Oral Session Abstracts

O1 PSNA: SOME REFLECTIONS

Eric E. Conn

Section of Molecular and Cellular Biology, University of California at Davis, Davis, CA 95616-8535, USA; [email protected]

A sabbatical leave in England during the summer of 1960 provided an opportunity to attend a meeting of the Plant Phenolics Group of Europe with Tony Swain and Jeffrey Harborne at Royal Holloway College on the west side of London.This was the first meeting I’d attended that was devoted to this specific group of compounds. I decided that a similar organization in the States would meet a specific need for workers in the field. To my surprise and delight, I found that efforts were already under way, details of which can be found in Dr. Stewart Brown’s 1992 history of the PSNA in Recent Advances in Phytochemistry (Volume 26). Today we can reflect upon the 50^th anniversary of this society and the exciting contributions that it continues to make.

S1.1 NATURAL PRODUCTS WITH BIOLOGICAL ACTIVITIES FROM THAI BIORESOURCES

Chulabhorn Mahidol

Chulabhorn Graduate Institute, Bangkok, 10210, Thailand; [email protected]

Research on natural products is essential for the discovery of lead compounds because of the incredible diversity of chemical structures that are produced by animals, plants, marine, and micro-organisms. It is considered that because of the diversities of the structures as well as the biological activities of their constituents, terrestrial plants, marine and micro-organisms offer a unique and renewable resource for the discovery of potential new drugs and biological entities.

Our interest in research of bioactive compounds from Thai medicinal plants and other sources has been long standing. In this presentation, we will focus on the bioactive compounds derived from various Thai plants, as well as from marine animals and microorganisms. The presentation will include the structure elucidation, chemistry, biological activity and, in some unique cases, the biosynthesis of the compounds isolated from these bioresources.

S1.2 PHYTOCHEMICALS AS SCAFFOLDS FOR NEW DRUG SYNTHESIS AND DISCOVERY

Michael B. Sporn

Department of Pharmacology, Dartmouth Medical School, Hanover NH 03755, USA; [email protected]

Humans have been ingesting phytochemicals for many centuries in order to prevent or treat illness. Even animals engage in this process, and the new science of “zoopharmacognosy” has been developed to study the agents and processes whereby non-humans self-medicate by selecting and eating plant materials to maintain health and avoid or treat disease. With the introduction of the skills of the synthetic organic chemist, we now have the potential to do even better than what exists in nature. Rational new drug design, based on natural phytochemical scaffolds that already are known to have potent and safe pharmacological activity is an important new area of medicinal chemistry. The activity of natural phytochemicals can be increased many hundred-fold by further chemical modification. Mechanistic research based on new knowledge of molecular targets and cellular regulatory networks now can add further validation to this approach.

S1.3 FROM YEW TO ME TO YOU: A PERSONAL HISTORY OF THE DISCOVERY AND DEVELOPMENT OF PLANT-DERIVED ANTICANCER AGENT, TAXOL

Mansukh C. Wani

RTI International, Organic and Medicinal Chemistry, Research Triangle Park, NC 27709,USA; [email protected]

Taxol, a secondary metabolite obtained from the wood bark of Taxus brevifolia, is found in the Pacific Northwest coastal region of the United States. It was isolated by the bioassay-guided fractionation of the crude plant material. The structure of Taxol was established by single crystal x-ray analysis. Taxol has a unique mechanism of antitumor activity. It inhibits cancer cell growth via stabilization of microtubules.

Currently, Taxol is approved for clinical use in the USA by the FDA for the treatment of refractory ovarian, breast, and non-small cell lung cancers and Kaposi’s Sarcoma. This presentation will describe the 30-year efforts which transformed this compound from an interesting plant secondary metabolite to a life-saving chemotherapeutic agent.

S1.4 HALF A CENTURY OF PROANTHOCYANIDIN/POLYPHENOL RESEARCH

Daneel Ferreira, Christina M. Coleman

Research Institute of Pharmaceutical Sciences and Department of Pharmacognosy, University of Mississippi, University, MS 38677,USA; [email protected]

The 5-deoxyproanthocyanidin pools of plants are extremely complex due to variation in oxygenation pattern and a variety of regio-/stereochemical and conformational phenomena. These structural complexities also confound the isolation and structure elucidation processes, especially NMR protocols where ¹H and ¹³C spin systems are often broadened and/or multiplied due to restricted rotation about the interflavanyl bond(s). Our research to understand the intricate structural, configurational, conformational, and chemical behavior began in the 1970s when we designed a synthesis protocol aimed at defining the linkage mode(s) and the absolute configuration of the constituent flavanyl moieties. Some key issues that emanated from our own and other studies, e.g., control of the regio- and stereochemistry of the interflavanyl bond formation process, the development of an electronic circular dichroism method to define the absolute configuration at C-4 of the chain extension unit and corroboration of the results via theoretically calculated ECD spectra, and the chemical manipulation of some crucial bonds in the proanthocyanidin architecture will be discussed.

S2.1 THE ROAD FROM MUNICH: MEINHART H. ZENK’S LEGACY

Norman G. Lewis

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA; [email protected]

Professor Meinhart Zenk is remembered as a true visionary and pioneer in many areas of plant metabolism, largely involving remarkable excursions into phenolic (including phenylpropanoid) metabolism and alkaloid biosynthesis, as well as phytoremediation.

We retrace his remarkable and fascinating scientific journey, taking into account that all scientific contributions must be judged from the time when they were made and the technologies available at the time. Driven by a passion for excellence and perfection, as well as an insatiable thirst for knowledge, his scientific legacy and persona have left an indelible mark. From a personal perspective, some of the pivotal steps and peaks scaled are retraced, and the legacy of the “Zenk School” is highlighted.

S2.2 FUNCTIONING DEPENDENT METABOLONS: THE “OXIME BOMB” IN CYANOGENIC GLUCOSIDE METABOLISM

Birger Lindberg Møller

University of Copenhagen, Faculty of Life Science, Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Frederiksberg, C 1871, Denmark; [email protected]

Cyanogenic glucosides are classical phytoanticipins. Following tissue disruption they release toxic hydrogen cyanide to provide protection of the plant from generalist herbivorous insects. However, numerous fungi are not deterred by hydrogen cyanide. Sorghum contains the cyanogenic glucoside dhurrin. Its synthesis from tyrosine is catalyzed by CYP79A1, CYP71E1 and UGT85B1 with (E)- and (Z)-p-hydroxyphenylacetaldoxime as intermediates. Experimental evidence indicate that the isomerization of the (E) to (Z) oxime is catalyzed by CYP71E1. CYP71E1 is highly labile and sensitive to oxygen. The reactive oxygen species generated at the site of infection as a plant defense response to fungal infection may thus inactivate CYP71E1. As a result, the dhurrin metabolon would dissociate resulting in the formation of an oxime as the final product. Oximes are toxic to fungal pathogens. A cyanogenic glucoside producing plant would thus be able to preferentially combat fungal infection if the metabolon catalyzing cyanogenic glucoside formation dissociated in a functioning dependent manner. This “moonlighting” function and reactivity of the oxime produced may give rise to the formation of a diverse array of conjugation products activating an innate immune response. Experimental studies of oxygen formation as a result of metabolon dissociation will be presented. Detonation of an “oxime bomb” to combat fungal infection would supplement the ability of cyanogenic glucoside containing plants to detonate a “cyanide bomb” when attacked by chewing insects. Other P450 based metabolons may serve similar functioning dependent properties.

S2.3 THE GREAT FOOTPRINTS OF THE LATE PROF. MEINHART ZENK IN ISOQUINOLINE ALKALOID BIOSYNTHESIS STUDIES

Fumihiko Sato

Graduate School of Biostudies, Kyoto University, Department of Plant Gene and Totipotency, Kyoto, 606–8502, Japan; [email protected]

Higher plants are rich sources of medicinal compounds. Many medicinal plants, however, are still harvested in the wild due to technical difficulties of cultivation, as well as for economic reasons. Due to the increased demands for quantities of these materials, production of metabolites by cell cultures has been intensively investigated. Prof. Zenk established many cell cultures of medicinal plants with high secondary metabolite productivity. This allowed him to investigate the respective biosynthetic pathways, and, most notably, isoquinoline alkaloid (IQA) pathways. One of his major contributions in IQA biosynthesis was the revision of the early steps of the reticuline biosynthetic pathway. His intensive biochemical studies further identified many biosynthetic enzymes. There is not enough space to describe them all. I simply note that he clarified the roles of many cytochrome P450s in alkaloid biosynthesis. His achievement is not limited to biosynthesis; Prof. Zenk also proposed the idea of transport and the regulation of gene expression through jasmonate, a key regulator in elicitor responses. These achievements clearly paved the way for the current developments of molecular biology, metabolic engineering, and synthetic biology in secondary metabolism, especially in IQA biosynthesis. I also note that Prof. Zenk’s interests were not restricted to plant metabolism. He identified morphine biosynthesis in human cells, and opened the door of plant natural product studies in human cells. The footprints of the late Prof. Zenk in IQA studies are, indeed, great, and we shall all miss him.

S2.4 BIOSYNTHETIC STUDY OF QUINOLIZIDINE ALKALOID – HISTORY AND RECENT ADVANCEMENT

Kazuki Saito,^1,2 Somnuk Bunsupa,¹ Mami Yamazaki^1,3

¹Chiba University, Graduate School of Pharmaceutical Sciences, Chiba, Japan 263–8522, ²RIKEN, Plant Science Center, Yokohama, Japan 230-0045, ³Japan Science and Technology, CREST, Kawaguchi, 332-0012, Japan; [email protected]

Quinolizidine alkaloid (QA) forms a major group of plant alkaloids that contain several hundred structurally related compounds distributed mostly in Leguminosae. QAs are biosynthesized through cyclization of a cadaverine unit, which is produced through action of lysine decarboxylase. The molecular mechanism underlying QA biosynthesis is poorly understood; only very few studies have been aimed at charactering the enzymes and regulatory mechanism for QA biosynthesis. We have cloned lysine/ornithine decarboxylase (L/ODC) in the first step of their biosynthesis from alkaloid-containing cultivars of Lupinus angustifolius by using PCR-select-subtraction and 5′/3′-RACE techniques. We also characterized L/ODC genes from Sophora flavescens and Echinosophora koreensis which produce QAs. These three purified recombinant L/ODCs showed decarboxylase activity toward two substrates, L-lysine and L-ornithine, with similar kinetic properties. The heterologous expression of L/ODC in Arabidopsis and tobacco resulted in the enhanced accumulation of cadaverine and tobacco alkaloids derived from lysine, indicating the actual function of this enzyme for formation of cadaverine and subsequent production of alkaloids. This is the first report on an L/ODC involved in QAs biosynthesis from plants.

S2.6 MY RESEARCH EXPERIENCE WITH PROF. MEINHART ZENK, THE PIONEER OF BENZYL-ISOQUINOLINE ALKALOID BIOSYNTHESIS

Wanchai De-Eknamkul

Chulalongkorn University, Dept. of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Bangkok, 10330, Thailand; [email protected]

It all started when I first met Prof. Zenk at his all-time favorite meeting: “The First Princess Chulabhorn International Congress on Natural Products”, held in Bangkok, Thailand in December 1987. Later, I had an opportunity to undertake my post-doc studies (1989–1990) in his laboratory in Munich. With the aim of elucidating the biosynthetic pathway of morphine, by focusing on the step of (S)-reticuline to (R)-reticuline conversion, I experienced both hard times and good times in working with him. It was a difficult task that the conversion step possibly involved different enzymatic reactions. However, Prof. Zenk impressively guided me to the right direction until finding the target enzyme eventually. I was proud to be a part of his research group and his success in clarifying one of the world’s most important biosynthetic pathways of natural products. While working with Prof. Zenk, I admired his abilities to predict and solve problems and his expertise in the field of biosynthesis of natural products. It is, thus, not surprising that he was also the first to resolve many other biosynthetic pathways of pharmaceutically-important benzylisoquinoline alkaloids, such as berberine, sanguinarine, protopine, papaverine, etc. From his remarkable contributions to the field of biosynthesis, Prof. Zenk is truly “The Pioneer of Benzylisoquinoline Alkaloid Biosynthesis”.

S2.7 CATHARANTHUS ROSEUS EXPRESSES A LEAF EPIDERMIS SPECIFIC AMYRIN SYNTHASE INVOLVED IN TRITERPENE BIOSYNTHESIS

Fang Yu,¹ Antje Thamm,¹ Darwin Reed,² Patrick Covello,²Vincenzo De Luca¹

¹Brock University, Department of Biological Sciences, St Catharines, ON L2S 3A1, Canada, ²National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, SK S7N 0W9, Canada; [email protected]

The plant kingdom produces many thousands of biologically active triterpenes that are derived from (3S)-oxidosqualene to generate over 80 different carbon skeletons. Different oxidosqualene cyclase (OSC) enzymes carry out the carbocation rearrangements responsible for this biological diversity. For example, Catharanthus roseus accumulates 2.5% of their leaf dry weights as the α-amyrin-derived ursane-type triterpene, ursolic acid, on the leaf surface. Sequencing of a leaf epidermis enriched cDNA library generated most of the mevalonic acid pathway as well as a new OSC gene with high amino acid sequence identities to amyrin synthases (CrAS) from other species. Functional expression of CrAS in Saccharomyces cerevisiae resulted in the production of α-amyrin and β-amyrin in an approximate 4 to 1 ratio. Transcription analysis showed that CrAS is predominantly expressed in the leaf epidermis of young Catharanthus leaves. These results strongly suggest that expression of triterpene biosynthesis genes is highly regulated during plant growth and development, that triterpenes appear to be produced in the specialized epidermis of young leaves, and that synthesis is closely associated with secretion on the leaf surface where they fulfill particular biological roles.

S2.8 MEMBRANE TRANSPORT OF ALKALOID IN PLANTS

Kazufumi Yazaki

Kyoto University, Research Institute for Sustainable Humanosphere, Uji, 611-0011, Japan; [email protected]

Plant alkaloids comprise an important group of secondary metabolites due to their strong and divergent biological activities. Most alkaloids have aromatic ring structures containing a nitrogen atom, which provides the basic property. Alkaloids often preferentially accumulate in a particular organ of the plant body, e.g., berberine in the inner bark of Phellodendron amurense, caffeine in the seeds of Coffea spp. In the 1980s, intensive studies on the accumulation mechanism of alkaloids in plant cells were done, mostly using plant cell cultures. In this field, Dr. Zenk first pointed out that the vacuolar transport of alkaloids was stereo-specific, and suggested the involvement of a specific transporter molecule for vacuolar sequestration of alkaloids, not simply by an ion-trap mechanism.

Many years later, we identified a couple of transporter proteins responsible for the membrane transport of alkaloids. Cjmdr1, an ATP-binding cassette transporter localized at the plasma membrane of Coptis japonica cells, can transport berberine, while this alkaloid is transported by proton antiport mechanism at tonoplast. To honor the pioneering works by Dr. Zenk, recent progresses in alkaloid transport will be discussed.

S2.9 XANTHOHUMOL FROM HOPS (HUMULUS LUPULUS): PHARMACOKINETICS AND EFFECTS ON BIOMARKERS OF METABOLIC SYNDROME IN RATS

LeeCole Legette,^1,2 Lian Ma,¹ Ralph L. Reed^1,2 Cristobal L. Miranda,^1,2 J. Mark Christensen,¹ Rosita R. Proteau,¹ Jan Frederik Stevens^1,2

¹Oregon State University, Pharmaceutical Sciences, Corvallis, OR 97331, USA, ²Oregon State University, Linus Pauling Institute, Corvallis, OR 97331, USA; [email protected]

Xanthohumol (XN) is the principal prenylated flavonoid of the hop plant, Humulus lupulus. It has received much attention due to its cancer chemopreventive, antiangiogenic, anti-inflammatory, antihyperlipidemic, and antihyperglycemic effects in vitro and in vivo. XN has been marketed as a dietary supplement. To determine whether biologically relevant concentrations of XN can be attained after oral administration, we conducted a pharmacokinetic study in male jugular vein-cannulated Sprague-Dawley rats. Rats received either an intravenous injection or oral gavage of XN at three dose levels. Plasma samples, treated with and without glucuronidase, were analyzed for XN and its metabolites (isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin) using LC-MS/MS. The combined bioavailability of conjugated and free xanthohumol was approximately 0.33, 0.13 and 0.11 for the low, medium and high dose of XN, respectively. The half-life of XN at the tested oral doses was in the range 18–30 hours. We also studied the chronic effects of dietary XN in a rat model of metabolic syndrome (Zucker fa/fa rats) by daily administration of XN per os at the three dose levels. The highest dose resulted in lower plasma glucose levels and decreased weight gain. This suggests that XN may be a viable therapeutic agent for the treatment of metabolic disorders and obesity.

S2.10 A BLUE ANIGOZANTHOS ROOT CULTURE AS AN INITIAL OF PHENYLPHENALENONE RESEARCH

Bernd Schneider

Max Plank Institute for Chemical Ecology, Beutenberg Campus, Hans Knöll Str. 8, Jena, 07745, Germany; [email protected]

Meinhart Zenk established and maintained in his Munich laboratory a collection of up to 1000 plant cell and organ cultures, which were used for “chasing the enzymes” [Phytochemistry 30, 3861–3863 (1991)] and genes of secondary metabolite biosynthesis.

Among these cultures, a unique blue-colored root culture attracted my special interest when I visited his lab as a Humboldt research fellow in the early 1990’s. On request, Meinhart Zenk allocated this particular culture to me with the comment that it probably accumulates so-called phenylphenalenones, which may be worthwhile to be investigated.

This was the starting point of a fruitful research area, first about phytochemistry and structure elucidation, then occurrence in the plant kingdom and tissue-specific distribution, biosynthetic experiments including ¹³C labelling and characterization of recombinant biosynthetic enzymes, some synthetic work, and finally bioactivity studies and ecological functions. Some highlights of our phenylphenalenone research will be presented.

Root culture of Anigozanthos preissii (Haemodoraceae)

S2.11 FUNGAL ORIGIN OF ERGOT ALKALOIDS IN DICOTYLEDONOUS PLANTS

Eckhard Leistner,¹ Ulrike Steiner²

Universität Bonn, Institut für Pharmazeutische Biologie, Bonn, 53115, Germany, ²Universität Bonn, Institut für Nutzpflanzenwissenschaften und Resourcenschutz (INRES)-Phytomedizin, Bonn, 53115, Germany; [email protected]

Convolvulaceous plants such as Ipomoea asarifolia and Turbina corymbosa are esteemed in southern Mexico as one of the principal hallucinogens for use in divinations as well as magico-religious rituals. The physiologically active principles are ergot alkaloids which are also known to be biosynthesized by clavicipitaceous fungi. The disjunct occurrence of ergot alkaloids in higher plants and fungi seemed to contradict the principle of chemotaxonomy that identical or at least similar natural products have a common evolutionary history and, thus, occur in taxonomically related plants or microorganisms.

Recently we have shown, however, that I. asarifolia and T. corymbosa are not the producer of ergot alkaloids but that the plants are colonized by the first ergot alkaloid producing clavicipitaceous fungi described that are apparently mutualistic symbionts of dicotyledonous plants. The fungi belong to a newly established genus which we named Periglandula with reference to the close association of the fungi with secretory glands of its plant hosts. The secretory glands are likely to be mediators of a metabolic dialogue between plant and fungus. Ergot alkaloids participate in this dialogue.

