Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Wheat lignans are phenylpropane dimers linked by β-β bonds with a 1, 4-diarylbutane structure. They are biosynthesized in the cell cytoplasm through action of enzymes of the phenylpropanoid pathway. Pinoresinol lariciresinol reductase (PLR) catalyzes the final steps of biosynthesis of wheat lignans. In epidemiological and clinical investigations, studies show that high plasma lignan amounts correlate with reduced risks of breast, colon, and prostate cancers. However, in some of the studies, the results are not consistent. More consistent results are observed when animal and cell culture models are used. Our previous studies in the Wang lab demonstrated that treatment of human colon cancer cells, SW480 with lignans results in a dose and time dependent inhibition of cancer cell growth. In the first paper, we investigated direct experimental cancer preventative characteristics of a wheat lignan, secoisolariciresinol diglucoside (SDG) vs. its metabolite enterolactone in human colon cancer SW480 cells. Treatment of cancer cells with 0-40 µM SDG or enterolactone resulted into inhibition of cancer cell growth as observed by reduction of cell numbers. The reduction appeared related to induction of S-phase cell cycle arrest rather than cytotoxic effect. Further analysis revealed that SDG was more stable in cell culture medium than enterolactone. HPLC-MS/ESI showed that enterolactone is the principle metabolite in cancer cells but undetectable SDG or its metabolites were in the cells treated with SDG. In the second paper, we investigated over expression of the PLR gene and enhancement of lignan levels in transgenic wheat. We transformed wheat cultivars (‘Bobwhite’, ‘Madison’, and ‘Fielder’ respectively) with the Forsythia intermedia PLR gene under the regulatory control of the maize ubiquitin promoter. Of the total 217 transgenic wheat lines, we successfully obtained 7 transformants with the inserted ubiquitin PLR gene as screened by PCR. Real-time PCR further indicated 109-117% PLR over expression over the transgenic control in 3 transformants of the 7 at T0 generation. In addition, the levels of SDG, as determined by HPLC was found to be significantly elevated in one of the 3 positive transgenic plants. To the best of our knowledge, this is the first study reported that genetically engineered wheat with over expressed PLR enzyme enhancing phytochemical lignan has been successfully achieved.

Plante de grande culture aux multiples applications, le lin accumule dans ses graines des métabolites spécialisés appelés lignanes aux propriétés phytooestrogène et antioxydante, bénéfiques en santé humaine et, dans ses feuilles, des lignanes de nature toxique. Pour l’utilisation et l’étude de ces composés phénoliques issus de la voie des monolignols, une technique d’éco-extraction assistée par les ultrasons a été développée et appliquée à un criblage de différentes variétés pour leur contenu en lignanes conférant une haute valeur ajoutée aux extraits et produits. Les potentiels verrous métaboliques de la voie de biosynthèse de ces composés ont fait l’objet d’investigations au niveau de la régulation transcriptionnelle des gènes responsables de la formation des énantiomères opposés de sécoisolaricirésinol dans la graine et les parties aériennes. La famille multigénique des protéines dirigeantes (DIR) du lin, premiers acteurs de cette voie, a été criblée au niveau génomique et transcriptionnel afin de déterminer des candidates pour fonctionner avec les pinorésinol laricirésinol réductases (PLR), enzymes bifonctionnelles catalysant la formation du sécoisolaricirésinol. L’étude de la régulation fine de l’expression des deux isoformes des gènes PLR a mis en évidence des profils d’inductibilité très contrastés et a conduit à l’identification d’éléments cis-régulateurs ainsi que de facteurs de transcription impliqués dans ces voies de régulation. L’ensemble des résultats convergent vers un rôle de défense des lignanes in planta et a permis de construire une vue d’ensemble des mécanismes complexes de régulation de leur biosynthèse et enfin de proposer des pistes pour l’amélioration de la production de ces molécules naturelles par une stimulation de leur accumulation et l’augmentation des rendements d’extraction par chimie verte. / As a crop with multiple purposes, flax accumulates lignans, specialized metabolites with health benefits, known for their phytoestrogenic and antioxidant properties in its seed, and toxic lignans in its leaves. In order to use and study these phenolic compounds derived from monolignols, an eco-friendly method based on ultrasound-assisted extraction was developed and applied to the screening of cultivars for their lignan content which confers high value to extracts and flaxseed by-product. Regulation of the lignan biosynthetic pathway was investigated at the transcriptional level for the genes responsible for the formation of secoisolariciresinol in seeds and aerial parts. The multigene family of dirigent proteins (DIR), first actors of this pathway, was explored by genomic and transcriptional approaches in order to select candidates to operate with pinoresinol lariciresinol reductases (PLR), bifunctional enzymes catalyzing secoisolariciresinol biosynthesis. The study of the transcriptional regulation of PLR genes evidenced very contrasting expression profiles and led to the identification of transcription factors acting as master regulators of this biosynthesis and their cis-regulatory target elements. Results allowed first to reinforce the hypothesis of the lignans defensive role in planta, second to afford an overall view of complex mechanisms occurring in the regulation of lignan natural production and finally to suggest leads to improve lignan yield by a stimulation of natural production and an enhancement of extraction yield using green chemistry methods.