using map-based cloning... A search of sequenced genes in these BAC’s found homology between gene MAA21_40 and a ubiquinone methyltransferase

23. Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase-Deficient High δ-Tocopherol Mutants Next, locate At-VTE3 using map-based cloning... Sequencing of this gene showed that hd2 mutant had a single base pair change causing a Glu to Lys substitution

24. Identification of an Allelic Series of 2-Methyl-6-Phytylbenzoquinol Methyltransferase-Deficient High δ-Tocopherol Mutants Next, locate At-VTE3 using map-based cloning... Four other high δ-tocopherol mutants also showed mutations in this gene For the other 13 mutants, it is assumed that there are other genes involved in regulation of tocopherol levels Therefore, MAA21_40 (henceforth referred to as At-VTE3) is a good candidate gene

25. Transgenic Restoration of Normal δ-Tocopherol Levels in hd2 Need to confirm that At-VTE3 is responsible for hd2 phenotype  Transform mutant with wild-type At-VTE3 gene and look for restoration of wild-type phenotype Used napin promoter, which is specific to seeds

26. Transgenic Restoration of Normal δ-Tocopherol Levels in hd2 Need to confirm that At-VTE3 is responsible for hd2 phenotype  Transform mutant with wild-type At-VTE3 gene and look for restoration of wild-type phenotype As a control, also transformed with CTP1 fused to Anabaena-VTE3 Used napin promoter, which is specific to seeds

27. Transgenic Restoration of Normal δ-Tocopherol Levels in hd2 Significant increase in γ-tocopherol levels in transformed hd2 mutants Controls

28. Demonstration that At-VTE3 Has 2-Methyl-6-Phytylbenzoquinol Methyltransferase Activity in E.coli Hypothesize that At-VTE3 encodes a plant MPBQ methyltransferase Tested this hypothesis by expressing wild-type and mutant proteins in E.coli

29. Demonstration that At-VTE3 Has 2-Methyl-6-Phytylbenzoquinol Methyltransferase Activity in E.coli Positive control Negative control Activity was seen in both Anab-VTE3 and At-VTE3 E.coli extracts while little activity was observed in the mutant, consistent with the hypothesis that At-VTE3 encodes the plant version of MPBQ methylation

30. Demonstration that At-VTE3 Has 2-Methyl-6-Phytylbenzoquinol Methyltransferase Activity in E.coli The authors also suggest that At-VTE3 is an essential gene since no mutants were isolated with complete lack of At-VTE3 activity

31. Effects of At-VTE3, At-VTE4 Expression on Tocopherol Composition Method Used soybean Transformed agrobacteria with vectors containing At-VTE3, At-VTE4, or both  Three total combinations of constructs Other things included in the construct: SSU 3’ UTR from pea CP4 selectable marker Mutagenized soy meristems, grow to maturation, harvest seeds. Analyzed the first generation using 8 transformed lines per construct

32. Was there a change in tocopherol composition? Eight lines selected per construct At-VTE3: α-, γ- up; β-, δ- down At-VTE4: α-, β- up: γ-, δ- down However, the double gene construct seeds shows dramatic increase in α-tocopherol % and decrease in δ-tocopherol % The VTE data that are comparable to WT lines are likely null transgenes

33. Was there a change in tocopherol composition? Analyze eight seeds in each of these two lines. VTE4-like

34. A more in-depth look at tocopherol composition, lines 27930 and 28096 VTE3 VTE3/VTE4 null transgenes typo

35. Why homologs were not found in higher plants BLAST of VTE3 ORF Comparison with cyanobacteria VTE3 genes Only ~35% conserved Four out of five mutations found in first exon first exon is crucial for enzymatic activity Transit peptide C-terminal anchor Figure 7

36. Recent controversy concerning Vitamin E Biochemical: vitamin E protects vitamin A and essential fatty acids from oxidation. (i.e., a good antioxidant) Johns Hopkins study published in Annals of Internal Medicine reported that high vitamin E dosage (400+ IU/day) is associated with a higher risk of dying. Vitamin E deficiency  characterized by nerve degeneration in the extremities. Do we get enough of it? Average American intakes 10 IU/day Supplementation (400 IU/day) has shown to be beneficial

37. Conclusion At-VTE3 encodes the enzyme 2-methyl-6-phytylbenzoquinol methyltransferase, supported by mutations in the gene causing raised levels of δ-tocopherol. This protein, when co-expressed with γ-tocopherol methyltransferase in transgenic soybean seeds, converts 95% of all tocopherols into the α form. Modifying soybean oil (70% of all oil intake in US) can increase vitamin E intake by population.