S2.12 HIGHLIGHTS ON MORPHINE RESEARCH WITH PROF. MEINHART H. ZENK

Michael Spiteller

Technical University of Dortmund, Institute of Environmental Research (INFU) of the Faculty of Chemistry, Chair of Environmental Chemistry and Analytical Chemistry, Dortmund, 44227, Germany; [email protected]

Over a decade Meinhart H. Zenk made significant contributions concerning the biosynthesis of morphine in plants and mammals. His work was characterized by sharpness of thought and experimental elegance, which constantly fascinated his pupils and colleagues. It was his particular talent to ornament the scientific discussion with convincing arguments presented with vast graciousness. We had the opportunity and honour to accompany Meinhart’s life’s work for a small distance. It all started on the 11th of June 2007, when he asked me to record a high resolution mass spectrum of papaverine. This initial measurement resulted in an intensive cooperation over the next three years concerning the biochemical precursors of morphine in plants and animals. The highlights of this cooperation are presented on the occasion of the Meinhart H. Zenk Memorial and emphasized with original citations from more than 300 e-mails, from which it is possible to follow the ups and downs and finally the successful publication of the results. Meinhart has left us with an important task: the significance of the result that mammals are able to carry out de novo synthesis of morphine from simple biochemical precursors for pain therapy will certainly occupy the fellow workers of M. H. Zenk in the years to come.

Grobe, M. et al. (2010) Proc. Natl. Acad. Sci., USA, 107, 8147–8152. Han, X. et al. (2010) Phytochemistry, 71, 1305–1312.

S2.13 ABSOLUTE CONFIGURATION OF SECONDARY METABOLITES via ELECTRONIC CIRCULAR DICHROISM

Daneel Ferreira and Christina Coleman

Department of Pharmacognosy and Research Institute of Pharmaceutical Science, School of Pharmacy, University of Mississippi, University, MS 38677, USA; [email protected]

Chiroptical methodology represents a powerful tool towards definition of the absolute configuration of stereogenic centers in secondary metabolites, irrespective of the physical state of the compound. The utility of the CD method has been strengthened with the introduction of TDDFT calculations that permit the theoretical calculation of electronic CD spectra to greatly enhance our ability to interpret ECD data in terms of absolute configuration. We will cover the configurational assignment of a significant array of natural products ranging from the caged xanthone, (–)-morellic acid (1), to conformationally labile polyphenols of the 1,1,3-triarylpropan-2-ol-type (2).

S2.14 “WHY DO LIVERWORTS BIOSYNTHESIZE MARCHANTINS, PUNGENT AND BITTER SUBSTANCES?”

Yoshinori Asakawa

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan; [email protected]

The most characteristic chemical phenomenon of liverworts is that most sesqui- and diterpenoids are enantiomers of those found in higher plants. It is very noteworthy that different species of the same genus like Frullania tamarisci and F. dilatata, each produces different sesquiterpene enantiomers. Liverworts are also rich sources of various types of bis-bibenzyls, such as marchantins, riccardins, plagiochins, and isoplagiochins, which are structurally similar to bis-bibenzylisoquinoline alkaloids such as tubocurarine, and bibenzyl cannabinoids as well as its related bibenzyls. Prof. Meinhart Zenk’s question was that why do liverworts produce potent pungent, bitter or muscle relaxing products when the author presented his lecture in Munich. The author predicted that they might be defense substances against insects and mammals. He had been quite interested in biosynthesis of marchantin-type bis-bibenzyls. Asakawa and Matsuda [1] proposed that cyclic bis-bibenzyls might be biosynthesized from bibenzyls that correspond chemically to dihydrostilbene. Zenk and Asakawa et al. [2] proved their hypothesis by feeding experiments of radioactive and ¹³C labeled phenylalanine and dihydro-p-coumaric acid. The A- and B-rings of the marchantin molecule are derived from the benzene ring of L-phenylalanine via trans-cinnamic acid and p-coumaric acid. Enzymatically hydrogenated dihydro-p-coumaric acid from p-coumaric acid condenses with three molecules of malonyl-CoA to form prelunularic acid which is aromatized to yield lunularic acid and possibly lunularin which is followed by condensation of lunularin or lunularic acid to form marchantin A. This is the first experimental result of biosynthesis of macrocyclic bis-bibenzyls obtained from liverworts. Since this work, more than 70 cyclic- or acyclic bis-bibenzyls and their monomer have been isolated from liverworts and their total synthesis and bioactivity reported.

1. Asakawa, Y., and Matsuda, R. (1982) Phytochemistry 21, 2143–2144.

2. Friederich, S., Maier, U. H., Deus-Neumann, B., Yoshinori, A., and H. Zenk, M. (1999) Phytochemistry 50, 589. Friederich, S., Rueffer, M., Asakawa, Y., and Zenk, M. H. (1999) Phytochemistry 52, 1195.

S2.15 REGIO- AND STEREOSELECTIVE INTERMOLECULAR OXIDATIVE PHENOL COUPLING IN FILAMENTOUS FUNGI

Christian Gil Girol,¹ Wolfgang Hüttel,¹ Silke Foegen,¹ Katja M. Fisch,² Jörn Piel,² Thorsten Heinekamp,³ Axel Brakhage,³ Michael Müller¹

Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, 79104, Germany, ²Institute of Organic Chemistry and Biochemistry, Universität Bonn, Bonn, 53121, Germany, ³Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology (HKI), Jena, 07745, Germany; [email protected]

More than 50 years ago, Barton and Cohen correlated the structures of many phenol-coupled natural products of higher plants to oxidative phenol coupling. In 1997 Lewis et al. reported that in some plant families, lignan formation, via oxidative phenol coupling, is a two-enzyme-process. The late Meinhart Zenk showed that P450 enzymes catalyze regio- and stereoselective intramolecular oxidative phenol coupling in the biosynthesis of several alkaloids in plants and mammals.

Our aim is directed toward characterizing the enzymes that are responsible for the regio- and stereoselective oxidative phenol coupling in fungi. For that reason we have chosen the filamentous fungi Penicillium citreo-viride and Aspergillus niger, producers of the dimeric metabolites vioxanthin and kotanin, respectively. In order to develop an assay system we synthesized the dimeric target molecules as well as the monomeric precursors semi-vioxanthin and demethylsiderin, the latter ones specifically ¹³C-labeled. Feeding studies revealed the polyketide origin and the regio- and atropselective dehydrodimerization of demethylsiderin. Progress towards the identification of the enzymes involved in these transformations will be discussed.

S2.16 THE MYB75 TRANSCRIPTION FACTOR PLAYS A CENTRAL ROLE IN REGULATING CARBON FLUX INTO CELL WALL-RELATED METABOLIC PATHWAYS IN ARABIDOPSIS THALIANA

Apurva Bhargava, Carl Douglas, Brian Ellis

University of British Columbia, Vancouver, BC V6T 1Z4, Canada; [email protected]

Formation of plant secondary cell walls, which represent a major metabolic sink for photosynthate, typically requires coordinated synthesis of large amounts of both carbohydrate and phenylpropanoid polymers. The broad outline of the transcriptional network that regulates the channeling of carbon into these products has begun to be revealed but the interactions that define its architecture remain largely unknown. We found that the Arabidopsis R2R3-class MYB transcription factor, MYB75, which is known to physically interact with other transcriptional regulators such as TT8 and bHLH012, also interacts with the KNOX-class transcription factor, KNAT7. Analysis of gain-of-function and loss-of-function mutants allows us to place MYB75 within a regulatory circuit that represses the activity of key players in secondary cell wall synthesis, both in the Arabidopsis inflorescence stem and in the developing seed coat, possibly as part of a mechanism to fine tune commitment to secondary cell wall biosynthesis according to available metabolic resources.

S2.17 MODERN TOOLS FOR ANCIENT MEDICINES: INVESTIGATING THE BIOSYNTHESIS OF BIOACTIVE COMPOUNDS IN IMPORTANT MEDICINAL PLANTS

David R. Gang

Washington State University, Institute of Biological Chemistry, Pullman, WA 99164, USA; [email protected]

The number and types of specialized compounds produced by any plant is very unique, producing considerable variation and resulting in distinct chemotypes. Many medicinal herbs possess chemotypes with distinctive biochemical profiles in specific tissues, such as rhizomes or trichomes. Using a biochemical genomics-based approach we have identified several classes of enzymes that play important roles in controlling the diversity of the terpenoids, such as turmerones and other sesquiterpenoids, and phenylpropanoid-derived compounds, such as gingerols and curcuminoids, in several medicinal plants. Specialized metabolism appears to be organized at times and in certain species (if not all) in a modular fashion: groups of metabolites that are biosynthetically linked can accumulate in a concerted manner in biosynthetic modules. In many cases, a large collection of related metabolites (e.g., sesquiterpenoids or gingerols/curcuminoids) accumulate that do not all belong to the same module. Indeed, subgroups of compounds accumulate in separate modules. This suggests that multiple enzymes must be involved in production of these compounds, which has been verified in our recent efforts. Identification of such enzymes and characterization of their individual functions are important steps in understanding how such complex arrays of metabolites can evolve in specific plant lineages. Evaluating such information is an important step in understanding how the great diversity of plants has evolved.

S2.18 COMPARATIVE ANALYSIS OF BENZOXAZINOID BIOSYNTHESIS IN MONOCOTS AND DICOTS: INDEPENDENT RECRUITMENT OF STABILIZATION AND BIO-ACTIVATION FUNCTIONS

Alfons Gierl, Monika Frey

Technical University of Munich, Plant Sciences, Freising, 85350, Germany; [email protected]

Stabilization by glucosylation and bio-activation by hydrolysis is essential for phytoanticipin function in plant defense. Benzoxazinoids represent protective and allelopathic phytoanticipins that are found in a multitude of species of the family Poaceae and occur sporadically in a single species of phylogenetically unrelated dicots. We have isolated and functionally characterized the benzoxazinoid-specific UDP-glucosyltransferase and β-glucosidase from the dicot Consolida orientalis (larkspur). A phylogenetic comparison of these enzymes with their counterparts in the grasses indicates convergent evolution by repeated recruitment of these functions during evolution. Protein modeling supports the phylogenetic analysis. The data indicate a great evolutionary flexibility in recruitment of these essential functions of secondary plant metabolism.

S2.19 HEAVY METALS; XENOBIOTICS; AND MORE

Erwin Grill

Technical University of Munich, Department of Plant Sciences, Freising, Germany, 85354; [email protected]

Plants are constantly challenged by a variety of noxious compounds, including heavy metal ions, microbial toxins and agrochemicals. The tripeptide glutathione (GSH) plays a central role in the detoxification of such compounds in plants. GSH alleviates metal ion-induced oxidative reactions. In addition, GSH is a precursor of phytochelatins (PCs), which bind and sequester heavy metals. A third role of GSH is to form conjugates with xenobiotics, which are subsequently catabolized and sequestered to the vacuole by the action of ATP-driven ABC-type transporters. PCs provide a basal form of metal-tolerance and -homeostasis even in some fungi and Caenorhabditis elegans. PCs are generated by the action of a specific dipeptidyl-transferase, PC synthase (PCS), which catalyses the repeated transfer of glutamylcysteinyl units of GSH onto GSH or PC. PCs bind the heavy-metal ions more avidly than the monothiol GSH. Interestingly, PCS also catalyses the turnover of GS-conjugates to glutamylcysteinyl-conjugates by removing the carboxylterminal glycine. The glutamyl-cysteinyl-conjugate formation of herbicides has been identified as a catabolic pathway characteristic for plants. Our studies with PCS-knockout lines of Arabidopsis reveal that PCS provide the cytosolic activity for glutamylcysteinyl-conjugate formation. Thus, PCS appear to fulfill at least two functions in plants; PC biosynthesis for heavy metal detoxification and the turnover of GS-conjugates.

S2.20 PHENOLICS IN THE SURFACE WAXES OF SECALE CEREALE: FORMATION AND ACCUMULATION OF CUTICULAR ALKYLRESORCINOLS

Reinhard Jetter, Ruonan Yao

University of British Columbia, Botany and Chemistry, Vancouver, BC V6T 1Z4, Canada; [email protected]

Alkylresorcinols are phenolic lipids found in diverse taxa of higher plants and at particularly high levels in grass species. They have strong anti-bacterial/anti-fungal activities and are often deposited at or near the surfaces of plants, making it plausible that they serve as a first line of defense against pathogens. However, direct evidence showing that surface alkylresorcinols are biosynthesized for a protective function at the surface is still insufficient. Earlier work in our lab had shown that in Secale cereale leaves very-long-chain alkylresorcinols (C19 to C27) accumulate mainly in the cuticular wax mixtures, near the tissue surface. Additionally, alkylresorcinol accumulation was found to be synchronized with production of other wax components during leaf ontogenesis. A homology-based approach has now been used to clone several genes potentially encoding CHS-like type III polyketide synthases. Heterologous expression of these genes in yeast showed that one of them indeed encodes an alkyl resorcinol synthases. A second alkylresorcinol synthase was cloned from Brachypodium distachyon, a closely related genetic model system. The rye alkylresorcinol synthase gene was strongly expressed in green leaves, only weakly in etiolated leaves, and not in roots. Leaf expression levels were found correlated with alkylresorcinol accumulation rates. All results together indicate that alkylresorcinols are indeed biosynthesized specifically for a defensive function associated with the wax lining the surface of grass leaves.

S2.21 HAIRY GENOMICS: STUDIES OF SECRETORY GLANDULAR TRICHOMES IN TOMATO AND RELATIVES

Robert L. Last,¹ A. Daniel Jones,¹ Eran Pichersky,² Cornelius S. Barry,¹ Jeongwoon Kim,¹ Anthony Schilmiller,¹ Kiyoon Kang,¹ Eliana Gonzales-Vigil¹

¹Michigan State University, Biochemistry and Molecular Biology, Chemistry, Horticulture and Plant Biology, East Lansing, MI 48824-1319, USA; ²University of Michigan, Molecular, Cellular and Developmental Biology, Ann Arbor, MI 48109-104, USA; [email protected]

Secreting Glandular Trichomes (SGTs) are epidermal protuberances that produce a wide variety of specialized metabolites in many plant species. For example, the tastes and smells of Mediterranean cooking herbs such as basil and oregano are from essential oils produced and stored in SGTs. Medicinal compounds such as the anti-malaria artemisinin and anti-emetic cannabinoids are also found in SGTs. These structures contribute to defense against biotic stress agents such as herbivores and pathogens.

The collaborative Solanum Trichome Project (www.trichome.msu.edu) is taking a combined chemistry, biochemistry, genomics and genetics approach to study the biosynthetic pathways in the SGTs of tomato and its close relatives. Targets include simple and modified terpenes, acylsugars and methylated flavonoids. Results of these studies will be described, including evidence that there is great diversity in the metabolites produced by Solanum SGTs, presumably in response to selective pressure imposed by biotic stress agents over evolutionary time.

S2.22 POST-GENOMIC ELUCIDATION OF PLANT NATURAL PRODUCT PATHWAYS

Toni M. Kutchan, Dan Ruzicka, Megan Rolf

Donald Danforth Plant Science Center, Saint Louis, MO 63132, USA; [email protected]

The study of the biosynthesis of plant alkaloids at the enzyme and gene level has greatly advanced in recent years. A number of genes are available from the monoterpenoid indole-, tetrahydrobenzylisoquinoline-, and structurally related to both of the previous classes, the terpenoid-isoquinoline alkaloid biosynthetic pathways. To date, however, only partial understanding of the formation of medicinal natural products at the enzyme and gene levels has been attained. The explosive increase in understanding of biology over the past two decades has been enabled by work on model genetic organisms. The study of selected species-specific medicinal natural products, however, requires investigation of those plant species that harbor all or most components of the focal biosynthetic pathway. Detailed genetic and biochemical information on these highly specialized species is often missing. Having comprehensive medicinal plant transcriptomes would greatly advance research on medicinal plant species. We now seek to generate and use transcriptome data to understand the complete formation, storage and regulation of plant-derived medicinal compounds at the enzyme and gene level. Results will be presented from efforts to date to produce deep transcriptome datasets from members of the Papaveraceae and to interrogate the datasets for candidate alkaloid biosynthetic genes.

S3.1 PHYTOCHEMICALS AND GENES FOR THEIR SYNTHESIS IN PEST MANAGEMENT

Stephen O. Duke, Franck E. Dayan, Charles L. Cantrell, Agnes M. Rimando, David E. Wedge, Zhiqiang Pan, Scott R. Baerson, Kumudini M. Meepagala

Agricultural Research Service, United States Department of Agriculture, Natural Products Utilization Research Unit, University, MS 38677, USA; [email protected]

Natural products and their derivatives represent almost 20% of the pest management products being sold worldwide. Phytochemical-derived compounds are strongly represented among insecticides, with less representation in other pesticides. This presentation will briefly review those compounds that have been successful and will provide examples of phytochemicals with promising activity as herbicides, insecticides, insect repellents, molluscicides, and fungicides. Genetically engineering synthesis of natural pesticides into crops will be discussed with focus on the example of sorgoleone, a natural herbicide from the genus Sorghum.

S3.2 IMPROVED CROP PRODUCTIVITY THROUGH MANIPULATION OF PHYTOHORMONE SIGNALING

Suzanne R. Abrams, Ken M. Nelson, L. Irina Zaharia

National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, SK Canada S7N 0W9; [email protected]

Manipulation of plant hormone metabolism and of hormone signaling pathways are powerful strategies to improve productivity of agricultural crops, for enhancing a plant’s tolerance to environmental stresses (drought, heat, cold stress), improving seedling vigour, modifying plant architecture, etc. Towards these ends, an in depth understanding of hormone-induced complex signaling networks in controlling specific developmental pathways or physiological responses in plants is critical. This presentation will focus on recent studies on stress and development in crop plants, integrating profiles of the metabolites of the hormones auxin, cytokinin, gibberellin and abscisic acid, with genomic and physiological data. Application of metabolism resistant plant hormone analogs for agricultural applications and to probe hormone induced gene expression will also be described.

S3.3 BIOSYNTHESIS OF GOSSYPOL IN COTTON: FROM FARNESYL DIPHOSPHATE TO (+)– AND (–)–GOSSYPOL

Tanya Wagner, Jinggao Liu, Lorraine Puckhaber, Alois Bell, Robert Stipanovic

U.S. Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, TX 77845, USA; [email protected]

Gossypol is a dimeric sesquiterpene that occurs in some members of the Malvaceae family. It occurs as an enantiomeric mixture in the foliage, seeds and roots of the cotton plant (Gossypium). It provides protection from insect and animal herbivory. Early studies demonstrated that gossypol is the product of cyclization of E,E-farnesyl diphosphate to (+)-δ-cadinene, which is converted to 8-hydroxy-(+)-δ-cadinene. Proposed intermediates beyond 8-hydroxy-(+)-δ-cadinene include desoxyhemigossypol and hemigossypol. At the time of its first discovery, hemigossypol was proposed to be the immediate precursor of gossypol; it was subsequently shown that hemigossypol is converted into gossypol by peroxidase, and 30 years later that a peroxidase in concert with a flower petal dirigent protein provides a 56% enantiomeric excess of (+)-gossypol. To complete the last step in the gossypol biosynthesis, a temporal study has now identified hemigossypol in developing cottonseed.

