Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.
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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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. · 0.94 Impact Factor
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ABSTRACT: While a meta-analysis is necessary to test the claim that the logic dominates the majority of studies, most studies by academic scholars on thinking and actions by executives appear to rely on cross-sectional surveys that use self-reports by executives via scaled (e.g., strongly disagree to strongly agree) instruments whereby one executive per firm completes the instrument and data are collected for 50–500 firms. Useable response rates in these studies are almost always below 30% of the distributions of the surveys. While these studies are sometimes worthwhile for learning how respondents assess concepts and relationships among concepts, Rong and Wilkinson’s perspective on the severe limits to the value of such studies rings true: such surveys reveal more about executives’ sensemaking processes than the actual processes. The limitations of using one-shot, one-person-per-firm, self-reports as valid indicators of causal relationships of actual processes are so severe that academics should do more than think twice before using such surveys as the main method for collecting data – if scholars seek to understand and describe actual processes additional methods are necessary for data collection. The relevant literature includes several gems of exceptionally high quality, validity, and usefulness in the study of actual processes; identifying these studies is a useful step toward reducing the reliance on one-shot self-report surveys.Australasian Marketing Journal (AMJ) 01/2011; 19(3):153-156.
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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; · 3.69 Impact Factor
Enhancing Lignan Biosynthesis by Over-expressing Pinoresinol Lariciresinol Reductase in
Allan K. Ayella1, Harold N. Trick2 and Weiqun Wang1
1 Department of Human Nutrition, Kansas State University, Manhattan, KS 66506, USA
2 Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA
Correspondence: Dr. Weiqun Wang, Department of Human Nutrition, Kansas State University,
Manhattan, KS 66506, USA
Abbreviations: HPLC, high performance liquid chromatography; MS, mass spectrum; PCR,
polymerase chain reaction; PLR, pinoresinol lariciresinol reductase; SDG, secoisolariciresinol
diglucoside; Ubi, ubiquitin.
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 APCPMin
P 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 ~17% increase of PLR
gene expression over the control in 2 of the 3 positive transformants at TB0
B 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 TB2
B than that
in the wild-type (117.9 ± 4.5 vs. 52.9 ± 19.8 µg/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.
Keywords: Lignans / Secoisolariciresinol Diglucoside / Pinoresinol Lariciresinol Reductase /
Transgenic Wheat / Cancer Prevention
Lignans are phenylpropane dimers linked by β-β bonds with a 1,4-diarylbutane structure [1, 2].
They occur naturally in a number of plant families, including the gramineae and oleaceae which
contain the monocots and eudicots, respectively [3, 4]. In monocots such as wheat, lignans are
mostly located in the aleurone layer of seeds , and in eudicots such as forsthysia, lignans occur
in the fruits and stems .
The main lignan in wheat bran is secoisolariciresinol diglucoside (SDG). When
consumed, SDG is oxidized by intestinal microflora to lignan metabolites, e.g., enterodiol and
enterolactone. The biological importance of lignans and lignan metabolites has been previously
reviewed [7-9]. Epidemiological studies show an inverse association between dietary intake of
lignans and the risk of cardiovascular disease [10, 11]. Lignans also have potential protective
roles against cancer in breast , prostate , and colon [14, 15]. A study done in rats showed
that exposure of 10% flaxseed (SDG-rich plant seeds) or the equivalent SDG levels during
suckling suppressed chemical carcinogen 7, 12-dimethylbenz(α)anthracene (DMBA)-induced
mammary tumorigenesis . In addition, in vitro cell culture studies demonstrated that
enterolactone and/or enterodiol reduce growth and metastasis of breast cancer cells .
Furthermore, lignan metabolites have been shown to reduce cell growth in human colon cancer
SW480 cells . It is interesting that the contents of lignans in wheat bran from various
cultivars are correlated with anti-tumorigenesis in spontaneous Min mice with mutant
adenomatous polyposis coli (APCPMin
P) [18-19]. Lignans are abundant in flaxseed but not quite in
wheat grains that usually contain about 4-50 µg/g . Enhancement of the SDG biosynthesis in
wheat plants, therefore, appears to be significant for cancer prevention.
