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Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.

Department of Human Nutrition, Kansas State University, Manhattan, KS 66506, USA.
Molecular Nutrition & Food Research (Impact Factor: 4.91). 12/2007; 51(12):1518-26. DOI: 10.1002/mnfr.200700233
Source: PubMed

ABSTRACT Lignans are phenylpropane dimers that are biosynthesized via the phenylpropanoid pathway, in which pinoresinol lariciresinol reductase (PLR) catalyzes the last steps of lignan production. Our previous studies demonstrated that the contents of lignans in various wheat cultivars were significantly associated with anti-tumor activities in APC(Min) mice. To enhance lignan biosynthesis, this study was conducted to transform wheat cultivars ('Bobwhite', 'Madison', and 'Fielder', respectively) with the Forsythia intermedia PLR gene under the regulatory control of maize ubiquitin promoter. Of 24 putative transgenic wheat lines, we successfully obtained 3 transformants with the inserted ubiquitin-PLR gene as screened by PCR. Southern blot analysis further demonstrated that different copies of the PLR gene up to 5 were carried out in their genomes. Furthermore, a real-time PCR indicated approximately 17% increase of PLR gene expression over the control in 2 of the 3 positive transformants at T(0) generation. The levels of secoisolariciresinol diglucoside, a prominent lignan in wheat as determined by HPLC-MS, were found to be 2.2-times higher in one of the three positive transgenic sub-lines at T(2 )than that in the wild-type (117.9 +/- 4.5 vs. 52.9 +/- 19.8 mug/g, p <0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.

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    • "Overproduction of downstream lignans through accelerated reduction of Pin and Lar has also been challenged in the other plant species. Ayella et al. (2007) reported that overexpression of a PLR isolated from F. intermedia contributed to the accumulation of SDG in transgenic wheat seeds; the average level of SDG in transgenic seeds (117.9 ng/mg) was slightly more than 2-fold higher than that in wild-type seeds (52.9 ng/mg). In addition, increased (+)-Sec content was achieved by chemically induced overexpression of an endogenous gene for PLR (LuPLR1) in flax seeds (Corbin et al 2013), which led to 2.5-fold increase in (+)-Sec concentrations in dried seeds (23.9 μg/mg). "
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    ABSTRACT: Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4'-feruloyl ethers. In-depth quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.
    Applied Microbiology and Biotechnology 07/2014; DOI:10.1007/s00253-014-5934-x · 3.81 Impact Factor
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    • "of exogenous compounds with improved pharmacological properties both in plants and in cell cultures (Allen et al. 2004; Ayella et al. 2007; Badejo et al. 2009; Butelli et al. 2008; Leonard et al. 2009; Ogita et al. 2003). In the previous study, we stably introduced CYP81Q1 and an RNA interference (RNAi) sequence against an Fk endogenous lignan-biosynthetic enzyme, pinoresinol/lariciresinol reductase (PLR), into Forsythia koreana (Fk) cultured cells (Kim et al. 2009). "
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    ABSTRACT: In a previous study, we reported the production of the exogenous lignan, sesamin, using the Forsythia koreana transgenic cells (CPi-Fk cells) in which an exogenous sesamin-synthase CYP81Q1 is stably expressed while an endogenous pinoresinol-lariciresinol reductase is suppressed by RNA interference. Here, we present the effects of light on the production of sesamin and an endogenous lignan pinoresinol which is a precursor of sesamin in CPi-Fk cells. CPi-Fk cells showed a 2.3-fold, 2.7-fold, or 1.6-fold increase in sesamin production after two-week irradiation with white fluorescent, blue LED, or red LED light, respectively, compared with the level obtained under the dark condition. Likewise, CPi-Fk cells showed an approximately 1.5 to 3.0-fold increase in pinoresinol (aglycone and glucosides) production. Furthermore, expression of the pinoresinol-glucosylating enzyme UGT71A18 was suppressed in CPi-Fk cells under blue or red light. Considering that white fluorescent light contains the blue wavelength and that CYP81Q1 fails to convert pinoresinol glucosides to sesamin, it is concluded that blue light plays a major role in the up-regulation of the production of sesamin by CPi-Fk via an enhancement of the production of pinoresinol aglycone and a reduction of UGT71A18. This is the first report on the elevation of lignan biosynthesis by light.
    Plant Biotechnology 01/2011; 28:331-337. DOI:10.5511/plantbiotechnology.11.0420a · 1.06 Impact Factor
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    ABSTRACT: Lignans are a large class of secondary metabolites in plants, with numerous biological effects in mammals, including antitumor and antioxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable with that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.
    Plant and Cell Physiology 11/2009; 50(12):2200-9. DOI:10.1093/pcp/pcp156 · 4.98 Impact Factor
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