S3.4 NATURAL PRODUCTS IN AGRICULTURE: THE ARMS RACE BETWEEN CRUCIFERS AND THEIR FUNGAL PATHOGENS

M. Soledade C. Pedras

University of Saskatchewan, Chemistry, Saskatoon, SK S7N 5C9, Canada; [email protected]

Plants and their microbial invaders are involved in an arms race that continues to cause sustainability issues for agriculture. The use of fungicides and pesticides to prevent crop losses causes pollution and health hazards that make these agricultural practices unacceptable. To decrease the enormous social disparities of our world, food availability and safety is critical. Therefore, continuing efforts to develop methodologies that allow agriculture practices to be sustainable are critical to improve our existence. To devise sustainable methods to prevent and deter cruciferous pathogens, their molecular interaction with crucifers, both cultivated and wild species, is under intense investigation. Cruciferous plants (e.g. canola, mustard, cauliflower, broccoli, turnip, thale cress) produce complex blends of secondary metabolites with diverse ecological roles, which include self-protection against microbial pathogens, pests and other sorts of stress, whereas their fungal pathogens produce phytotoxic metabolites and macromolecules that facilitate plant invasion. Although many of the natural products involved in crucifer defense reactions are detoxified by fungal pathogens, these fungal detoxifications can be stopped. That is, inhibitors (paldoxins) of these transformations could protect plants by boosting their natural chemical defenses and prevent pathogen growth. The fundamental aspects and challenges of this strategy to treat plant fungal diseases will be presented.

S3.5 INSECTICIDES BASED ON PLANT NATURAL PRODUCTS: LONG ON PROMISE, SHORT ON PRODUCTS

Murray B. Isman

University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC Canada V6T1Z4; [email protected]

The spectacular success of the synthetic pyrethroid insecticides in agricultural, structural pest control and public health for over three decades beginning in the mid-1970s, and the more recent success of the neonicotinoid insecticides (since the mid-1990s) has long provided the impetus for the discovery of novel plant natural products with insecticidal properties. Although a number of plant natural products with truly insecticidal actions have been discovered, these are rare in nature and their commercial potential even more rarely realized. The only commercially successful botanical insecticides developed in the past two decades have been those based on neem seed extracts, containing the remarkable triterpenoid, azadirachtin, and those based on certain plant essential oils, typically comprised largely of more ubiquitous monoterpenoids and sesquiterpenoids. Prolonged attempts to synthesize or otherwise produce azadirachtin on a viable commercial scale have proven unsuccessful. Azadirachtin (and natural analogs) is the primary active insecticidal constituents of neem, and commercial neem insecticides are based on semi-refined extracts enriched in azadirachtin. Certain plant essential oils widely used in the flavouring and fragrance industries have been formulated for insect control, but in this case efficacy is seldom correlated with one or more putative active constituents. In contrast, efficacy appears to depend on internal ‘synergy’ among both active and putatively inactive constituents. Evidence for this phenomenon of synergy in insects and mites will be presented.

O3.1 SULFUR VOLATILES IN ALLIUM CANADENSE AND A. TUBEROSUM

Russell L. Rouseff, John M. Smoot, Rajinder S. Mann, William S. Castle, Lukasz L. Stelinski

University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, FL 33850, USA; [email protected]

Headspace volatiles from wild onion (A. canadense L.) and garlic chives (A. tuberosum Rottl.) were examined using GC-MS and GC-PFPD (pulsed flame photometric detector, sulfur mode). Although both species contain numeroussulfur volatiles, only crushed garlic chive leaves and oil repelled the Asian citrus psyllid, Diaphorina citri Kuwayama, which is the insect vector for the fatal citrus greening or huanglongbing disease¹. Since sulfur volatiles have been shown to repel this insect², the purpose of this study was to examine the sulfur volatiles in both species to help determine which might have biological activity. Twelve sulfur volatiles were identified in garlic chive and 18 sulfur peaks were observed in the wild onion. Eight volatiles were common to both. Identified volatiles consisted primarily of thiols and sulfides with various methyl, allyl, and propyl substitutions. Low temperature and gradient temperature injections were performed to determine if thermal artifacts were formed.

1. Mann, R. S.; Rouseff, R. L.; Smoot, J. M.; Castle, W. S.; Stelinski, L. L., Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Bulletin of Entomological Research 2011,101, 89–97.

2. Onagbola, E. O.; Rouseff, R. L.; Smoot, J. M.; Stelinski, L. L., Guava leaf volatiles and dimethyl disulphide inhibit response of Diaphorina citri Kuwayama to host plant. J. Appl. Entomol. 2011, 135, 404–414.

O3.2 L-METHIONINE CATABOLISM INTO SULFUR AROMA VOLATILES IN MELON FRUIT

Itay Gonda,^1,2 Einat Bar,¹ Noga Sikron,² Vitaly Portnoy,¹ Ya’akov Tadmor,¹ Arthur A. Schaffer,¹ Nurit Katzir,¹ Yosef Burger,¹ Aaron Fait,² Efraim Lewinsohn¹

¹Institute of Plant Sciences, Newe Ya’ar Research Center, Agricultural Research Organization, Vegetable Crops, Ramat Yishay, Israel 30095, ²Ben-Gurion University of the Negev, Jacob Blaustein Insts. for Desert Research, Midreshet Ben-Gurion, Dept. of Dryland Biotechnology, Sedeh Boker, Israel 84990; [email protected]

Volatiles derived from essential amino acids have a strong impact on melon and other fruit aromas. Different plants utilize different biochemical routes to produce such volatiles. We have previously shown that amino acid aminotransferases are key factors in the production of aromatic- and branched-chain-amino-acid-derived aroma volatiles in developing melon fruit (Gonda et al., 2010, J. Exp. Bot. 61: 1111–1123). We present evidence that in fruit melon slices, ¹³C₅- L-methionine was incorporated into sulfur aroma volatiles following two different labeling patterns. We also present results indicating that melon fruit possess L-methionine aminotransferase as well as L-methionine gamma lyase enzymatic activities. Data mining from transcriptomic databases yielded two sequences that putatively code for such proteins. The sequences are currently being tested for their biochemical role by functional expression in E. coli. Further, metabolic flux analysis will be carried out to generate information on the active pathways in the fruit. Integrating this with existing genomic, transcriptomic, enzymatic and metabolomic data, will contribute to deciphering the metabolic pathways of the conversion of essential amino acids into aroma volatiles in melon fruits.

O3.3 PLANT DEFENSE ACTIVATORS AS ELICITORS OF OAT AVENANTHRAMIDE BIOSYNTHESIS

Mitchell L. Wise

USDA, ARS, Cereal Crops Research Unit, Madison, WI 53726, USA; [email protected]

Oats produce a group of phenolic secondary metabolites termed “avenanthramides”. Among food crops these metabolites are unique to oat. In addition to their biological role as phytoalexins, the avenanthramides are potent antioxidants in vitro and have potential as nutraceuticals. In cellular assays and animal models they demonstrate potent anti-inflammatory activity through inhibition of nuclear factor kappa beta. Although produced constitutively in the oat grain, the levels of avenanthramides tend to be highly variable and the levels are strongly influenced by environment, genotype and genotype × environment interactions. Recent work in my laboratory has shown that avenanthramide levels in vegetative tissue, and to some extent in the grain, can be enhanced by treatment with plant defense activators such as acibenzolar-S-methyl (benzothiadiazoles, BTH) and isonicotinic acid (INA). Treatment of the plants with BTH or INA produced a strong up-regulation of avenanthramide biosynthesis within 48 hours. This response tends to be fairly long lasting (days to weeks). The dynamics of avenanthramide biosynthesis in various tissues of oat plants will be described in detail. Genotypic variation in avenanthramide production will also be described.

O3.4 IDENTIFICATION OF THE MOSQUITO BITING DETERRENT CONSTITUENTS FROM THE INDIAN FOLK REMEDY PLANT, JATROPHA CURCAS

Charles L. Cantrell,¹ Abbas Ali,² Stephen Duke,¹ Ikhlas Khan²

¹United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, MS 38677, USA, ²University of Mississippi, National Center for Natural Products Research, University, MS 38677,USA; [email protected]

An investigation of the Indian folk remedy plant, Jatropha curcas, was performed to identify the constituents responsible for the mosquito biting deterrent activity of the oil. J. curcas seed oil is burned in oil lamps in India and parts of Africa to repel biting insects, primarily mosquitoes. The seed oil was thoroughly analyzed by ¹H NMR, ¹³C NMR, HPLC-RI, and GC-FID to identify the constituents in the oil. Identified constituents, both free fatty acids and triglycerides, were evaluated for activity in Aedes aegypti biting deterrent assays. Furthermore, an oil condensation trap was used to demonstrate that free fatty acids or triglycerides are partially volatilized during the combustion process. These compounds were found to be responsible for the biting deterrency of the burned oil. Specifically, oleic, palmitic, linoleic, and stearic acids were all active at 25 nmol/cm² above that of solvent control in A. aegypti biting deterrent assays. Oleic, palmitic, and linoleic acids were all more active than stearic acid in the same bioassay. Evaluation of the triglycerides containing each of these fatty acids revealed that tripalmitin, tristearin, trilinolein, and triolein all demonstrated significant activity above a solvent control at 10 μg/cm², whereas tripalmitin was the most active. Due to literature reports suggesting larvicidal activity of the oil, J. curcas seed oil and its free fatty acid constituents were also evaluated against 1-day old A. aegypti larvae up to 500 ppm. Oleic acid was the only fatty acid having larvicidal activity against 1-day old AP nutrients. aegypti larvae with an LC₅₀ of 47.9 ppm.

O3.5 CORRELATION BETWEEN TEA LEAF AGE AND CHEMICAL CONTENT AND SHADE LEVELS

Ran Song,^1,2 Dovi Kelman,¹ Kimberley Johns,^1,3 Anthony D. Wright¹

¹College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA, ²Yale University, New Haven, CT, 06511, USA, ³Sheffield Hallam University, City Campus, Sheffield, S1 1WB, United Kingdom; [email protected]

This study investigated tea leaf samples from a Hawaiian tea plantation and the relationship between tea leaf age, the relative concentrations of three naturally occurring compounds commonly found in them, L-theanine, caffeine and epigallocatechin gallate (EGCG), all of which are reported to have positive effects on human health, shade levels and FRAP determined antioxidant activity. An HPLC method was developed that utilized a reversed phase C-18 stationary phase and a mobile phase composed of water and acetonitrile to quantify the relative amounts of the three natural products. The outcome of the analyses showed that the concentration of L-theanine and caffeine decreased as leaf age increased moving from bud to first and then second leaf, while EGCG concentration increased in moving from the bud to first, second and lower leaves. The influence of shade on the relative concentrations of these three compounds in tea leaves was also investigated and shown to have a positive correlation with EGCG levels. Antioxidant activity, as determined using the FRAP assay system, was found to correlate positively with increasing EGCG levels. This is the first investigation of its type and also of tea samples from Hawaii. The presented findings show that certain chemical components of tea can potentially be used as markers for the age, quality and authenticity of various teas now and into the future.

O3.6 IMPACTS OF CLIMATE CHANGE ON ALLOCATION OF N TO CYANOGENIC GLYCOSIDES

Roslyn Gleadow,¹ Birger Møller,² Timothy Cavagnaro,¹ Rebecca Miller,¹ Peter Stuart,³ Alan Neale,¹ Cecilia Blomstedt,¹ John Hamill¹

¹Monash University, School of Biological Sciences, Melbourne, Victoria, 3800, Australia, ²University of Copenhagen, Department of Plant Biology and Biotechnology, Copenhagen, DK-1871, Denmark, ³University of Queensland, School of Agriculture and Food Science, Brisbane, Queensland 4072, Australia; [email protected]

The allocation of resources to bioactive products is likely to change as a result of climate change, affecting plant nutritive value. Cyanogenic glucosides, which break down to release toxic HCN, are synthesized by about 5% of all plants as well as many insects. In humans, epidemics of the neurological disorder Konzo are more common during periods of drought. Similarly, animals grazing on forage sorghum can die if plants are young, highly fertilized or water stressed. Toxicity of cassava and other C₃plants (e.g. clover) is higher in plants grown at elevated CO₂whereas drought effected-toxicity in sorghum (C₄) is moderated by higher concentrations of atmospheric CO₂. We created an EMS mutagenized population of sorghum and identified individuals with reduced, enhanced or zero dhurrin. Allocation of N to different metabolites in different genotypes is compared with growth using a high through-put phenomics in order to help develop predictive models.

S4.1 GENETIC AND EPIGENETIC MECHANISMS OF COLON CANCER CHEMOPREVENTION IN HUMANS BY WHOLE BERRIES AND BERRY CONSTITUENTS

Gary D. Stoner,¹ Li-Shu Wang,¹ Mark Arnold,² Carol Burke,³ Tong Chen,² Yi-Wen Huang¹

¹Medical College of Wisconsin, Medicine and Obstetrics and Gynecology, Milwaukee, WI 53226, USA, ²The Ohio State University, Surgery and Internal Medicine, Columbus, OH 43210, USA, ³Cleveland Clinic Foundation, Gastroenterology, Cleveland, OH 44195, USA; [email protected]

Our laboratories have been evaluating the ability of freeze-dried berries to prevent gastrointestinal tract cancers in animals and in humans. Most studies have used black raspberries (BRBs), due to their high antioxidant potential and their high content of anthocyanins and fiber. In rodent studies, the consumption of BRB powder, at concentrations of 2.5, 5 and 10% (w/w) of a synthetic diet, results in a 40–70% inhibition of carcinogen-induced cancer in the rat esophagus and colon. Mechanistically, BRBs exhibit a broad range of chemopreventive effects on a cellular level including inhibition of cell proliferation, inflammation, angiogenesis, and stimulation of apoptosis, cell adhesion, and differentiation, and they protectively modulate the expression levels of genes associated with all of these cell functions. Based upon these preclinical observations, we have conducted a series of pilot clinical trials of BRBs in patients at high risk for cancer. The oral administration of BRB powder (45g/day) to Barrett’s esophagus patients for 6 months led to a reduction in parameters of oxidative stress, but minimal effects on the lesion itself. Oral administration of strawberry powder (60 g/day) to 37 Chinese patients with esophageal dysplasia led to histologic regression of 80% of the dysplastic lesions and reduced levels of iNOS, COX-2, and phospho-NF-κB-p65 proteins. Treatment of 20 colorectal cancer patients with BRB powder for about 3 weeks led to a reduction in cell proliferation and demethylation of suppressor genes in the Wnt signaling pathway in colorectal tumors. Finally, a trial in 14 patients with familial adenomatous polyposis showed that daily treatment with rectal BRB suppositories for 9 months caused a 36% regression of rectal polyps. These preliminary trials indicate that berries have significant promise for chemoprevention of esophageal and colon cancer in humans.

S4.2 NATURAL TRITERPENOIDS AS SCAFFOLDS FOR NEW DRUG SYNTHESIS

Michael Sporn

Dartmouth Medical School, Hanover, NH 03755, USA; [email protected]

There are many triterpenoids that exist in nature that have interesting biological activities. However, most of these triterpenoids are not potent enough to be practically used in clinical medicine as preventive or therapeutic agents. Thus, it is necessary to enhance activity by chemical modification of the naturally occurring triterpenoid scaffold. We will discuss the application of this principle, as exemplified by the synthetic oleanane triterpenoids that have been made in the Department of Chemistry at Dartmouth College by Professor Gordon Gribble and his colleagues, and then tested for biological activity in my own laboratory in the Department of Pharmacology at Dartmouth Medical School. Many of these agents have profound anti-inflammatory activity, by virtue of their ability to modulate the synthesis of enzymes involved in the inflammatory process. They also have similar anti-oxidative effects, again by inducing the synthesis of enzymes that destroy reactive oxygen species. One such derivative of oleanolic acid, namely CDDO-methyl ester (bardoxolone methyl), is now in Phase III clinical trial for treatment of advanced kidney disease in diabetic patients.

S4.3 THE CLINICAL PROMISE OF SULFORAPHANE (SF)

Paul Talalay

Johns Hopkins University School of Medicine, Dept. of Pharmacology & Molecular Sciences, Baltimore, MD 21205, USA; [email protected]

Isolation of sulforaphane (SF) from broccoli as the principal transcriptional inducer of a network of cytoprotective genes provided a rationale for the health benefits of high plant consumption (especially crucifers). It established that aerobic cells contain elaborate gene networks for defense against damage by oxidative stress, electrophiles, inflammation, and radiation. Upregulation of these genes, mediated by the Keap1-Nrf2-ARE signaling pathway, protects cells against these damaging processes which lead to neoplastic and other chronic diseases. Broccoli sprouts deliver standardized doses of glucoraphanin (the glucosinolate precursor of SF) which undergoes hydrolysis to its active metabolite SF by plant myrosinase, or by microflora of the gastrointestinal tract. Microbial conversion efficiency varies enormously among individuals (2–40%), raising important health implications. SF and all other inducers are thiol reagents that target the cysteine-rich intracellular sensor Keap1, and upregulate a wide variety of genes. But SF also affects a broader range of functions that include induction of heat shock proteins, modulation of NFκB-dependent inflammatory pathways, and suppression of histone deacetylase. The magnitudes of these responses probably depend on inducer levels, cell type and prevailing stress conditions. Insight into the multiple cytoprotective effects of sulforaphane, and the ease with which this dietary component can be delivered to humans has generated considerable interest in translating these findings into the clinic. Many clinical trials currently in progress are targeting malignancies (breast, prostate, bladder, skin), asthma, COPD, and radiation damage.

S4.4 CANCER PREVENTION BY δ- AND γ-TOCOPHEROLS

Chung Shu Yang, Guangxun Li, Fei Guan, Jihyeung Ju, Gang Lu, Nanjoo Suh

Rutgers, The State University of New Jersey, Chemical Biology, Ernest Mario School of Pharmacy, Piscataway, NJ 08854-8020, USA; [email protected]

Epidemiological studies have shown an inverse correlation between vitamin E intake and cancer risk. However, recent large-scale human trials with high doses of α-tocopherol (α-T) have produced disappointing results. This points out the need for a better understanding of the biological activities of different forms of tocopherols. Using a tocopherol mixture that is rich in γ-T (γ-TmT, containing 57% γ-T, 24% δ-T and 13% α-T), we have demonstrated the inhibition of cancer formation and growth in animal models for colon, lung, mammary gland and prostate cancers. δ-T was found to be more active than γ-T in the inhibition of cancer cell growth in culture and xenograft tumors as well as in AOM-induced colon carcinogenesis in rats, whereas α-T was ineffective. The blood and tissue levels of δ-T were low and those of α-T were high, but the levels of side-chain degradation metabolites of δ-T were high, suggesting that metabolites of δ-T contribute to the inhibitory activity. The inhibitory activity was associated with the quenching of reactive oxygen and nitrogen species as well as anti-inflammatory activities. We suggest the use of γ-TmT, δ-T or γ-T, rather than pure α-T, for cancer prevention (Supported by NIH grants CA141756, CA122474 & CA133021 and the John Colaizzi Chair Endowment Fund).

S4.5 LUNG CANCER PREVENTION: REVERSE MIGRATION STRATEGY

Kathryn Ann Gold,¹ Edward S. Kim,¹ J. Jack Lee,² Ignacio I. Wistuba,^1,3 Carol J. Farhangfar,⁴ Waun Ki Hong^1,4

¹University of Texas MD Anderson Cancer Center, Department of Thoracic/Head and Neck Medical Oncology, Houston, TX 77030, USA, ²University of Texas MD Anderson Cancer Center, Department of Biostatistics, Houston, TX 77030, USA, ³University of Texas MD Anderson Cancer Center, Department of Pathology, Houston, TX 77030, USA, ⁴University of Texas MD Anderson Cancer Center, Division of Cancer Medicine, Houston, TX 77030, USA; [email protected]

Despite extensive research, there are no known effective chemoprevention agents for lung cancer. Clinical trials in the past, using agents without a clear target in an unselected population, have shown interventions to be ineffective or even harmful. We propose a new approach to drug development in the chemoprevention setting: Reverse migration; that is, drawing on our experience in the treatment of advanced cancer to bring agents, biomarkers, and study designs into the prevention setting. Our institution has experience with biomarker-driven clinical trials, as in the recently reported Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, and we now propose to bring this trial design into the prevention setting.

S4.6 ARTHUR NEISH YOUNG INVESTIGATOR AWARD LECTURE

A NOVEL ROLE FOR SULFHYDRYL-REACTIVE ACTIVATORS OF TRANSCRIPTION FACTOR NRF2: HSF1-DEPENDENT UPREGULATION OF Hsp70

Ying Zhang,¹ Young-Hoon Ahn,² Vittorio Calabrese,³ Philip A. Cole, ²Albena T. Dinkova-Kostova^1,2

¹University of Dundee, Medical Research Institute, Dundee, DD1 9SY, United Kingdom, ²Johns Hopkins University School of Medicine, Department of Pharmacology and Molecular Sciences, Baltimore, MD 21205, USA, ³University of Catania, Department of Chemistry, Catania, 95100, Italy; [email protected]

Transcription factors NRF2 and HSF1 provide broad defence mechanisms by regulating the expression of several hundred genes that encode proteins with versatile cytoprotective functions. The identification of small-molecule inducers of these pathways is actively being pursued as a strategy to prevent or delay disease onset and to extend healthy lifespan. Such compounds, many of which are abundant in plants, and their synthetic analogues protect against the otherwise detrimental consequences of the toxic, neoplastic, and pro-inflammatory effects of xenobiotics and endogenous substances in experimental models of carcinogenesis, cardiovascular disease, and neurodegeneration. We found that structurally distinct small-molecule NRF2 activators, all of which react with sulfhydryl groups, but differ in potency by 15,000-fold, upregulate Hsp70, a prototypic HSF1-target gene, thus implicating the heat shock response as a target for NRF2 activators. Hsp70 upregulation requires functional HSF1, but is NRF2-independent. In addition, a sulfoxythiocarbamate inducer conjugates to the negative regulator of HSF1, Hsp90. The differential concentration-dependence of the two responses suggests that activation of NRF2 precedes that of HSF1. These findings support the future development of potent “dual” activators of this type as mechanism-based comprehensive cytoprotective agents.

S4.7 SYNTHETIC TRITERPENOIDS AND CHEMOPREVENTION: BIOLOGICAL ACTIVITIES AND MOLECULAR TARGETS

Karen Liby

Dartmouth Medical School, Department of Medicine, Hanover, NH 03755, USA; [email protected]

Synthetic oleanane triterpenoids are multifunctional agents with potent anti-inflammatory, anti-proliferative, and pro-apoptotic activities in vitro. Although they are not conventional cytotoxic molecules, the triterpenoids can prevent and treat cancer in a variety of experimental animal models. Both CDDO-methyl ester (CDDO-Me) and CDDO-ethyl amide (CDDO-EA) significantly decrease the number, size, and histopathology of lung adenocarcinomas in A/J mice challenged with vinyl carbamate. CDDO-Me also delays the development of estrogen receptor negative mammary tumors induced by overexpression of the ERbB2 oncoprotein or by deletion of the BRCA1 tumor suppressor gene. Both CDDO-Me and CDDO-EA also extend survival in a transgenic mouse model of pancreatic cancer, driven by mutations in Kras and p53. In addition to their profound effects on the redox status of cells, the triterpenoids directly interact with regulatory proteins containing reactive cysteines. Keap1, IKK, STAT3, PTEN, and mTOR are all validated molecular targets of the triterpenoids, and all of these proteins and their associated downstream signaling pathways are highly relevant targets for the prevention of cancer.

Supported by NIH R01 grant CA78814, the Breast Cancer Research Foundation, the Sidney Kimmel Foundation for Cancer Research, the American Cancer Society, and Reata Pharmaceuticals, Inc.

S4.8 CANCER CHEMOPREVENTION WITH ANTI-INFLAMMATORY PHYTOCHEMICALS

Young-Joon Surh

Seoul National University, College of Pharmacy, Seoul, 151–742, South Korea; [email protected]

A new horizon in cancer chemoprevention research is the recent discovery of molecular links between inflammation and cancer. Modulation of cellular signaling involved in chronic inflammatory response by anti-inflammatory agents hence provides a rational and pragmatic strategy in molecular target-based chemoprevention and cytoprotection. Many substances derived from herbs and spices have been found to activate this particular redox-sensitive transcription factor, thereby potentiating cellular antioxidant or detoxification capacity. It is noteworthy that there is a good correlation between anti-inflammatory activity of some chemopreventive/cytoprotective agents and their ability to induce antioxidant gene expression. The current research in my laboratory concerns evaluation of chemopreventive and cytoprotective effects of some edible antioxidative and anti-inflammatory phytochemicals and elucidation of their underlying molecular mechanisms. Our research program has attempted to unravel common events mediated by transcription factors, such as NF-κB, STAT3 and Nrf2, and their regulators, involved in the cellular signaling network for molecular target-based chemoprevention with selected dietary and medicinal phytochemicals.

S4.9 THE PHENOMENA OF RESVERATROL

John M. Pezzuto

College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA; [email protected]

Cancer chemoprevention entails the ingestion of dietary or pharmaceutical agents that can prevent, delay or reverse the process of carcinogenesis. We have been actively engaged in the systematic discovery and characterization of natural product chemopreventive agents. The typical approach involves identifying active crude substances, such as extracts derived from terrestrial plants or marine organisms, utilizing in vitro bioassay systems, followed by isolation of pure active components. As part of this project, an extract obtained from a nonedible Peruvian legume, Cassia quinquangulata Rich. (Leguminosae), was evaluated and found to be active as an inhibitor of cyclooxygenase. The active component was identified as resveratrol. A surprisingly broad spectrum activity was observed, indicative of potential to inhibit carcinogenesis at the stages of initiation, promotion and progression. This discovery has led to many additional research efforts. There are now around 4000 papers concerning various aspects of resveratrol action, some of which have generated controversy. The molecule is unusually promiscuous and specific mechanisms remain elusive. Rapid and near complete metabolism add to the conundrum. Recently, we have exploited the broad spectrum of activities mediated by this simple stilbene to design derivatives with much greater potency and specificity. An overview of the field and a personal perspective will be presented. (Supported by NCI program project P01 CA48112).

S4.10 KEAP1-NRF2 SIGNALING AS A TARGET FOR CANCER PREVENTION BY NATURAL PRODUCTS

Thomas Kensler,^1,2 Nobunao Wakabayashi,¹ Li Yang,¹ Abena Agyeman,² Kala Visvanathan²

¹University of Pittsburgh, Pharmacology & Chemical Biology, Pittsburgh, PA 15261, USA, ²Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA; [email protected]

Health reflects the ability of an organism to adapt to stress. Stresses – metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses – not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis, other forms of toxicity, and inflammation whilst disruption of the pathway exacerbates these outcomes. Protection against these stresses is manifest in multiple ways: (i) prevention of macromolecular damage through induction of electrophile detoxication and antioxidative enzymes, as well as dampening of inflammatory processes, (ii) induction of macromolecular damage repair/removal systems including the proteasome, DNA repair and autophagy, and (iii) activation of tissue repair/regeneration pathways. These cytoprotective effects of Nrf2 reflect responses mediated by direct activation of downstream effector genes and through cross-talk with other signaling networks contributing to cellular plasticity. The Keap1-Nrf2 pathway can also be induced by thiol-reactive small molecules including dithiolethiones, isothiocyanates and triterpenoids that demonstrate protective efficacy in preclinical chemoprevention models and in clinical trials.

S4.11 CANCER CHEMOPREVENTION BY TARGETING THE EPIGENOME – STATE OF THE ART AND FUTURE CHALLENGES

Clarissa Gerhauser

German Cancer Research Center, Epigenomics and Cancer Risk Factors, 69120 Heidelberg, Germany; [email protected]

The term epigenetics refers to modifications in gene expression caused by heritable, but potentially reversible, changes in chromatin structure. Major epigenetic mechanisms include DNA methylation, histone acetylation and methylation, and non-coding (micro) RNAs. Given the fact that epigenetic modifications occur early in carcinogenesis, they have been identified as promising new targets for prevention strategies. Recent years have provided a wealth of information on the potential impact of chemopreventive agents on epigenetic mechanisms (reviewed in Huang, Plass, Gerhauser, Curr. Drug Targets, 2010). Food components targeting the epigenome include micronutrients (folate, selenium, retinoic acid, vitamin E), butyrate, polyphenols (from green tea, apples, coffee, and other dietary sources), genistein and soy isoflavones, curcumin, ellagitannin, indol-3-carbinol, lycopene from tomatoes, and sulfur-containing compounds from Allium and cruciferous vegetables. Their effects on the epigenome have potential impact on multiple mechanisms relevant for cancer prevention, including detoxification, cell cycle progression, signal transduction, apoptosis induction, and others. In vivo studies that demonstrate the functional relevance of epigenetic mechanisms for health promoting efficacy of natural products are still limited. Future projects will identify best strategies for chemopreventive intervention with micronutrients and dietary food components, taking into account the importance of epigenetic mechanisms for gene regulation.

S4.12 DIETARY CANCER CHEMOPREVENTIVE PHYTOCHEMICALS: SIGNALING AND EPIGENETICS IN BLOCKING CARCINOGENESIS INITIATION VERSUS TUMOR PROGRESSION

Ah-Ng Tony Kong

Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA; [email protected]

Diverse dietary phytochemicals can prevent diseases including cancer. These dietary “antioxidants”, can trap RONS, also trigger cellular signaling events including “proteins thiol modifications” leading to expression of cellular defense genes or other cellular effects. We study dietary phenolic antioxidants, isothiocyanates, tocopherols, ω-3 fatty acids and herbal medicines, which are effective against various many animal carcinogenesis models. These compounds could modulate kinases, activate Nrf2 signaling, and induce cellular defense genes HO-1, GST, NQO1, and GCS. Integrating results from Nrf2-/- mice with microarray bioinformatics, other genes including apoptosis, cell adhesion, cell growth, kinases, electron transport, transcription factors, and ubiquitination, are also Nrf2-mediated, leading to the overall cellular protective effects against oxidative/carcinogenic damages. These Nrf2-/- mice are more prone to carcinogen-induced skin, colon and other cancers. Incidentally, in the prostate TRAMP tumors, as cancer progresses, a shut-down of Nrf2 via CpG methylation of the promoter region, attenuating Nrf2-mediated genes, which were reversed by dietary PEITC, curcumin and tocopherols. Hence, daily intake of these phytochemicals would induce Nrf2-mediated anti-oxidative stress genes either directly or indirectly through epigenetic pathways, reduce inflammation, induction of apoptosis/autophagy of initiated tumor cells during early lesions. (Supported by NIH grants).

S4.13 DIETARY PREVENTION OF COLON CARCINOGENESIS AND DISCOVERY OF PREDICTIVE BIOMARKERS

Nancy H. Colburn,¹ Gerd Bobe,^1,2 Roycelynn Mentor-Marcel,^1,2 Terryl J. Hartman,³ Robb Chapkin,⁴ Elaine Lanza,⁵ John Milner,⁶ Young Kim,⁶ Amanda Cross,⁷ Matthew R. Young¹

¹CCR, NCI, NIH, Laboratory of Cancer Prevention, Frederick, MD 21702, USA, ²NCI, Preventive Oncology Program (Cancer Prevention Fellowship), Rockville, MD, USA, ³Pennsylvania State University, State College, PA, USA, ⁴Texas A & M University, College Station, TX, USA, ⁵Cytonix, VA, USA, ⁶NCI, Division of Cancer Prevention, Rockville, MD, USA, ⁷NCI, Division of Cancer Epidemiology and Genetics, Rockville, MD20852, USA; [email protected]

Our group has established multiple collaborations to prevent carcinogenesis by dietary intervention and to discover predictive molecular indicators. We have identified potential molecular targets and biomarkers of efficacious response to dry bean-based diets in intervention studies to prevent colon carcinogenesis in humans and mice. These studies are based on the observation that high dry bean intake decreased advanced colorectal adenoma recurrence in humans and protected against chemically induced colon carcinogenesis in obese mice. Both ethanol extract (containing flavonols and other soluble compounds) and residue (containing fiber) as well as whole cooked beans were active in attenuating carcinogenesis in the Ob/Ob mice. IL-6 has been identified as a potential biomarker and molecular target. Serum IL-6 concentrations were elevated in participants of the Polyp Prevention Trial (PPT) who developed high risk or advanced adenomas and were lower in participants consuming a flavonol-rich diet to which dry beans primarily contribute. Similarly, serum IL-6 concentrations were elevated in obese, carcinogen-induced mice with pre-neoplastic lesions and were lower in mice fed the dry bean-rich diets. RNA concentrations of IL-6 in colon tissue were elevated in mice receiving the carcinogen and were attenuated in mice fed the dry bean-rich diets (Mentor-Marcel et al., 2009). In a short-term human feeding (LIFE) study, we identified from fecal colonocyte microarray analysis sets of 3 genes that could be used as potential indicators of risk or exposure to dietary dry beans. The LIFE study, which paralleled the diets found efficacious in the PPT demonstrated that a legume-enriched low-glycemic index diet produces improvements in biomarkers of insulin resistance and inflammation as well as improvements in serum lipid profiles and plasma leptin. Indicators of insulin resistance and inflammation also improved in the 4-year Polyp Prevention Trial. Current studies are identifying serum metabolites as indicators in the PPT and LIFE studies.

S4.14 STUDIES FOR CANCER PREVENTION: A PATH FROM TEA TO CAFFEINE TO EXERCISE

Allan H. Conney, You-Rong Lou, Yao-Ping Lu

Rutgers University, The Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA; [email protected]

Oral administration of green or black tea to SKH-1 mice inhibited UVB-induced skin carcinogenesis, but the decaffeinated teas were inactive and caffeine had a strong inhibitory effect. Mechanistic studies indicated that caffeine inhibited UVB-induced carcinogenesis by increasing UVB-induced p53 and by inhibiting UVB-induced increase in the ATR/Chk1 pathway thereby enhancing UVB-induced apoptosis. Caffeine had no effect on apoptosis in normal epidermis. Since caffeine administration increased locomotor activity and decreased tissue fat, we evaluated the effect of voluntary exercise (running wheel in the cage) on UVB-induced apoptosis. Exercise, like the effect of caffeine, also enhanced UVB-induced apoptosis, decreased tissue fat and inhibited UVB-induced carcinogenesis. Caffeine or running wheel exercise-related inhibition of UVB-induced carcinogenesis was associated with increased apoptosis in the tumors but not in areas away from tumors. A combination of caffeine and exercise had a much stronger stimulatory effect on UVB-induced apoptosis and a stronger inhibitory effect on UVB-induced carcinogenesis than either treatment alone. Since both regimens decreased tissue fat, we evaluated the effect of removal of the parametrial fat pads on UVB-induced apoptosis and found that UVB-induced apoptosis was enhanced, suggesting that tissue fat may secrete anti-apoptotic factors. These results suggest that tissue fat may enhance carcinogenesis by inhibiting apoptosis in DNA-damaged precancer cells and in cancer cells.

S4.15 CANCER CHEMOPREVENTION: MISSION ACCOMPLISHED IN RODENT MODELS BUT WHY NOT SO IN HUMANS

Hasan Mukhtar

University of Wisconsin-Madison, Department of Dermatology, Madison, WI 53706, USA; [email protected]

I define cancer chemoprevention as slowing the process of carcinogenesis. Based on our experience with hundreds of chemopreventive agents in animal models, the process of carcinogenesis could be slowed by many agents in every spontaneous, induced and transgenic model. Thus, we can say that the mission of cancer chemoprevention has been successfully accomplished in rodent models. However, the picture of cancer chemoprevention in humans is not that glowing. The results of prospective randomized human trials of chemopreventive agents have in many cases been less impressive or have conflicted with the results of animal and observational studies. First of all, understandably any successful animal protocol for cancer chemoprevention in rodent models has never been duplicated in human settings. For reasons unclear and unfortunately any human large scale cancer chemoprevention trial done to date before initiation has never been first validated in a reliable rodent model. In addition, the timing of the intervention during multistep carcinogenesis, the complexity of dietary interactions, baseline levels in a given individual or population, duration of the study and the dose-response effects further complicates translation of animal data to human settings. It is also possible that because of our lifestyle of consuming many chemopreventive agents present in fruits and vegetables and other sources, we have already reaped some benefits of cancer chemoprevention. Thus, we might have to settle for only modest delays in cancer occurrence as a result of intervention. Some thoughts will be presented for further slowing the process of carcinogenesis in the human population.

S4.16 NATURAL-AGENTS MECHANISMS AND PERSONALIZING MARKERS FOR CANCER PREVENTION

Scott M. Lippman

University of Texas MD Anderson Cancer Center, Department of Thoracic/Head and Neck Medical Oncology, Houston, TX 77030, USA; [email protected]

Mechanism-based, personalized approaches with natural agents are on the march in cancer prevention. The natural agents discussed here represent recent advances in molecular targeting and predictive markers for natural-agent cancer prevention. Promising preclinical/clinical results with green tea and its polyphenolic flavonoid constituent EGCG (epigallocatechin gallate) include randomized trials in the head and neck and prostate. Elegant new mechanistic data indicate that EGCG binds to the peptidyl prolyl cis/trans isomerase Pin1, which is required for EGCG effects on cell growth, c-Jun activation, and NF-kB and AP-1-mediated transcription regulation. EGCG analogs that target Pin1 more specifically could be developed, then tested in phase 0 trials, such as that recently conducted with an indole-3 carbinol compound (derived from cruciferous vegetables; primarily inhibits Akt), which was the first phase 0 trial for chemoprevention. Promising preclinical/early clinical results of myoinositol include inhibiting phospho-Akt and regulating PI3K expression signatures in the lungs of smokers. With promising preclinical/early clinical activity through effects on AMPK and IGF1R signaling, metformin (derived from French lilac) recently was found to have germline predictive markers. Selenium did not prevent prostate cancer in SELECT or high-grade PIN patients, but pharmacogenetics suggest genotypes that predict selenium benefit. Developing standard personalized chemoprevention, with either natural or other agents, is becoming clearer with studies of molecular targets and predictive markers. The recent mechanistic study of EGCG is a model of molecular-targeted research in this field.

O4.1 ANTI-CANCER EFFECT OF SCALLION EXTRACT AGAINST COLON TUMOR

Ning-Sun Yang,¹ Chih-Chun Wen,^1,2 Palanisamy Arulselvan,¹ Chun-Wen Lan¹

¹Academia Sinica, Agricultural Biotechnology Research Center, Taipei, 115, Taiwan, ²Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, 404, Taiwan; [email protected]

Colorectal cancer is a common malignancy and a leading cause of cancer death worldwide. Diet is known to play an important role in the etiology of colon cancer and, recently, dietary chemoprevention has received increasing attention for the prevention and alternative treatment of colon cancers. Scallion is used as a spice/vegetable worldwide, and as a traditional Chinese medicine in treating a variety of diseases. The possible beneficial effects of scallion on mouse colon cancer were evaluated in this study. The in vivo anti-tumor effects of scallion extracts were assessed in a subcutaneously inoculated CT-26 colon tumor model in BALB/c mice. Tumor tissues were subjected to western blotting and immunohistochemistry for analysis of key inflammatory markers, and ELISA for analysis of cytokines. A specific preparation of scallion extracts, orally fed to test mice at 50 mg/kg b.w./day, resulted in a significant suppression of tumor growth and enhanced the survival rate of test mice. Dosage of specific scallion extract, when translated into human application, is equivalent to 10 g fresh weight/70 kg b.w./day. This amount of scallion is highly acceptable as a dietary uptake for both Asian and Western food cultures. At the molecular level, scallion extracts inhibited the key inflammatory markers COX-2 and iNOS, and suppressed the expression of various cellular markers known to be involved in tumor apoptosis (apoptosis index), proliferation (cyclin D1 and c-Myc), angiogenesis (VEGF and HIF-1α), and tumor invasion (MMP-9 and ICAM-1), when compared with vehicle control-treated mice. Our findings may warrant further investigation on the use of common scallion as a chemopreventive dietary agent to lower the risk of colon cancers.

O4.2 GINGER SUPPLEMENTATION AND THE EXPRESSION OF NF-KB IN THE NORMAL-APPEARING COLORECTAL MUCOSA OF PATIENTS AT HIGH RISK OF COLORECTAL CANCER: RESULTS FROM A PILOT RANDOMIZED, CONTROLLED TRIAL

Suzanna M. Zick,¹ Roberd M. Bostick,² Mack Ruffin,¹ Kim Turgeon,¹ Benjamin D. Wright,¹ Dean Brenner¹

¹University of Michigan, Family Medicine, Internal Medicine, Ann Arbor, MI 48105,USA, ²Emory, School of Public Health, Atlanta, GA, USA; [email protected]

Background: Ginger has been proposed as a promising candidate for colorectal cancer (CRC) prevention. To assess the potential of ginger for modulating inflammation in normal human colorectal mucosa, we measured the expression of NF-kB, a transcription factor involved in the control of inflammation, in normal-appearing colon mucosa of patients at high risk for CRC in a pilot, randomized, double-blinded, placebo-controlled, clinical trial.

Methods: A total of 20 patients were treated with either 2.0 g (eight 250 mg capsules) of encapsulated ginger (standardized to 5%-gingerols) or placebo for 28 days. Overall expression and distributions of NF-kB in colorectal crypts in biopsies of normal-appearing colon mucosa were detected and measured using automated immunohistochemistry and quantitative image analysis. For quality control, 10% of slides were randomly selected for repeat measurements, which yielded an intra-rater reliability of 0.96.

Results: In the ginger group relative to the placebo group, NF-kB expression decreased 25.8% (p = 0.08) along the full length of the crypts, with a 29.4% (p = 0.08) decrease in the upper 40% (differentiation zone) of the crypts, and 23.7% (p = 0.08) in the lower 60% (proliferative zone) of the crypts relative to the placebo group.

Conclusions: These results suggest that ginger may reduce inflammation in the colorectal epithelium.

O4.3 GINGER SUPPLEMENTATION AND THE EXPRESSION OF BAX IN THE NORMAL-APPEARING COLORECTAL MUCOSA OF SPORADIC COLORECTAL ADENOMA PATIENTS: RESULTS FROM A PILOT RANDOMIZED, CONTROLLED TRIAL

Jessica Citronberg,¹ Roberd M. Bostick,¹ Mack Ruffin,³ D. Kim Turgeon,² Dean E. Brenner,² Benjamin D. Wright,³ Suzanna M. Zick³

¹Emory University Rollins School of Public Health, Atlanta, GA 30322, USA, ²University of Michigan, Internal Medicine, Ann Arbor, MI 48109, USA, ³University of Michigan, Family Medicine, Ann Arbor, MI 48109, USA; [email protected]

Background: Ginger has been proposed as a promising candidate for colorectal cancer (CRC) prevention. To assess the potential of ginger for modulating apoptosis in the normal human colorectal mucosa, we measured the expression of bax, an apoptosis promoter, in the normal-appearing colon mucosa of patients at high risk for CRC in a pilot, randomized, double-blinded, placebo-controlled, clinical trial.

Methods: A total of 20 patients were treated with either 2.0 g (eight 250 mg capsules) of encapsulated ginger (standardized to 5%-gingerols) or placebo once daily for 28 days. Overall expression and distributions of bax in colorectal crypts in biopsies of normal-appearing colon mucosa were detected and measured using automated immunohistochemistry and quantitative image analysis. For quality control, 10% of slides were randomly selected for repeat measurements, which yielded an intra-rater reliability of 0.98.

Results: In the ginger group, relative to the placebo group, bax expression decreased 15.6% (p = 0.81) along the full length of the crypts, 6.6% (p = 0.38) in the upper 40% (differentiation zone) of the crypts, and 21.7% (p = 0.77) in the lower 60% (proliferative zone) of the crypts; however, there was a 19% increase (p = 0.80) in the proportion of the expression of bax in the upper 40% relative to the whole crypt.

Conclusions: These preliminary results suggest that ginger may reduce apoptosis in the colorectal epithelium overall while shifting more of its expression from the proliferative zone of the crypt to the luminal, or differentiation zone.

O4.4 POTENTIAL ROLE OF GINSENG FOR CANCER CHEMOPREVENTION

Chong-Zhi Wang, Guang-Jian Du, Jacqueline Somogyi, Chun-Su Yuan

University of Chicago, NCCAM Center for Herbal Research on Colorectal Cancer, Chicago, IL 60637, USA, and University of Chicago, Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, Chicago, IL 60637, USA; [email protected]

The clinical management of cancer invariably involves diverse conventional modalities, including surgery, radiation, and chemotherapy. However, the complexity of human cancer requires some alternative management to improve the therapeutic efficacy of conventional treatment and/or the quality of life of cancer patients. Medicinal botanicals have recently gained more attention for cancer management. Numerous effective anticancer drugs have been developed from botanicals, and identifying new herbal sources to develop ideal chemoprevention remains an essential step in advancing the treatment of colorectal cancer. In this study, potential roles of ginseng herbs, especially American ginseng and notoginseng, in cancer chemoprevention are evaluated. The major pharmacologically active constituents of ginsengs are ginsenosides, which can be mainly classified into protopanaxadiol and protopanaxatriol groups. The recognized active anticancer compounds from American ginseng and notoginseng are ginsenosides Rg3, Rh2, and protopanaxadiol. The structure-activity relationship between their chemical structures and pharmacological activities is discussed. Sugar molecules within a ginsenoside have a high impact on cancer cells. Anticancer activities increase with the decrease of sugar number. In addition, we observed that various steaming temperatures and time treatments of the ginseng herbs can change their ginsenoside profiles and enhance their anticancer activities. This heat treatment process may increase the efficacy of ginseng in cancer chemoprevention. (This work was supported in part by the NIH/NCCAM grants AT003255, AT004418 and AT005362).

O4.5 BLUEBERRY DIET AND BLUEBERRY BIOACTIVES INHIBIT LUNG CANCER AND ENHANCE THE ACTIVITY OF PACLITAXEL

Ramesh C. Gupta,¹ Hina Kausar,² Jeyaprakash Jeyabalan,² Farrukh Aqil²

¹University of Louisville, Pharmacology & Toxicology and the James Graham Brown Cancer Center, Louisville, KY 40202, USA, ²University of Louisville, James Graham Brown Cancer Center, Louisville, KY 40202, USA; [email protected]

Unlike significant progress made in the prognosis of certain cancers, the prognosis for lung cancer remains grim. We describe efficacy of blueberry powder and bioactives for their anti-lung cancer activity, and for their chemosensitizing effects. Nude mice were provided AIN-93M diet or diet supplemented with blueberry powder (7.5%, w/w) and then inoculated with highly aggressive human lung cancer H1299 cells. This intervention showed significant inhibition of the tumor burden progressively. To determine if blueberry anthocyanidins (anthos) were the main bioactives, H1299 cells were treated in culture with blueberry anthos individually and their equimolar mixture. These compounds showed a time- and dose-dependent inhibition of the cell growth, with much greater effect observed with the antho mixture. Nude mice carrying H1299 cell xenograft when treated i. p. with either delphinidin (1.5 mg/mouse) or a native mixture of blueberry anthos (0.5 mg/mouse) resulted in 60–65% inhibition of the tumor burden. Further, nude mice upon i. p. treatment with suboptimal doses of the chemotherapeutic drug paclitaxel and blueberry anthos elicited significant tumor inhibition only in the combination groups. Analyses of H1299 cells from the various treatments by Western blot revealed that the synergistic effects observed were due to the attack of anthos either on overlapping and/or distinct targets associated with cell proliferation, apoptosis, inflammation and metastasis.

O4.6 ANTIOXIDANT EFFECTS OF LYCOPENE IN MEN WITH PROSTATE CANCER OR BENIGN PROSTATE HYPERPLASIA: A RANDOMIZED CONTROLLED TRIAL

Richard B. van Breemen,¹ Roohollah Sharifi,² Marlos Viana,³ Phyllis E. Bowen,⁴ Maria Stacewicz-Sapuntzakis⁴

¹University of Illinois, College of Pharmacy, Medicinal Chemistry and Pharmacognosy, Chicago, IL 60612, USA, ²University of Illinois at Chicago, Department of Surgery, Chicago, IL 60612, USA, ³University of Illinois at Chicago, Department of Ophthalmology and Visual Science, Chicago, IL 60612, USA, ⁴University of Illinois at Chicago, Department of Kinesiology and Nutrition, Chicago, IL 60612, USA; [email protected]

Consumption of tomato products is associated with a decreased risk of prostate cancer, and lycopene, the red carotenoid in the tomato, is a potent antioxidant that might contribute to this chemoprevention activity. A double-blind, randomized, placebo-controlled trial of 120 men, 90% African American veterans, with either prostate cancer or benign prostate hyperplasia was carried out to investigate whether oral administration of lycopene increases lycopene levels in blood and prostate tissue and lowers markers of oxidative stress. Urology patients were randomly assigned to receive either 30 mg/d of lycopene as a tomato oleoresin or placebo for 21-days prior to prostate biopsy for possible diagnosis of prostate cancer. For the men receiving lycopene, the mean lycopene concentration in plasma increased 2-fold compared to placebo and 2-fold in prostate tissue. There was a trend in the reduction the DNA oxidation product 8-oxo-deoxyguanosine in men diagnosed with benign prostate hyperplasia but not in men diagnosed with cancer. Lipid peroxidation measured as malondialdehyde in plasma was negatively correlated with lycopene levels.

O4.7 RESVERATROL AND ITS ANALOGUES AS POTENTIAL EPIGENETIC AGENTS FOR CHEMOPREVENTION AND THERAPY IN PROSTATE CANCER

Steven J. Dias¹, Kun Li¹, Agnes M. Rimando², Anait S. Levenson^1,3

¹University of Mississippi Medical Center, Cancer Institute, Jackson, MS 39216, USA, ²University and US Department of Agriculture, Natural Products Utilization Unit, Agricultural Research Services, Jackson, MS 38677, USA, ³University of Mississippi Medical Center, Pathology, Jackson, MS 39216, USA; [email protected]

Epigenetic silencing of tumor suppressor gene(s) is a contributing factor to the pathogenesis of prostate cancer (PCa).Reversal of epigenetic silencing is therefore a potentially desirable modality of targeted chemoprevention and therapy for PCa. We report on the epigenetic HDAC inhibitory activity of resveratrol (Res) through inhibition of metastasis-associated protein 1 (MTA1), which plays a critical role in PCa progression. Resveratrol down-regulates the MTA1 protein by promoting its degradation and dissociation from the NuRD (nucleosome remodeling and deacetylation) repressor complex, which results in reverse deacetylation of tumor suppressor p53. Further, silencing MTA1 by genetic approaches (shRNA) in combination with Res treatment significantly enhances p53 acetylation and subsequent apoptosis in PCa cells. In the present study, we compared and contrasted the anti-proliferative and MTA1-regulation of Res and its natural (PTER, PIC and 3M-Res) and synthetic (2Ac-Res, 3Ac-Res and DMSA) analogues in a panel of PCa cells: LNCaP, Du145 and PC3M. Results indicate that selected Res analogues have higher biopotency in inhibiting MTA1 and androgen receptor (AR) therefore promising greater efficacy as potential “epigenetic” chemopreventive and therapeutic agents in PCa.

O4.8 ELUCIDATION OF STRUCTURAL/FUNCTIONAL CHANGES UPON MODIFICATION OF KEAP1 C151, A PRIMARY TARGET OF BOTANICAL CHEMOPREVENTIVE AGENTS

Aimee L. Eggler,¹ Evan Small,³ Chenqi Hu,² Richard B. van Breemen,² Andrew D. Mesecar¹

¹Purdue University, Biological Sciences, West Lafayette, IN 47907, USA, ²University of Illinois at Chicago, Department of Medicinal Chemistry and Pharmacognosy, Chicago, IL 60612, USA, ³University of Illinois at Chicago, Biochemistry, Chicago, IL 60612, USA; [email protected]

Many botanical agents, including sulforaphane from Cruciferae species, activate the transcription factor Nrf2. Numerous cytoprotective genes are upregulated by Nrf2, comprising a promising therapeutic strategy for the prevention of cancer and other diseases. At basal conditions, Nrf2 is repressed by the cysteine-rich Keap1 protein, which targets Nrf2 for ubiquitination by a Cul3-mediated ubiquitination complex and subsequent degradation. Modification of Keap1 cysteines, in particular C151, by sulforaphane and other compounds leads to Nrf2 activation, by downregulating Nrf2 ubiquitination and degradation. We find C151 to be highly modified by sulforaphane and other promising chemopreventive botanical compounds. We and others have shown by co-immunoprecipitation experiments that modification of C151 disrupts the Keap1-Cul3 interaction. We sought to understand the structural and functional changes that occur upon modification of Keap1 C151. Interestingly, we find that when Keap1 C151 is modified, in addition to a decrease in the Keap1-Cul3 affinity, there is a conformational change in the Keap1-Cul3 complex that does form. We propose that this conformational change reduces the ability of the Keap1-Cul3 complex to target Nrf2 lysines for ubiquitination.

O4.9 EXTRACTS OF COLLARD GREENS DOWN-REGULATE THE EXPRESSION OF HER2/NEU PROTEIN AND mRNA IN MCF-7 AND SK-BR3 BREAST CANCER CELLS

Tracie D. Locklear,^1,2 Udeshi Patel,¹ Kapil K. Soni,¹ David Kroll,² Gail B. Mahady¹

¹University of Illinois at Chicago, Pharmacy Practice, Chicago, IL 60612, USA, ²Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Pharmaceutical Sciences, Durham, NC 60612, USA; [email protected]

Cruciferous vegetables such as broccoli, cabbage, and brussel sprouts have been shown in animal studies, as well as clinical trials, to have chemopreventative effects. We have been investigating plants with potential chemopreventive activity from the Lumbee Tribe of North Carolina, including Brassica oleracea var. acephala L., or ‘collard greens’, a vegetable common in the Lumbee diet. Our preliminary data show that MeOH extracts of collards enhanced secreted human placental alkaline phosphatase (SEAP) reporter activity. However, the ethyl acetate liquid fraction (EtOAc, 20 μg/ml) reduced SEAP expression and expression of endogenous pS2, an estrogen dependent gene, when administered with 10 nM estradiol. Additionally, both crude MeOH extract and the EtOAc fraction inhibited the activity of HER-2 tyrosine kinase completely at 20 μg/ml. The inhibition of HER2/erbb2 protein expression was corroborated by In-Cell Western analysis in SK-BR3 cells. The EtOAc partition inhibited the expression of HER2 protein by 30% at 37 μg/ml in SKBR3 cells, and became significantly cytotoxic at higher concentrations. These results indicate that collard greens may potentially convey chemoprotection when consumed regularly. In both MCF-7 and SK-BR3 breast cancer cells, the MeOH extract and the EtOAc partition at 20 μg/ml down-regulated the expression of the mRNA for the HER-2 receptor. These data suggest that collard greens have chemopreventative effects and may be developed as new anti-HER-2 agents.

O4.10 SUPPRESSION OF CYCLOOXYGENASE-2 AND INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION BY 4-[(2′-O-ACETYL-α-l-RHAMNOSYLOXY)BENZYL] ISOTHIOCYANATE IN LPS-STIMULATED RAW 264.7 CELLS

Eun-Jung Park, Sarot Cheenpracha, Leng Chee Chang, Tamara P. Kondratyuk, John M. Pezzuto

College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720,USA; [email protected]

4-[(2′-O-Acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate (RBITC) from Moringa oleifera Lamarck suppressed the expression of COX-2 and iNOS at both the protein and mRNA levels through the inhibition of phosphorylation of the extracellular-signal-regulated kinase and the stress-activated protein kinase, as well as ubiquitin-dependent degradation of inhibitor κBα (IκBα). In accordance with IκBα degradation, nuclear accumulation of NF-κB, and subsequent binding of NF-κB to the NF-κB cis-acting element, was attenuated by treatment with RBITC. These data suggest RBITC should be included in the dietary armamentarium of isothiocyanates potentially capable of mediating anti-inflammatory or cancer chemopreventive activity.

S5.1 BIOSYNTHESIS OF ISOPRENE UNITS VIA THE MEP PATHWAY: ELECTRON AND PROTON TRANSFERS IN THE FORMATION OF IPP AND DMAPP

Michel Rohmer, Lionel Charon, Catherine Grosdemange-Billiard, Myriam Seemann, Jeanne Toulouse, and Denis Tritsch

Université de Strasbourg/CNRS, Institut de Chimie, 67070 Strasbourg Cedex, France; [email protected]

Methylerythritol cyclodiphosphate (MEcPP, 1) reductase (GcpE) and hydroxymethylbutenyl diphosphate (HMBPP, 2) reductase (LytB), the last two enzymes of the mevalonate-independent MEP pathway, catalyze respectively the reductions of MEcPP into HMBPP and HMBPP into isopentenyl diphosphate 4 and dimethylallyl diphosphate 5. These reactions involve the transfer of two electrons provided by the [4Fe-4S] cluster of the enzymes and the protonation of an allylic anion intermediate 3. Experiments performed with the isolated enzymes suggest that the electron source for the Fe/S cluster is in bacteria NADPH/flavodoxin/flavodoxin reductase for both enzymes and in plants photosynthesis via ferredoxin in the light or NADPH/ferredoxin reductase/ferredoxin in the dark for GcpEpE. Incubation of [1-²H]glucose into the terpenoids of the bacterium Zymomonas mobilis was in accordance with the role of the flavine of flavodoxine as proton donor in the LytB catalyzed reduction.