Genetic engineering is one of the ways for genetic crop manipulation in order to enhance
phytochemical synthesis, which has already been shown in many cases to improve agronomic
and nutritional aspects of crop plants [20, 21]. The biosynthetic pathways to SDG occur via
coupling of two coniferyl alcohol molecules to afford pinoresinol (Figure 1). Then pinoresinol
undergoes sequential reduction by pinoresinol-lariciresinol reductase (PLR) to generate
lariciresinol and secoisolariciresinol [3, 22]. Since PLR catalyzes the last steps of the lignan
biosynthesis, it is postulated that over-expression of PLR gene by genetic engineering may
enhance lignan contents. Although PLR gene had already been isolated from various woody
plants such as Forsthysia intermedia [3, 23-24], the only known isolated PLR enzyme in
monocots has been found from flaxseeds . In wheat, however, the PLR gene and
corresponding protein(s) have not been reported yet.
The purpose of this study is to enhance SDG biosynthesis in transgenic wheat by genetic
transformation of Forsthysia intermedia PLR gene. To the best of our knowledge, this is the first
study trying to apply genetic engineering wheat for enhancement of lignan biosynthesis.
2 Materials and Methods
2.1 DNA constructs
PLR cDNA (1.2 kb, GenBank accession number U81158) encoding (+)pinoresinol-
(+)lariciresinol reductase in Forthysia intermedia was kindly provided by Dr. Norman Lewis at
Washington State University (Pullman, WA). Forsthysia PLR cDNA was initially cloned into
pGEM® T Easy vector (Promega, Madison, WI). During amplification of PLR by PCR, BglI
sites were appended to the 5’ and 3’ ends, respectively. The PCR product for PLR gene was then
obtained following digestion by restricted enzyme BglI. The sequence of the Forsthysia PLR
gene was confirmed at the Gene Sequencing Facility, Department of Plant Pathology, Kansas
State University (Manhattan, KS). The PLR gene was then inserted into BamHI site (compatible
ends with BglI) in pAHC17 plasmid under the control of the maize ubiquitin (Ubi) promoter (2.1
kb) as described by Christensen and Quail . Restriction digestion with PstI, EcoRI, and
BamHI, respectively, were used to confirm the correct directional insertions (data not shown).
The PCR products by both primer sets (PLR F & R and Ubi-PLR F & R as denoted in Table 1
and illustrated in Figure 2) were further confirmed by sequencing at the Gene Sequencing
Facility, Department of Plant Pathology, Kansas State University (Manhattan, KS). The new
constructed plasmid designated as pAHCUbi-PLR contains the Ubi promoter, opening reading
frame from the Forsthysia cDNA encoding PLR, and nopaline synthase (nos) terminator region
(Figure 2). In addition, plasmid pAHC20 contains the bar gene (2.0 kb) under the control of the
maize ubiquitin promoter-intron . The bar gene confers resistance to the herbicide
glufosinate (Liberty®, Aventis, Research Triangle Park, NC). Both pAHCUbi-PLR and pAHC20
plasmids were used for wheat co-transformation.
2.2 Transformation procedure
Both pAHCUbi-PLR and pAHC20 plasmids were co-bombarded into embryogenic calli of
wheat plants (Triticum aestivum L. cv. ‘Bobwhite’, ‘Madison’, and ‘Fielder’, respectively). The
method of co-transformation and selection of transgenic events have been described by Anand et
al . Briefly, the premature seeds were surface sterilized with 20% sodium hypochlorite and
0.02% TWEEN-20. Immature embryos were then aseptically excised on CM4 medium to initiate
somatic embryo formation. Somatic embryos that were proliferated in CM4+ osmoticum (0.2 M
mannitol, 0.2 M sorbitol) were co-bombarded with pAHC20 and pAHCUbi-PLR plasmids at 1:1
ratio by using the particle inflow gun.