Abstract
Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.
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Abbreviations
- DMGGBQ:
-
2,3-dimethyl-5-geranylgeranyl-1,4-benzoquinol
- DMPBQ:
-
2,3-dimethyl-5-phytyl-1,4- benzoquinol
- DMSBQ:
-
2,3-dimethyl-5-solanesyl-1,4-benzoquinol
- HPPD:
-
4-hydroxyphenylpyruvate dioxygenase
- HGGT:
-
homogentisate geranylgeranyltransferase
- HPT:
-
homogentisate phytyltransferase
- HST:
-
homogentisate solanesyl transferase
- MPBQ:
-
2-methyl-6-phytyl-1,4-benzoquinol
- MGGBQ:
-
2-methyl-6-geranylgeranyl-1,4-benzoquinol
- MSBQ:
-
2-methyl-6-solanesyl-1,4-benzoquinol
- MT1:
-
MPBQ/MGGBQ/MSBQ methyltransferase
- MT2:
-
γ-tocopherol methyltransferase
- P-8:
-
plastochromanol-8
- TC:
-
tocopherol cyclase
References
Arango Y, Heise KP (1998) Tocopherol synthesis from homogentisate in Capsicum annuum L. (yellow pepper) chromoplast membranes: evidence for tocopherol cyclase. Biochem J 336:531–533
Azzi A, Ricciarelli R, Zingg JM (2002) Non-antioxidant molecular functions of alpha-tocopherol (vitamin E). FEBS Lett 519:8–10
Balz M, Schulte E, Thier HP (1992) Trennung von Tocopherolen und Tocotrienolen durch HPLC. Fat Sci Technol 94:209–213
Borowski T, Bassan A, Siegbahn PE (2004) 4-Hydroxyphenylpyruvate dioxygenase: a hybrid density functional study of the catalytic reaction mechanism. Biochemistry 43:12331–12342
Chan SS, Monteiro HP, Schindler F, Stern A, Junqueira VBC (2001) Alpha-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases. Free Radic Res 35:843–856
Collakova E, DellaPenna D (2001) Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol 127:1113–1124
Collakova E, DellaPenna D (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol 131:632–642
Dahnhardt D, Falk J, Appel J, van der Kooij TA, Schulz-Friedrich R, Krupinska K (2002) The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis. FEBS Lett 523:177–181
Denoya CD, Skinner DD, Morgenstern MR (1994) A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli. J Bacteriol 176:5312–5319
Falk J, Andersen G, Kernebeck B, Krupinska K (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves. FEBS Lett 540:35–40
Falk J, Brosch M, Schäfer A, Braun S, Krupinska K (2005) Characterization of transplastomic tobacco plants with a plastid localized barley 4-hydroxyphenyl-pyruvate dioxygenase. J Plant Physiol 162:738–742
Fritze IM, Linden L, Freigang J, Auerbach G, Huber R, Steinbacher S (2004) The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase. Plant Physiol 134:1388–1400
Fuqua WC, Coyne VE, Stein DC, Lin CM, Weiner RM (1991) Characterization of melA: a gene encoding melanin biosynthesis from the marine bacterium Shewanella colwelliana. Gene 109:131–136
Garcia I, Rodgers M, Pepin R, Hsieh TF, Matringe M (1999) Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco. Plant Physiol 119:1507–1516
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994
Hofius D, Sonnewald U (2003) Vitamin E biosynthesis: biochemistry meets cell biology. Trends Plant Sci 8:6–8
Hummel R, Norgaard P, Andreasen PH, Neve S, Skjodt K, Tornehave D, Kristiansen K (1992) Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles. J Mol Biol 228:850–861
Ischebeck T, Zbierzak AM, Kanwischer M, Dörmann P (2006) A salvage pathway for phytol metabolism in Arabidopsis. J Biol Chem 281:2470–2477
Karunanandaa B, Qi Q, Hao M, Baszis S, Jensen P, Wong YHH, Jiang J, Venkatramesh M, Gruys KJ, Moshiri F, Post-Beittenmiller D, Weiss JD, Valentin HE (2005) Metabolically engineered oilseed crops with enhanced seed tocopherol. Met Eng 7:384–400
Kleber-Janke T, Krupinska K (1997) Isolation of cDNA clones for genes showing enhanced expression in barley leaves during dark-induced senescence as well as during senescence under field conditions. Planta 203:332–340
Kumar R, Raclaru M, Schüsseler T, Gruber J, Sadre R, Lühs W, Zarhloul KM, Friedt W, Enders D, Frentzen M, Weier D (2005) Characterisation of plant tocopherol cyclases and their overexpression in transgenic Brassica napus seeds. FEBS Lett 579:1357–1364
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lenne C, Matringe M, Roland A, Sailland A, Pallett K, Douce R (1995) Partial purification and localization of p-hydroxyphenylpyruvate dioxygenase activity from cultured carrot cells. In: Mathis P (eds) Photosynthesis: from light to biosphere. Kluwer Academic Publishers, Dordrecht The Netherlands, pp␣285–288
Lichtenthaler HK (1998) The plants’ 1-deoxy-d-xylulose-5-phosphate pathway for biosynthesis of isoprenoids. Fett Lipid 100:128–138
Lichtenthaler HK, Prenzel U, Douce R, Joyard J (1981) Localization of prenylquinones in the envelope of spinach chloroplasts. Biochim Biophys Acta 641:99–105
Marshall PS, Morris SR, Threlfall DR (1985) Biosynthesis of tocopherols: a re-examination of the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol. Phytochemistry 24:1705–1711
Marwede V, Schierholt A, Möllers C, Becker HC (2004) Genotype × environment interactions and heritability of tocopherol contents in Canola. Crop Sci 44:728–731
Matringe M, Sailland A, Pelissier B, Rolland A, Zink O (2005) p-Hydroxyphenylpyruvate dioxygenase inhibitor-resistant plants. Pest Manag Sci 61:269–276
Menhaj AR, Mishra SK, Bezhani S, Kloppstech K (1999) Posttranscriptional control in the expression of the genes coding for high-light-regulated HL#2 proteins. Planta 209:406–413
Munné-Bosch S, Falk J (2004) New insights into the function of tocopherols in plants. Planta 218:323–326
Olejnik D, Gogolewski M, Nogala-Kalucka M (1997) Isolation and some properties of plastochromanol-8. Nahrung 41:101–104
Padidam M, Cao Y (2001) Elimination of transcriptional interference between tandem genes in plant cells. Biotechniques 31:328–334
Porfirova S, Bergmüller E, Tropf S, Lemke R, Dörmann P (2002) Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis. Proc Natl Acad Sci USA 99:12495–12500
Quinn PJ (2004) Is the distribution of α-tocopherol in membranes consistent with its putative functions? Biochemistry 69:74–84
Rippert P, Scimemi C, Dubald M, Matringe M (2004) Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance. Plant Physiol 134:92–100
Rippka R (1988) Isolation and purification of cyanobacteria. Methods Enzymol 167:3–27
Rodriguez-Concepcion M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089
Rohmer M (1998) Isoprenoid biosynthesis via the mevalonate-independent route, a novel target for antibacterial drugs? Prog Drug Res 50:135–154
Ruetschi U, Odelhog B, Lindstedt S, Barros-Soderling J, Persson B, Jornvall H (1992) Characterization of 4-hydroxyphenylpyruvate dioxygenase. Primary structure of the Pseudomonas enzyme. Eur J Biochem 205:459–466
Ruetschi U, Rymo L, Lindstedt S (1997) Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD). Genomics 44:292–299
Sadre R, Paus H, Frentzen M, Weier D (2003) Characterisation of enzymes involved in tocopherol biosynthesis. In: Murata N (ed) Advanced research on plant lipids. Kluwer Academic Publishers, Dordrecht, pp 253–256
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press
Sattler SE, Cheng Z, DellaPenna D (2004) From Arabidopsis to agriculture: engineering improved vitamin E content in soybean. Trends Plant Sci 9:365–367
Savidge B, Weiss JD, Wong YH, Lassner MW, Mitsky TA, Shewmaker CK, Post-Beittenmiller D, Valentin HE (2002) Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol 129:321–332
Schledz M, Seidler A, Beyer P, Neuhaus G (2001) A novel phytyltransferase from Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis. FEBS Lett 499:15–20
Schneider C (2005) Chemistry and biology of vitamin E. Mol Nutr Food Res 49:7–30
Schulz A, Ort O, Beyer P, Kleinig H (1993) SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase. FEBS Lett 318:162–166
Shintani D, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2100
Soll J (1987) α-Tocopherol and plastoquinone synthesis in chloroplast membranes. Methods Enzymol 148:383–392
Soll J, Schultz G, Joyard J, Douce R, Block MA (1985) Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch Biochem Biophys 238:290–299
Spiekermann P (2005) Isolierung und Charakterisierung von Genkopien der mikrosomalen Oleatdesaturase FAD2 aus Brassica napus L. sowie Entwicklung transgener Rapspflanzen mit erhöhtem ölsäuregehalt im Samen. PhD thesis, Hamburg University
Thies W (1997) Quantitative Bestimmung der Tocopherole durch HPLC. Angew Bot 71:62–67
Threlfall DR, Whistance GR (1971) Biosynthesis of isoprenoid quinones and chromanols. In: Goodwin T (ed) Aspects of terpenoid chemistry and biochemistry. Academic Press, Liverpool UK, pp 357–404
Traber MG, Sies H (1996) Vitamin E in humans: demand and delivery. Annu Rev Nutr 16:321–347
Tsegaye J, Shintani D, della Penna D (2002) Overexpression of the enzyme p-hydroxyphenylpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis. Plant Physiol Biochem 40: 913–920
Valentin HE, Qi Q (2005) Biotechnological production and application of vitamin E: current state and prospects. Appl Microbiol Biotechnol 68:436–444
Wyckoff EE, Pishko EJ, Kirkland TN, Cole GT (1995) Cloning and expression of a gene encoding a T-cell reactive protein from Coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen. Gene 161:107–111
Yamauchi J, Iwamoto T, Kida S, Masushige S, Yamada K, Esashi T (2001) Tocopherol-associated protein is a ligand-dependent transcriptional activator. Biochem Biophys Res Commun 285:295–299
Zarhloul MK, Friedt W, Khoschkhoi Yazdi MR, Lühs W (1999) Genetic transformation and shoot regeneration ability of resynthesised Brassica napus line ‘RS 306’. Cruciferae Newsl 21:59–60
Acknowledgements
The authors thank Christof Stoll for performing statistical analysis and Ingrid Klawiter, Marion Heitmann, Petra Degen, Gabi Ciobanu, Ingrid Frantz, Violeta Cadar, Sonja Sahner and Stavros Tzigos for technical assistance.
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Mirela Raclaru and Jens Gruber contributed equally
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Raclaru, M., Gruber, J., Kumar, R. et al. Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis. Mol Breeding 18, 93–107 (2006). https://doi.org/10.1007/s11032-006-9014-5
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DOI: https://doi.org/10.1007/s11032-006-9014-5