S5.2 UNEARTHING THE BIOSYNTHETIC DIVERSITY IN THE STEROL METABOLOME

W. David Nes

Texas Tech University, Chemistry and Biochemistry, Lubbock, TX 79409, USA; [email protected]

Eukaryotes exhibit diversity in the composition of membrane sterols. This is largely due to the sterol side chain construction of various combinations of alkyl groups, which can be synthesized by a family of structurally similar enzymes known as sterol C24-methyltransferases (24-SMTs). Discovery trails to early 24-alkyl sterols involving geochemistry, phylogenetic distribution studies and comparative genomic and mechanistic analyses of 24-SMTs reveal remnants of ancient phytosteranes that date back to the Precambrian eon, phyla-specific differences in the length of the side chain (C8 to C11) and configuration of the C24-alkyl(idene) group (α/β- and cis-trans-orientations) and evolutionary accommodation in substrate differences to form single versus multiple products. Notably, fitness requirements determined in a range of organisms show the importance of targeted 24-alkyl sterol homeostasis (type and amount of compound) in growth and/or reproduction and therefore implicates a mating of 24-alkyl sterol structure to function in systems biology. This talk covers the secrets of sterol side chain complexity unearthed in the topics outlined above.

S5.3 BIOSYNTHESIS OF BIOACTIVE ACYCLIC DITERPENOIDS AND PHYTOSTEROLS IN CROTON STELLATOPILOSUS

Damrong Kongduang,^1,2 Juraithip Wungsintaweekul,² Worapan Sitthithaworn, ³Wanchai De-Eknamkul¹

¹Chulalongkorn University, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Bangkok, 10330, Thailand,² Prince of Songkla University, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, HatYai, Songkhla, 90112, Thailand,³ Srinakharinwirot University, Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmacy, Nakonnayok, 26120, Thailand; [email protected]

Croton stellatopilosus, a Thai medicinal plant containing an antipeptic plaunotol, has been used in forms of whole plants, callus and cell suspension cultures as models for biosynthetic studies of acyclic diterpenoids and phytosterols. Plaunotol is a simple linear acyclic diterpene alcohol derived from 4 isoprene units whereas the phytosterols, β-sitosterol and stigmasterol, are from the same monomer of 6 units. Various techniques, including feeding experiments, enzymology, subcellular compartmentalization molecular cloning and metabolic profiling, have been used to clarify the nature of the biosynthesis of both isoprenoid groups. Interesting results have been obtained with respect to their origin of isoprene units, degree of metabolite exchange between the mevalonate pathway and the deoxyxylulose pathway and the transcription profiles of their biosynthesis.

S5.4 POLYKETIDE SYNTHASES AND TETRAKETIDE-PYRONE REDUCTASES OF ARABIDOPSIS THALIANA ARE INVOLVED IN SPOROPOLLENIN BIOSYNTHESIS

Benjamin Lallemand,¹ Etienne Grienenberger,^1,2 Sung Soo Kim,² Thierry Heitz,¹ Carl J. Douglas,² Michel Legrand¹

¹IBMP du CNRS, Université de Strasbourg, Strasbourg, 67000, France, ²University of British Columbia, Department of Botany, Vancouver, BC V6T 1Z4, Canada; [email protected]

Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB and then reduced by TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) and TKPR2 to yield α-pyrone polyketides required for pollen development and sporopollenin biosynthesis. Genes encoding the enzymes are present in all plants surveyed to date, thus suggesting they participate in an ancient conserved biochemical pathway.

S5.5 ENGINEERING PLANT POLYKETIDE SYNTHASES

Ikuro Abe

University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo, 113-0033, Japan; [email protected]

The superfamily of type III polyketide synthases (PKSs) produce a variety of plant secondary metabolites with remarkable structural diversity and biological activities. In the last 10 years, there have been significant advances in understanding the structures and functions of the enzymes. Because of the remarkable catalytic potential and the substrate promiscuity, the structurally simple type III PKSs can be an excellent platform for engineering to design and develop supra-natural enzyme with novel catalytic functions. In this presentation, our recent progress of engineering of plant type III PKS enzymes by structure-based and precursor-directed approach will be discussed.

S5.6 THE ROLE OF ASCORBATE IN THE ACCLIMATION OF LEAVES TO HIGH LIGHT

Nicholas Smirnoff

Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom; [email protected]

Arabidopsis thaliana leaves accumulate ascorbate (vitamin C) and anthocyanins over a similar time course when transferred from low to high light conditions. Ascorbate and anthocyanin deficient mutants are more susceptible to photodamage in high light (HL). Ascorbate deficient mutants (vtc1 and vtc2) have severely decreased anthocyanin accumulation in HL. However, flavonol glycosides, also produced from the flavonoid biosynthesis pathway, show much smaller HL-induced increases and are unaffected in vtc mutants. HL induces increased transcript levels of flavonoid biosynthesis enzymes and the transcription factors that control anthocyanin synthesis (e.g. PAP1 and 2). Induction of these transcripts is decreased in vtc mutants suggesting that ascorbate status influences the signal perception or transduction processes upstream of PAP1 and 2. HL-induced ascorbate biosynthesis appears to be controlled to a large extent at the first committed step of the biosynthesis pathway catalysed by GDP-L-galactose phosphorylase (VTC2 and VTC5). Both VTC2 and anthocyanin gene expression may be controlled via photoreceptors such as cryptochrome and the HY5 transcription factor or via poorly understood chloroplast/photosynthesis-derived signals. Investigations of these signal transduction pathways are underway to provide information on how ascorbate accumulation is controlled and how ascorbate in turn influences anthocyanin accumulation. The roles of ascorbate both as a photoprotectant in its own right and as a proposed redox buffer that modulates the acclimation of leaf metabolism to light intensity will be discussed.

S5.7 CATHARANTHUS ROSEUS AS A NON-MODEL MODEL SYSTEM FOR MIA BIOSYNTHESIS

Vincenzo De Luca, Sayaka Masada-Atsumi, Dylan Levac, Vonny Salim

Brock University, Biological Sciences, St. Catharines, ON L2S 3A1, Canada; [email protected]

Approximately 20% of plant species contain alkaloids with a broad range of physiological properties that protect them from various types of herbivores & pathogens. Within this abundant group of nitrogen containing secondary metabolites the monoterpenoid indole alkaloids (MIAs) make up a large and most diverse class of compounds that are characteristically found within the Apocynaceae, the Loganiaceae, & the Rubiaceae plant families. Their complexity MIA chemistry is matched by their remarkably diverse effects on living organisms that has led to their use as drugs for preventing malaria (quinine) & for treating neurological disorders (reserpine), cancer (camptothecin, vinblastine and vincristine) & as vasodilators (yohimbine) in human beings. Since the MIAs of Catharanthus roseus are among the best characterize with respect to their chemistry, biochemistry, & molecular biology, our laboratory has continued to develop this model non-model system by broadening the scope of our genomic research & pathway discovery to a number of other medicinally important MIA producing plant species that accumulate distinct classes of these compounds. The approach involves directed metabolic profiling of each plant species to identify the best MIA biosynthesis tissues. Selected tissues are then used to harvest mRNA enriched in MIA biosynthesis & transcripts are processed for large scale sequencing. The data produced from several selected species can then be used to assemble genes with putative functions for incorporation into bioinformatic databases in order to perform comparative genomics & for identification of interesting MIA pathway candidate genes. Progress made will be described.

S5.8 STEREOSELECTIVE LIGNAN BIOSYNTHESIS ENGENDERED BY DIRIGENT PROTEINS IN ARABIDOPSIS THALIANA

Kye-Won Kim, Syed G. Moinuddin, Laurence B. Davin, Norman G. Lewis

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA; [email protected]

Regiospecific and/or stereoselective coupling reactions are often key steps in natural product biosynthesis. Our discovery of a (+)-pinoresinol forming dirigent protein (DP) in Forsythia intermedia, and the observation of (–)-lariciresinol accumulation in Arabidopsis thaliana root tissue, motivated us to search for the corresponding (–)-pinoresinol forming DP in this plant species. Using a GUS-reporter gene strategy and in silico analysis, we provisionally identified AtDP6 as a possible candidate for this physiological role in the DP multi-gene family in Arabidopsis. AtDP6 (At4g23690) was then established to engender stereoselective lignan biosynthesis both in vitro using heterologous proteins from both insect and tomato cell culture systems, as well as in vivo using reverse genetics. Up- and down-regulated AtDP6 mutants also clearly showed alteration of their enantiomeric compositions and contents of both pinoresinol and lariciresinol. Another homolog, AtDP5 (At1g64160) closest to AtDP6, was also established to be a (–)-pinoresinol forming DP based on our in vitro, analyses with recombinant protein. Additionally, in order to begin to understand how such different stereoselectivities are controlled, sequence analyses, protein structure modeling and site-directed mutagenesis approaches were employed to identify putative substrate binding sites and amino acid residue(s) or domains which not only bind incoming substrates, but also differentially orientate them in such a way as to engender distinct (+)- and (–)-pinoresinol forming DP stereoselectivities.

S5.9 CYANOGENIC GLUCOSIDES AND THE P450S INVOLVED IN THEIR FORMATION IN PLANTS AND INSECTS

Birger Lindberg Møller

University of Copenhagen, Faculty of Life Science, Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Frederiksberg, C1871, Denmark; [email protected]

For more than 420 million years, plants, insects and their predators have co-evolved based on a chemical arms race including deployment of refined chemical defense systems by each player. Cyanogenic glucosides are produced by numerous plants (e.g. sorghum, barley, cassava, clover, flax, almonds) whereas some specialized insects as part of this arms race are able to sequester cyanogenic glucosides from their food plant as well as to carry out de novo biosynthesis. The classes of intermediates involved in de novo synthesis are identical in plants and insects whereas the enzymes involved have been derived by convergent evolution. The genes encoding the biosynthetic enzymes in plants are clustered on the genome and in different higher plant lineages appear to have been repeatedly and independently recruited from members of similar gene families. Following tissue disruption, the cyanogenic glucosides are hydrolyzed and release toxic hydrogen cyanide to protect the plant or insect from generalist herbivorous insects or predators. Cyanogenic glucosides serve numerous additional metabolic functions in addition to defense. They may function as storage reservoirs of nitrogen and sugar and as quenchers of reactive oxygen species. Forage sorghum contains the cyanogenic glucoside dhurrin and following adverse growth conditions, the amounts of HCN released may be toxic to grazing livestock. In collaboration with Australian researchers, biochemical screens and TILLING approaches have been used to identify a single amino acid change in the CYP79A1 enzyme in sorghum that resulted in an inactive enzyme and acyanogenic plants. Acyanogenic mutants have also been obtained in Lotus japonicus. In synthetic biology approaches, we have shown that P450 catalyzed hydroxylations may be linked to photosystem I and driven by light.

C.K. Blomstedt et al. (2012) Plant Biotech. J., 10, 54–66.

S5.10 THE BIOSYNTHESIS OF CYCLIC PEPTIDES IN THE CARYOPHYLLACEAE

Patrick S. Covello, Janet A. Condie, Goska Nowak, Carla J.S. Barber, Sheila Chiwocha, Darwin W. Reed, John Balsevich

National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, SK S7N 0W9, Canada; [email protected]

Plants within the Caryophyllaceae produce a wide range of cyclic peptides (CPs) consisting of 5-9 proteinogenic amino acids. Many of these CPs have interesting bioactivity in mammalian systems. Despite this, there is very little information on the in planta function and biosynthesis of CPs in the Caryophyllaceae. A Saponaria vaccaria expressed sequence tag collection was investigated for information about CP biosynthesis. This revealed genes that appear to encode CP precursors which are cyclized to mature CPs. Expression of CP precursor genes in transgenic roots resulted in the production of cyclic peptides. Recent efforts to understand the enzymes involved in CP biosynthesis will be discussed.

O5.1 DITERPENE BIOSYNTHESIS IN RICINUS COMMUNIS − MODIFICATION OF CASBENE BY A CYTOCHROME P450 MONOOXYGENASE

James Kirby, Minobu Nishimoto, Genevieve Park, Jeffrey L. Fortman, Jay D. Keasling

University of California, Berkeley, California Institute of Quantitative Biosciences (QB3), Berkeley, CA 94720, USA; [email protected]

Many Euphorbiaceae plants are known to synthesize diterpenes of medicinal interest, such as the latent HIV-1 activator prostratin (12-deoxyphorbol 13-acetate), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. We have previously reported that casbene is the key diterpene olefin produced by several members of this family, including castor bean (R. communis). Many of the functionalized diterpene products found in these plants could in theory be synthesized from casbene but their biosynthetic routes have not been elucidated. In order to investigate steps downstream from casbene synthesis, we undertook an investigation into cytochrome P450 monooxygenase candidates from the Euphorbiaceae. Utilizing a metabolically engineered strain of yeast for heterologous expression, we have found a P450 from R. communis that efficiently oxidizes casbene. This work has provided insights into heterologous production of oxidized diterpenes and improved our understanding of diterpene biosynthesis in the Euphorbiaceae.

O5.2 A FAMILY OF SQUALENE SYNTHASES IN POTATO

Jim G. Tokuhisa,¹ Alice M. Mweetwa,² William H. Wadlington,¹ Chris G. Wolberg¹

¹Virginia Tech, Department of Horticulture, Blacksburg, VA 24061, USA, ²University of Zambia, School of Agricultural Sciences, Lusaka, Zambia; [email protected]

Squalene synthase (EC 2.5.1.2.1; SQS) catalyzes the condensation of farnesyl diphosphate to form squalene and is located at a critical juncture in isoprenoid metabolism. In plants, SQS activity contributes to the formation of phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). Unlike most eukaryotes, higher plants have more than one gene coding for SQS. S. chacoense accumulates transcript for at least three genes encoding SQS homologs. The pattern of transcript accumulation in the plant differs for each gene. The predicted polypeptides have 74 to 83% identity and have differences in the active sites of the enzyme. Current research focuses on characterizing SQS activity by heterologous expression and generating hairy root cultures transformed with specific antisense gene constructs to test the function of the three genes in sterol metabolism. Each of the three genes contained an intron in the 3′-UTR. We are generating gene constructs with premature stop codons with and without the intron to test the role of the intron in nonsense-mediated decay of mRNA.

O5.3 CRYSTAL STRUCTURE ANALYSIS OF THE TYPE III POLYKETIDE SYNTHASE THAT PRODUCES CURCUMINOID

Hiroyuki Morita, Ikuro Abe

Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, 113-0033, Japan, and JST, CREST, Tokyo, 102-0075, Japan; [email protected]

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C₆-C₇-C₆ diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5 Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C₆-C₃ coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H₂O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C₆-C₇-C₆ scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin.

O5.4 EXTRACELLULAR GLYCOSIDASES OF PYTHIUM IRREGULARE

Dimitre A. Ivanov, Mark A. Bernards

University of Western Ontario, Department of Biology & the Biotron, London, ON N6A 5B7, Canada; [email protected]

The ginseng (Panax quinqufolius L.) pathogen Pythium irregulare (Buis) is able to selectively metabolize the 20(S)-protopanaxadiol ginsenosides Rb1, Rb2, Rc, Rd, and gypenoside XVII in vitro via extracellular glycosidases, leading to the formation and partial assimilation of ginsenoside F2. To determine whether there is a correlation between the activity of ginsenoside metabolizing β-glucosidases and the pathogenicity of P. irregulare towards ginseng, the production of ginsenoside-specific glycosidases and pathogenicity of various isolates of P. irregulare were determined. For this, 10 isolates of P. irregulare were selected on the basis of their genetic variability and the host plant they were isolated from (including ginseng), and obtained from the Canadian Collection of Fungal Cultures. These isolates were cultured in vitro, in the presence of ginsenosides and the level of ginsenoside-specific glycosidase activity in their extracellular proteins was measured. Meanwhile ginseng seedlings were inoculated with the same suite of P. irregulare isolates and scored for disease symptoms to estimate the relative pathogenicity of each isolate towards ginseng plants. When combined this data shows a positive correlation between glycosidase activity in P. irregulare and the pathogenicity of this organism towards ginseng.

O5.5 DECIPHERING MOLECULAR MECHANISMS OF LIGNIN PRECURSOR TRANSPORT

Ke-Wei Zhang, Yu-Chen Miao, Chang-Jun Liu

Brookhaven National Laboratory, Biology Department, Upton, NY 11973, USA; [email protected]

Lignin is the second most abundant terrestrial biopolymer after cellulose. It reinforces and waterproofs cell walls of vascular plants, therefore, is essential for the viability of plants. However, the presence of lignin in cell wall impedes the efficient utilization of cellulosic fibers in agricultural and industrial processes. Lignin precursors, the monolignols, are synthesized within the cytoplasm of the cell. Thereafter, these monomeric precursors are translocated into the cell wall, where they are polymerized and integrated into the wall matrix. While the biosynthesis of monolignols is relatively well understood, our knowledge on transport of these monomers is sketchy. To explore the molecular mechanisms underlying monolignol transport, we used isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, to examine the uptake of monolignols and their derivatives. We found that the transport of lignin precursors across plasmalemma and sequestration of them into vacuoles are ATP-dependent primary-transport processes. Therefore, transport across cell membrane likely involves ATP-binding cassette (ABC) transporters. By gene expression correlation analysis, we then identified a set of ABC transporter genes that their expressions potentially link to lignin biosynthesis. By in vitro biochemical analysis using a dedicated transporter expression system and genetic disturbance of gene expression, we identified one specific ABC transporter acting as exporter of monolignols and responsible for the tissue-specific lignin deposition. The detailed analysis will be presented and discussed.

O5.6 BIOSYNTHESIS AND STEREOCHEMISTRY OF 9,9′-DEOXYNEOLIGNANS IN SAURURUS CHINENSIS

Takeshi Katayama, Toshisada Suzuki, Keisuke Makino, Kenji Denda

Kagawa University, Faculty of Agriculture, Miki-cho, Kagawa 761-0795, Japan; [email protected]

9,9′-Deoxyneolignans were isolated mainly from Myristicaceae, Lauraceae, and Piperaceae plants. However, their biosynthesis is unknown. The present report describes stereochemistry and biosynthesis of D7′-4,7-dihydroxy-3,3′-dimethoxy-8-O-4′-neolignans, whose erythro and threo isomers are named as machilins C and D, respectively, in Saururus chinensis (Saururaceae). (1) The neolignans were prepared as racemic standards by dehydrogenation of isoeugenol with horseradish peroxidase and hydrogen peroxide. The diastereomeric ratio (erythro:threo) was 16:84. (2) Machilin D (threo) was isolated from the underground parts of the plant with an enantiomeric ratio [(+):(–)] of 2:98. Machilin C (erythro) was not isolated. (3) To determine the enantiomeric ratio of the preferred diastereomer (machilin D), its terminal double bond was saturated by catalytic reduction with 10% palladium carbon and chiral HPLC of the resulting dihydromachilin D was done. (4) Dehydrogenation of isoeugenol with a soluble enzyme preparation from the plant in the presence of hydrogen peroxide gave machilins C and D in the ratio of 14:86, respectively. This machilin D was racemic. (5) Dehydrogenation of isoeugenol with an insoluble enzyme preparation from the plant in the presence of hydrogen peroxide afforded the neolignans in the ratio of 16:84, respectively. The enantiomeric ratio of the resulting machilin D (as dihydro form) was (+):(–) = 2:3 (20% e.e.). Stereoselective formation of (–)-machilin D by an enzyme activity in S. chinensis was suggested.

O5.7 A STRESS-INDUCED RICE ENZYME THAT EQUILIBRATES GLUCOSYL CONJUGATES

James R. Ketudat Cairns,¹ Sukanya Luang,¹ Kunnika Phasai,¹ Rodjana Opassiri,¹ Takashi Akiyama,² Nobuhiro Sasaki,³ Yoshihiro Ozeki,³ Yuki Matsuba³

¹Suranaree University of Technology, Institute of Science, Schools of Biochemistry & Chemistry, Nakhon Ratchasima, 30000, Thailand, ²National Agricultural Research Center for the Hokkaido Region, Sapporo, Hokkaido 062–8555, Japan, ³Tokyo University of Agriculture and Technology, Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo, 184–8588, Japan; [email protected]

Os9BGlu31, a rice (Oryza sativa L.) member of a monocot-specific subcluster of glycoside hydrolase family 1 (GH1) was found to transfer glucose to carboxylic acids and alcohols, rather than hydrolyze 4-nitrophenyl β-D-glucopyranoside (4NPGlc). The enzyme could also transfer β-D-fucoside and β-D-xyloside, but not other sugars from 4NP glycosides. Among natural glucose conjugates, 1-O-β-D-feruloyl-glucose, 1-O-β-D-4-hydroxybenzoyl-glucose, 1-O-β-D-4-coumaroyl-glucose, 1-O-β-D-vanillyl-glucose, 1-O-β-D-sinapoyl-glucose, phlorizin, apigenin 7-O-glucoside and GA4 glucosyl ester could also act as donors. Of natural substrates available, 4-coumaric acid (k_cat/K_m= 33 s⁻¹ mM⁻¹) and ferulic acid (k_cat/K_m= 25 s⁻¹ mM⁻¹) showed the highest efficiency as acceptors, while a wide range of molecules could act as acceptors with lesser efficiency. The OsBGlu31 gene was most highly expressed in the first day of germination and upon ethephon, abscisic acid, jasmonate, auxin and drought treatments of seedlings, suggesting a role in germination and stress response.

O5.8 STRUCTURAL BIOLOGY STUDY OF PLANT NATURAL PRODUCT BIOSYNTHESIS

Hui Shao, Lenong Li, Haiyun Pan, Luis L. Escamilla-Trevino, Luzia Modolo, Jack W. Blount, Richard A. Dixon, Xiaoqiang Wang

Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK 73401, USA; [email protected]

Plants synthesize a large number of natural products which play important roles in plant defense against microorganisms and herbivores and also have significant health benefits for animals and humans. The biosynthesis of plant natural products is very complex with many different types of enzymes involved in a variety of different chemical reactions. We are working on three types of enzymes involved in plant natural product biosynthesis, including glycosyltransferases for glycosylation, reductases involved in reduction reactions, and cytochrome P450s involved in hydroxylation and dehydration. We have determined crystal structures of several uridine diphosphate glycosyltransferases, NADPH-dependent reductases, and cytochrome P450s. These structures provide essential insights into their structure-function relationships and catalytic mechanisms in their complex biosynthetic processes. Structure-based mutagenesis and the further functional studies were carried out to explore the roles of key residues for catalysis and substrate specificity, and to further decipher the mechanisms. These studies also provide a basis to manipulate enzyme activity and substrate specificity and the biosynthetic processes for enhancing plant disease resistance or producing more or new health-promoting chemicals.

O5.9 MOLECULAR SENSORS IN PLANT THIOL METABOLISM

Joseph M. Jez

Washington University, Department of Biology, St. Louis, MO 63130, USA; [email protected]

Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur state and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism highlights nature’s ability to engineer pathways that respond to multiple inputs and cellular demands under a range of conditions. Recent work on the cysteine and glutathione biosynthesis pathways in plants reveals that macromolecular changes in protein structure play a pivotal role in changing enzyme activities in response to cellular signals. For example, formation of the cysteine regulatory complex by the biosynthetic enzymes, serine acetyltransferase and O-acetylserine sulfhydrylase is critical for modulating activity of the pathway. Likewise, the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase, uses a thiol-based structural switch to sense cellular redox state and to modulate enzyme activity. Ultimately, biochemical regulation of plant metabolic pathways through sensing changes in cellular state may be more general than previously believed.

O5.10 ELUCIDATING THE NETWORK TO POLYMETHOXYLATED FLAVONES IN SWEET BASIL (OCIMUM BASILICUM L.) GLANDULAR TRICHOMES

Anna Berim, David Hyatt, David R. Gang

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA; [email protected]

Sweet basil (Ocimum basilicum L.) accumulates significant amounts of polymethoxylated flavones in its leaves. The metabolic network involved in production of this array of extensively modified compounds has not been elucidated. Analysis of an EST database from the peltate glandular trichomes of four different basil lines that accumulate not only different volatile metabolite bouquets, but also different flavone profiles, afforded a number of candidate flavone O-methyltransferase (FOMT) genes. Recombinant FOMTs display distinct substrate preferences and product specificities that can account for most detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Apparent K_M values in the low micromolar range and specific gene expression profiles support the involvement of specific FOMTs in the biosynthesis of specific flavones in the different sweet basil lines. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regiospecificities. The roles of these individual residues were confirmed by site-directed mutagenesis. A parallel study of flavone A-ring hydroxylases allowed us to delineate the network from apigenin to salvigenin, gardenin B and nevadensin, the major polymethoxylated flavones that accumulate in sweet basil.

O5.11 THE FIRST STEP OF PROANTHOCYANIDIN GALLOYLATION INVOLVES GLUCOSYLTRANSFERASES

Fida Khater, David Fournand, Sandrine Vialet, Emmanuelle Meudec, Véronique Cheynier, Nancy Terrier

¹INRA, UMR Sciences pour l’Enologie, Montpellier, 34060, France; [email protected]

Grape proanthocyanidins (PA) play a major role in organoleptic properties of wine. They are accumulated mainly in skin and seed during the early stages of berry development. Despite the recent progress in understanding PA biosynthesis, the mechanisms involved in PA galloylation are still not elucidated in plants.

Two Myb transcription factors controlling the PA pathway in grapevine have recently been identified and ectopically over-expressed in an homologous system. In addition to already known PA genes, three genes coding for glucosyltransferases were significantly induced in hairy roots over-expressing those Myb factors (Terrier et al., Plant Physiology, 2009). The three glucosyltransferases display high sequence similarities with other plant glucosyltransferases able to catalyze formation of glucose esters. Studies of the in vitro properties of these 3 enzymes were performed through production of recombinant proteins and they are able to catalyze the formation of 1-O-acyl-Glc esters of phenolic acids but are not active on flavonoids and stilbenes. The transcripts are expressed in the early stages of grape berry development, mainly in skins and seeds. The results presented here suggest that these enzymes could be involved in PA galloylation.

O5.12 STUDIES ON THE BIOSYNTHESIS OF DEOXYNOJIRIMYCIN IN BACILLUS AMYLOLIQUEFACIENS

Lorraine F. Clark, Jodie V. Johnson, Nicole A. Horenstein

University of Florida, Chemistry, Gainesville, FL 32611, USA; [email protected]

Deoxynojirimycin (DNJ) is an analog of glucose containing a nitrogen atom in place of the endocyclic ring oxygen of the pyranosyl ring. This natural product, and related azasugars have been long known as inhibitors of glycosidase and glycosyltransfer enzymes, and more recently, have become of keen interest for use as molecular chaperones. Though azasugars have been widely popular and have defined the paradigm for molecular features of many glycosidase inhibitors, the biosynthetic pathway had remained unreported for many years. We report here our progress on the identification of genes involved in deoxynojirimycin biosynthesis in Bacillus amyloliquefaciens, and studies on their function. Initially guided by genomic annotation, blast analyses and chemical logic, we identified a cluster of three genes, gabT1, yktC1 and gutB1 that were likely candidates for the pathway. Knockout of gabT1 resulted in abolition of DNJ production based on kinetic and mass spectrometric analyses. Chemical complementation with a putative biosynthetic intermediate, downstream of the mutation site, restored DNJ production. Further, transformation of E. coli with these three genes resulted in production of a key biosynthetic intermediate, mannojirimycin. Further aspects of work including functional studies of pathway enzymes and the prospects for identification of the remainder of the pathway will be discussed.

S6.1 DIETARY SUPPLEMENTS AND NATURAL HEALTH PRODUCTS: PHYTOCHEMISTRY AND METABOLOMICS AS TOOLS FOR EVIDENCE-BASED DECISIONS ABOUT THEIR USE

Michael Heinrich

Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia and Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, London, WC1N 1AX, United Kingdom; [email protected]

Different (and mostly limited) levels of evidence are available for widely used health products. Using examples from our own metabolomic research and from our critical reviews of the scientific evidence, I discuss new opportunities for an evidence-based assessment of such products.

Euterpe oleracea Mart. (açai) with reportedly high levels of polyphenols (anti-oxidants) has become a poster child of the power of the internet. In vitro and in vivo evidence on its “effectiveness” is very limited and mostly inconclusive (Heinrich et al. 2011). Garcinia mangostana L. (mangosteen) yields “liquid botanical supplements”, but evidence for their health benefits of is still lacking. Central to the species biological activity are xanthones. A serious weakness is the lack of clinical data (Obolskiy et al. 2009). Serenoa repens (Bartrum) Sm. (saw palmetto) is used in the treatment of Benign Prostatic Hyperplasia. Overall, the clinical evidence is much better, but very often the composition of the extracts used is not known and is variable. A metabolomic analysis (Booker, Suter and Heinrich, unpublished) identified oleic acid and caproic acid ethyl ester as potential marker compounds.

A more rigorous systematic strategy which integrates phytochemical and pharmacological is needed in order to assess the health claims of such products. With the rapid rise of internet-driven marketing, we also need strategies to prioritise products that need to be assessed.

• Heinrich, M., T. Dhanji, I. Casselman (2011) Phytochem. Lett. 4:10 - 21.

• Obolskiy, D., I. Pischel, N. Siriwatanametanon, M. Heinrich (2009) Phytother. Res. 27:1047–1065.

S6.2 METABOLOMIC EVALUATION OF SEVERAL ANTICANCER AGENTS OF PLANT ORIGIN

Wei Jia,^1,2,3 Guoxiang Xie^1,3

¹University of North Carolina at Greensboro, Nutrition, Kannapolis, NC 28081, USA, ²Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China, ³Shanghai Jiao Tong University, Shanghai, 200240, China; [email protected]

Emerging evidence indicates that altered cellular metabolism is the defining characteristic of nearly all cancers regardless of cellular or tissue origin. Therefore, a view of cancer as primarily a metabolic disease suggests that metabolomic technology can be applied in cancer research for the investigation of pharmacological effects as well as mechanism of action. We recently evaluated the cancer-preventive effect of several plant-derived compounds, quercetin, resveratrol, salvianolic acid B (Sal-B, isolated from Salvia miltiorrhiza Bge, Danshen), and breviscapine (a flavonoid isolated from Herba Erigerontis), which were previously reported to have inhibitory effect against the malignant transformation of precancerous lesion. A precancerous colorectal lesion model with azoxymethane (AOM) treated male wistar rats was investigated to evaluate dietary resveratrol and quercetin treatment, alone or in combination. Additionally, a 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral carcinogenesis model in hamster was used to evaluate the Sal-B and breviscapine treatments. The global metabolic variations in sera and tissues of model groups and treatment groups were characterized by gas chromatography time of flight mass spectrometry (GC-TOFMS). The dynamic changes of metabolic profiles indicate that these natural compounds were able to attenuate chemical-induced metabolic perturbation, which is consistent with the findings of significantly decreased lesion/ carcinoma incidences in the treatment groups. Differentially expressed metabolites involved glycolytic intermediates, amino acids, TCA intermediates, fatty acids and nucleosides.

S6.3 COPALCHI AND OTHER SELECTED ANTIDIABETIC PLANTS FROM MEXICO

Rachel Mata, Fernando Brindis, Sol Cristians

Universidad Nacional Autónoma de Mexico, Facultad de Química, Mexico City, DF 04510, Mexico; [email protected]

There is currently no cure for diabetes, but the condition can be managed so that patients can live a relatively normal life. Oral medications are available to lower blood glucose in type-II diabetics but most of these products possess side effects after prolonged use. In consequence, the search for new therapeutic agents for treating type-II DM, including plants used in folk medicine has increased notably in recent years. Mexico is rich in medicinal plants highly prized by the population for the treatment of diabetes and, according to a recent review there are about 306 species from 235 genera and 93 families used as hypoglycemic agents. Copalchi, Hintonia latiflora (Rubiaceae), Ligusticum porteri (Apiaceae), and Brickellia cavanillesii (Asteraceae) are some of these. A concise overview on these species will be presented including phytochemistry aspects, quality control methods, as well as preclinical safety and efficacy parameters, stemming from our own work.

S6.4 TRADITIONAL OCEANIC CROPS FOR IMPROVED NUTRITION, NUTRACEUTICALS AND NATURAL HEALTH PRODUCTS

Susan Murch

University of British Columbia, Kelowna, BC V1V 1V7, Canada, and National Tropical Botanical Garden, Kalaheo, HI 96741, USA; [email protected]

Breadfruit, Artocarpus altilis (Parkinson) Fosberg, has been a staple food and traditional crop in the Pacific for more than 3,000 years and is widely cultivated in the Caribbean and other tropical regions. In 1787 Captain W. Bligh sailed the Bounty to Tahiti to collect breadfruit trees for food in the Caribbean. A single breadfruit tree produces 150–200 kg of fruit per year and the fruit can be eaten fresh or processed into fermented products or flours. There are hundreds of cultivars of breadfruit selected by indigenous peoples across Oceania that have been collected and curated at the Breadfruit Institute germplasm bank of the National Tropical Botanical Garden, Hawaii. Individual varieties vary in specific vitamins, nutrients, proteins, salt tolerance, and medicinal phytochemicals. The process of bringing breadfruit to modern markets required the negotiation of international agreements for equitable benefit sharing, development of mass-propagation technologies for large scale production of disease free plants, and studies to identify nutrient-rich varieties, develop new products and create opportunities for not-for-profit distribution.

S6.5 GLYCYRRHIZA URALENSIS: UNUSUAL CHEMISTRY AND UNUSUAL APPLICATIONS

Stefan Gafner,¹ Chantal Bergeron,¹ Jacquelyn Rae Villinski,¹ Markus Godejohann,² Pavel Kessler,² John Henry Cardellina,³ Daneel Ferreira,⁴ Karine Feghali,⁵ Jacynthe Desjardins,⁵ Daniel Grenier⁵

¹Tom’s of Maine, R&D, Kennebunk, ME 04043, USA, ²Bruker-Biospin, R&D, Rheinstetten, Baden-Württemberg 76287, Germany, ³Reeves Group, Walkersville, MD 21793, USA, ⁴University of Mississippi, Department of Pharmacognosy and National Center for Natural Products Research, University, MS 86773, USA, ⁵Université Laval, Groupe de Recherche en Ecologie Buccale, Québec City, QC G1V 0A6, Canada; [email protected]

The phytochemical investigation of a supercritical fluid extract of Glycyrrhiza uralensis led to the isolation of 20 known isoflavonoids and coumarins, as well as glycycarpan, a new pterocarpan. The presence of two isoflavan-quinones, licoriquinone A and licoriquinone B, in a fraction subjected to gel filtration on Sephadex LH-20 is believed to be due to metal-catalyzed oxidative degradation of licoricidin (1) and licorisoflavan A (2). The licorice extract as well as 1 and 2 were able to reduce volatile sulfur compounds (VSCs) production by Porphyromonas gingivalis, Prevotella intermedia, and Solobacterium moorei as well as in a human saliva model. Although the extract and isolates did not inhibit the proteolytic activity of bacteria, they blocked the conversion of cysteine into VSCs by P. intermedia. Compounds 1 and 2 also showed potent antibacterial activities, causing a marked growth inhibition of the cariogenic species Streptococcus mutans and Streptococcus sobrinus at 10 μg/mL and the periodontopathogenic species P. gingivalis (at 5 μg/mL) and P. intermedia (at 5 μg/mL for 1 and 2.5 μg/mL for 2). Only 1 moderately inhibited growth of Fusobacterium nucleatum at the highest concentration tested (10 μg/mL).

S6.6 MULTILAB METHOD VALIDATION OF BLUEBERRY LEAF EXTRACT BY NMR: QUALITATIVE AND QUANTITATIVE

Josh Hicks,¹ Sarah Luchsinger,¹ Brian Killday,¹ Pavel Kostikin,¹ Asim Muhammad,² John Arnason,² Fabrice Berrue,³ Chris Kirby,⁴ Kevin Knagge,⁵ Jianping Zhao,⁶ Ikhlas Khan,⁶ Tanja Goedecke,⁷ Guido Pauli,⁷ Wenbin Luo,⁸ Alan Benesi,⁸ Wendy Popplewel,⁹ Kirk Gustafson,⁹ Tobias Karakach,¹⁰ Ian Burton,¹⁰ Mark O’Neil-Johnson,¹¹ Kim Colson¹

¹Bruker BioSpin, Billerica, MA 01821, USA, ²U. Ottawa, Ottawa, ON K1N 6N5, Canada, ³U. of PEI, Charlottetown, PE C1A4P3, Canada, ⁴Agriculture and Agri-Food Canada, Charlottetown, PE C1A 4N6, Canada, ⁵Murdock Research Institute, Kannapolis, NC 28081, USA, ⁶U. of Mississippi, Oxford, MS 38677, USA, ⁷U. Illinois at Chicago, Chicago, IL 60680, USA, ⁸Penn State U., University Park, PA 16802, USA, ⁹NCI, Frederick, MD 21702, USA, ¹⁰NRC, Halifax, NC B3H 3Z1, Canada, ¹¹Sequoia Sciences, St. Louis, MO 63114, USA; [email protected]

The ability to quickly assay natural product extracts for new compounds or higher levels of known compounds or confirm the natural source or origin can enhance the selection of natural products for ensuring efficacy, safety, origin, and quality. Similarly, the ability to perform these assays reproducibly on different instruments and at multiple locations frees users from time consuming sample validation on multiple instruments independently. NMR is well documented as an analytical technique which provides structurally definitive and quantitative data simultaneously in a single NMR experiment. NMR’s high reproducibility across platforms allows qualitative assessment (chemometric modeling) of highly complex samples such as botanical extracts, which enables data comparison at different sites. Concepts and results of a 12 site multi-site reproducibility study are presented, which aims to identify and quantify compounds directly from the spectra of raw blueberry leaf extract and qualitatively identify the natural source of the raw plant material.

O6.1 HUO-LUO-XIAO-LING DAN (HLXL) PROTECTS AGAINST EXPERIMENTAL ARTHRITIS BY MODULATING ANTIGEN-INDUCED CELLULAR AND HUMORAL RESPONSES

Ying-Hua Yang,¹ Rajesh Rajaiah,¹ David Y. Lee,² Zhongze Ma,² Hua Yu,¹ Harry H. Fong,³ Lixing Lao,⁴ Brian M. Berman,⁴ Kamal D. Moudgil¹

¹University of Maryland School of Medicine, Microbiology and Immunology, Baltimore, MD 21201, USA, ²McLean Hospital, Harvard Medical School, Mailman Research Center, Belmont, MA 02478, USA, ³University of Illinois at Chicago, Department of Medicinal Chemistry and Pharmacognosy, Chicago, IL 60612, USA, ⁴University of Maryland School of Medicine, Center for Integrative Medicine, Baltimore, MD 21201, USA; [email protected]

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint damage. Proinflammatory cytokines, antibodies, matrix-degrading enzymes and nitric oxide mediate the pathogenic events in arthritis. Accordingly, we examined whether an herbal formula, HLXL, which has long been used in traditional Chinese medicine for the treatment of arthritic conditions, targets these mediators of inflammation leading to attenuation of arthritis. We tested HLXL in the rat adjuvant-induced arthritis model of human RA. We demonstrated that oral administration of HLXL (2.3 g/kg) to Lewis rats after the onset of arthritis significantly reduced the severity of arthritis compared with the water-fed controls. Interestingly, HLXL-fed rats revealed a lower concentration of the proinflammatory cytokines interleukin-17 (IL-17) and IL-1β but a higher concentration of the immunoregulatory cytokine IL-10 in recall response to antigen than controls. HLXL feeding also suppressed the serum levels of antigen-specific antibodies as well as nitric oxide. These results provide a strong rationale for further testing and validation of the use of HLXL in patients with RA.

O6.2 NEW MODELS IN TRANSLATIONAL PHYTOTHERAPY: DIETARY PHYTOCHEMICALS IN FUNCTIONAL NUTRITION MANAGEMENT AND PHARMACONUTRITION

Cedric B. Baker,¹ Eloy Rodriquez²

¹Mercer University, College of Pharmacy and Health Sciences, Department of Pharmacy Practice, Atlanta, GA 30341-4155, USA, ²Cornell University, Department of Plant Biology, Laboratory of Chemical Biology-Ecology & Medical Ethnobotany, Ithaca, NY 14853, USA; [email protected]

The new fields of Functional Nutrition Management (FNM) and Pharmaconutrition (PN) deal with the transdisciplinary integration of the multifarious dietary phytochemicals in functional foods, medical foods, and botanical dietary supplements. Total evidence review from epidemiology, in vitro tests, and clinical trials supports health benefits from multicomponent dietary phytochemical mixtures, whole foods, and dietary patterns, but overall appears to be lacking in isolated individual dietary phytochemicals. The need for a new model that addresses this disparity in health benefits leads us to propose a novel way to develop therapies based on a dietary phytochemical model that integrates chemical ecology with traditional medical systems from a functional nutrition and pharmaconutrition perspective with medicinal foods and food components. A case study of the Zingiberaceae plant family in this integrative context will explore ginger (Zingiber officinale), tumeric (Curcuma longa), galangal (Alpinia galangal), and Thai finger root/krachai (Boesenbergia pandurata), and their respective phytochemical profiles (e.g., gingerols, shogaols, diarylheptanoids/curcuminoids, chalcones, and other phenolic compounds/flavonoids) with known functional and pharmacological bioactivity.

O6.3 TOXICOLOGICAL MECHANISMS OF BOTANICAL DIETARY SUPPLEMENTS

Dale G. Nagle,¹ Fahkri Mahdi,¹ Sandipan Datta,¹ Jun Li,¹ Lin Du,¹ Troy J. Smillie,² Ikhlas A. Khan,^1,2,3 Mika B. Jekabsons,⁴ Yu-Dong Zhou¹

¹University of Mississippi, Department of Pharmacognosy, School of Pharmacy, University, MS 38677, USA, ²University of Mississippi, National Center for Natural Products Research and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, University, MS 38677, USA, ³King Saud University, Department of Pharmacognosy, College of Pharmacy, Riyadh, Saudi Arabia, ⁴University of Mississippi, Department of Biology, University, MS 38677, USA; [email protected]

Reports indicate that a variety of botanical dietary supplement (BDS) products are associated with hepatotoxicity or other forms of idiosyncratic toxicity. Extracts from more than 350 species of plants and other organisms used in traditional Chinese, Indian, African, and Western herbal medicine were evaluated for their ability to disrupt mitochondrial function in cultured cells. Extracts from 15 different plant species suppressed mitochondrial respiration. Extracts from other species potently uncoupled oxidative phosphorylation. Several plant species that have been associated with severe hepatic or cardiac toxicity contain compounds that have been reported to interfere with mitochondrial function. While the pharmaceutical industry has recently come to recognize the importance of implementing measures to assess the potential mitochondrial toxicity of new drug leads, no such methods have been widely used to evaluate the botanical constituents found in herbal medicines. These results indicate that certain plant extracts, including some that were previously thought to be safe, contain components that induce mitochondrial dysfunction and may be responsible for potential BDS-induced liver or heart toxicity.

O7.1 PTEROSTILBENE INCREASES PPARα GENE EXPRESSION, ACTIVATES AMPK, AND SUPPRESSES EXPRESSION OF GENES INVOLVED IN HEPATIC LIPID METABOLISM AND GLUCONEOGENESIS

Agnes M. Rimando,¹ Guang Ren,² Suresh Mathews²

¹U.S. Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, MS 38677, USA, ²Auburn University, Department of Nutrition, Auburn, AL 36849, USA; [email protected]

Pterostilbene (a dimethylether analog of resveratrol) at 10, 20 and 50 μM dose-dependently increased PPARα gene expression in H4IIE rat hepatoma cells, consistent with earlier studies that showed it is a PPARα agonist, and provided greater increases in PPARα gene expression than 100 and 200 μM fenofibrate. Expectedly, pterostilbene increased gene expression of fatty acyl-CoA oxidase and carnitine palmitoyltransferase-1, enzymes involved in fatty acid catabolism. Phosphorylation of 5′-adenosine monophosphate-activated protein kinase (AMPK), known to regulate fatty acid metabolism, was increased by pterostilbene dose-dependently (at 10, 20 and 50 μM); 50 μM pterostilbene activated AMPK to a greater extent than AICAR (0.5 mM) or metformin (2 mM). AMPK is also known to repress transcription of phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), enzymes involved in hepatic gluconeogenesis. In this study, dexamethasone was used to induce gluconeogenesis, and insulin as positive control to suppress gluconeogenesis. Similar to insulin, pterostilbene dose-dependently decreased gene expression of PEPCK and G6Pase. Additionally, pterostilbene decreased glucose production in H4IIE cells. This is the first study to show pterostilbene activates AMPK, regulates FA metabolism, and suppresses gluconeogenesis, providing mechanistic support for its use in management of dislipidemia and type 2 diabetes.

O7.2 THE ISOFLAVONOID, EQUOL IMPROVES SEVERE AND MODERATE BPH SYMPTOMS IN MID-AGED CAUCASIAN MEN: CLINICAL EVIDENCE

Edwin D. Lephart,^1,2 Ronald L. Rizer³

¹Brigham Young University, PDBio & The Neuroscience Center, Provo, UT 84602, USA, ²Homeopathic Holdings LLC, Pleasant Grove, UT84062, USA, ³Thomas J. Stephens & Associates, Inc., Clinical Research, Colorado Springs, CO 80915, USA; [email protected]

Equol, the major metabolite of the isoflavone daidzein, specifically binds and blocks the hormonal action of 5α-dihydrotestosterone (DHT) in vitro and in vivo (Reprod Biol Endo, 2011, 13; 9:4). Equol can at the same time bind estrogen receptor beta that has important implications for prostate disorders. The objective of this clinical study was to provide proof of principle that equol at a low effective dose improves health symptoms of benign prostatic hyperplasia (BPH). A proprietary non-racemic equol mixture (6 mg) was taken twice per day for 4 weeks by 18 healthy males between 49 and 60 years old that have BPH according to the International Prostate Symptoms Score (IPSS) form before treatment started (baseline). Institutional Board Review approval, informed consent, medical histories and qualification produces were followed. At baseline (0 weeks), at 2 weeks and then at 4 weeks all subjects completed the IPSS forms indexing 7 BPH parameters; total scores and self-perceived quality of life changes. Data was analyzed by repeated ANOVA. In brief, the overall IPSS scores in both groups significantly improved from baseline levels at 0 weeks compared to 2 or 4 weeks with equol treatment. In the severely symptomatic group (n = 8): each IPSS indicator and the quality of life significantly improved by 4 weeks with equol treatment. These results demonstrate great promise for equol at a low effective dose to improve BPH symptoms in men. Funding: HoHo

O7.3 NONPSYCHOACTIVE CONSTITUENTS FROM CANNABIS SATIVA (MARIJUANA): THERAPEUTIC POTENTIAL IN INFLAMMATORY DISORDERS AND DIABETES

Pal Pacher

National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Section on Oxidative Stress Tissue Injury, Laboratory of Physiologic Studies, Bethesda, MD 20852, USA; [email protected]

Medical marijuana plant (Cannabis sativa) has been used for centuries for various therapeutic indications in human medicine. Previously it was thought that its only active constituent is the δ-9-tetrahydrocannabinol (THC). THC is a nonselective agonist of both cannabinoid receptors 1 and 2 (CB1, CB2). While CB2 activation in immune cells results in potent anti-inflammatory effects, CB1 stimulation in the central nervous system (CNS) decreases pain, but leads to undesirable psychoactive consequences, thus limiting the therapeutic potential of THC and marijuana. However, numerous recent studies, including those from our laboratories have demonstrated that several previously considered inactive constituents of C. sativa, such as cannabidiol or δ-9-tetrahydrocannabivarin (THCV), which do not exert psychoactive effects via CB1 activation, may exert very potent anti-inflammatory and antioxidant properties, and attenuate the disease progression in preclinical models of diabetes/diabetic complications, ischemic-reperfusion injury, inflammation, and cancer, just to mention a few. Examples of the powerful anti-inflammatory and tissue protective effects of these natural plant derived constituents from preclinical disease models will be presented, and their tremendous therapeutic potential will be discussed.

O7.4 SESQUITERPENES FROM HOLOSTYLIS RENIFORMIS

Lucia M. X. Lopes,¹ Marcos D. P. Pereira,¹ Tito da Silva,¹ Antoniana U. Krettli²

¹Institute of Chemistry, São Paulo State University, UNESP, Organic Chemistry, Araraquara, SP 14800-900, Brazil, ²Institute René Rachou, FIOCRUZ, Laboratory of Malaria, Belo Horizonte, MG 30190-002, Brazil; [email protected]

The hexane extract of Holostylis reniformis roots yield oily fractions which were analyzed by GC/MS and ¹H and ¹³C NMR spectroscopy. Palmitic acid, guaiol, 1-epi-cubenol, 10-epi-eudesmol, γ-eudesmol, bulnesol, elemol, and β-eudesmol were the main constituents in the oil. In addition, the new megastigmane (1) and nine-membered lactone (2), together with the 4,5-seco-guaiane (3), with an unusual carbon skeleton, were isolated from root extracts and their structures were determined by spectroscopic analyses. (FAPESP, CNPq).

O7.5 TRIPTOLIDE AMELIORATES INSULIN RESISTANCE IN OBESE DIABETIC MICE

Jennifer Huang,¹ Debora Esposito,¹ Pablo Kizelsztein,¹ Ilya Raskin,¹ Slavko Komarnytsky^1,2

¹Biotech Center, Rutgers University, New Brunswick, NJ 08901, USA, ²Plants for Human Health Institute, NCSU, Kannapolis, NC 28081, USA; [email protected]

Diabetes arises because of failed insulin action, however the role of inflammation in insulin resistance remains unclear. To determine if targeting systemic inflammation ameliorates diabetes, we orally administered 50 μg/kg triptolide every 3 d for 9 wk to high fat diet-induced obese C57BL/6J mice. Triptolide (1) is a diterpene triepoxide isolated from a traditional Chinese medicinal plant (Tripterygium wilfordii) with anti-inflammatory and immunosuppressive properties. Mice treated with triptolide exhibited higher glucose disposal by oral glucose tolerance test with no significant changes in body weight, lean body mass, or fat mass. We next determined changes in macrophage recruitment and cytokine signature of adipose tissue in these animals. Visceral fat from triptolide-treated mice showed decreased expression of inflammatory markers TNFα, IL-6, and monocyte chemoattractant protein (MCP). These results show the importance of adipose tissue inflammation and gene regulation in systemic glucose metabolism and insulin sensitivity.

O7.6 ANTIOXIDANT ACTIVITY OF HAWAIIAN MACRO-ALGAE

Ellen Kromkowski Posner,¹ Karla J. McDermid,¹ Anthony D. Wright,² Dovi Kelman²

¹University of Hawaii at Hilo, Department of Marine Science, Hilo, HI 96720, USA, ²University of Hawaii at Hilo, College of Pharmacy, Department of Pharmaceutical Sciences, Hilo, HI 96720, USA; [email protected]

Marine macro-algae are known to contain a wide variety of bioactive compounds, many of which have commercial applications in pharmaceutical, medical, cosmetic, nutraceutical, food and agricultural industries. Natural antioxidants, found in many macro-algae, are important bioactive compounds that play an important role against various diseases and ageing processes through protection of cells from oxidative damage. In this respect, relatively little is known about the bioactivity of Hawaiian macro-algae that could be a potential natural source of such antioxidants. The antioxidant activity of organic extracts of 27 species of Hawaiian macro-algae from 25 different genera was determined. The activity was determined by employing the FRAP (Ferric Reducing Antioxidant Power) assay. Of all of the algae tested, the extract of Turbinaria ornata was found to be the most active. Bioassay-guided fractionation of this extract led to the isolation of a variety of different carotenoids as the active principles. These results show, for the first time, that numerous Hawaiian macro-algae exhibit antioxidant activity, a property that could lead to an application in one of many useful healthcare or related products.

O7.7 MEDICINAL PLANTS OF HIGH POTENCY AND THEIR APPLICATIONS IN TRADITIONAL MEDICINE

Varima Wongpanich, Srisomporn Preeprame

Faculty of Pharmaceutical Sciences, Khon Kaen University, Division of Pharmacognosy and Toxicology, Khon Kaen, 40002, Thailand; [email protected]

Ethnopharmacological information has a role to play as one of the approaches to drug discovery and a number of medicinal plants are well recognized to provide active principles for modern medicine. However, many failed along the empirical pipeline of development because of their toxicity. In this work, we review 12 medicinal plants recognized in Thai Traditional Medicine as potent medicinal ingredients, 10 of which are mentioned possibly as fatal. Five medicines are Euphorbiaceous products, the others are derived from Anacardiaceae, Cannabaceae, Convolvulaceae, Lauraceae, Papaveraceae, Solanaceae, and Strychnaceae. Their indications are not only strictly described, but applications also require some special traditional technique prior to incorporating to the formulated preparations which will be discussed herein. Other aspects of concern with respect to attitudes towards modern and traditional medicines will be described in this presentation.

O7.8 NOVEL TRITERPENOID DERIVATIVES FROM EUCOMIS BICOLOR (HYACINTHACEAE: HYACINTHOIDEAE)

Jaspreet Kaur Sihra,¹ Dulcie A. Mulholland,¹ Moses K. Langat,¹ Neil R. Crouch,² Jean-Marc Nuzillard,³ Alfred E. Thumser⁴

¹University of Surrey, Chemical Sciences, Faculty of Health and Medical Sciences, Guildford, GU2 7HX, United Kingdom, ²South African National Biodiversity Institute, Ethnobotany Unit, Durban, 4007, South Africa, ³University of Reims, Pharmacognosy Laboratory, Reims, 51687, France, ⁴University of Surrey, Biological Sciences, Faculty of Health and Medical Sciences, Guildford, GU2 7XH, United Kingdom; [email protected]

The phytochemical investigation of the dichloromethane extract of Eucomis bicolor has yielded five novel compounds (1-5). The triterpenoid derivatives have been screened against several tumour cells.

O7.9 DISCOVERY OF KANGAROO ISLAND MEDICINAL PLANTS USING HONEYBEES

Colin Charles Duke,¹ Rujee Kyokajee Duke,² Van Hoan Tran,¹ Abdallah Abu-Mellal,¹ Nooshin Koolaji¹

¹University of Sydney, Faculty of Pharmacy, Sydney, NSW 2006, Australia, ²University of Sydney, Faculty of Medicine, Department of Pharmacology, Sydney, NSW 2006, Australia; [email protected]

Kangaroo Island with its unique endemic flora is a potential source of medicinal plants. With very limited introduced flora, the island is well suited to employ honeybees (Apis mellifera) to source out the medicinal plants. Honeybees collect resins or exudates from plants to produce bee glue (propolis) to seal and disinfect their hives. Propolis has been well recognized for its medicinal property since ancient civilization, and still retains current worldwide popularity as a traditional medicinal product. As a sanctuary for Ligurian honeybees (Apis mellifera ligustica), Kangaroo Island has strict quarantine regulations with well established beekeeping practice including propolis production.

Methods were established to use honeybees to identify the plant sources of novel bioactive natural products. Single plant source propolis, resin/exudate from the plants and propolis carried on bee legs were matched, mainly through comparison of ¹H-NMR spectra analytical profiles. Our survey of distribution of source plants has identified chemotypes and their abundance on the island.

Our studies showed that the composition of propolis from Kangaroo Island is novel, containing many new chemical entities as the main constituents. In particular, novel prenylated cinnamates and prenylated tetrahydroxystilbenes were identified and biologically evaluated.

O7.12 BIOPRODUCTS FROM THE CANADIAN FOREST

Mamdouh M. Abou-Zaid, Domenic A. Lombardo, Linda Buchan, Melanie Coppens, Scott Hopkin

Natural Resources Canada, Canadian Forest Services, Great Lakes Forestry Centre, Sault Ste. Marie, ON P6A 2E5, Canada; [email protected]

Canada’s boreal forest is a “natural synthetic manufacturing factory” continuously working on the production of enormous numbers of complex organic substances playing a variety of roles and possessing diverse bioactivities. Each species has a unique phytochemical profile with characteristic classes of substances, a phenomenon known as phytochemical diversity and redundancy. Their biosyntheses, the metabolic sequences leading to the production of the various classes of natural products, are thoroughly interconnected.

Canadian forest flora provides for a diverse and rich source of bioactive natural product compounds. Among the compounds of interest are those of the phenolic, polyphenol, and flavonoids classes of compounds. Extracts of indigenous plant species have been screened for antioxidant (maple syrup), and antifungal and anticancer activities. Those plants whose whole foliage extracts exhibited preliminary activity were subjected to a bioassay-guided fractionation and isolation of the active constituents from the plant extracts.

This presentation describes our laboratory’s on-going research interests involving the screening, fractionation, isolation, purification and structural elucidation of the antioxidant/antifungal-active compounds from Acer, Pinus, Taxus, Chimaphila, and Abies species, and considers their role in health promotion and disease prevention.

O7.13 CULTURED CAMBIAL MERISTEMATIC CELLS AS A SOURCE OF PLANT NATURAL PRODUCTS

Eun-Kyong Lee,² Young-Woo Jin,² Joong Hyun Park,² Young Mi Yoo,² