ABSTRACT
Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol soybean with transgenic high alpha-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-fold higher than the average WT soybean seed vitamin E activity.
Subject(s)
Genetic Enhancement/methods , Glycine max/genetics , Glycine max/metabolism , Seeds/genetics , Seeds/metabolism , Soybean Oil/metabolism , Tocopherols/metabolism , Gene Expression Regulation, Plant/physiology , Soybean Oil/chemistry , Tocopherols/chemistryABSTRACT
Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis gamma-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of gamma-tocopherol to alpha-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed alpha-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.
Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Seeds/genetics , Seeds/metabolism , alpha-Tocopherol/metabolism , Gene Expression Regulation, Plant/physiology , Gene Targeting/methods , Genetic Enhancement/methods , Genetic Markers , Plants, Genetically Modified/metabolism , Protein Engineering/methods , Recombinant Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/physiology , Zinc Fingers/geneticsABSTRACT
Plant sterols and their hydrogenated forms, stanols, have attracted much attention because of their benefits to human health in reducing serum and LDL cholesterol levels, with vegetable oil processing being their major source in several food products currently sold. The predominant forms of plant sterol end products are sitosterol, stigmasterol, campesterol and brassicasterol (in brassica). In this study, 3-hydroxysteroid oxidase from Streptomyces hygroscopicus was utilized to engineer oilseeds from rapeseed (Brassica napus) and soybean (Glycine max), respectively, to modify the relative amounts of specific sterols to stanols. Each of the major phytosterols had its C-5 double bond selectively reduced to the corresponding phytostanol without affecting other functionalities, such as the C-22 double bond of stigmasterol in soybean seed and of brassicasterol in rapeseed. Additionally, several novel phytostanols were obtained that are not produced by chemical hydrogenation of phytosterols normally present in plants.
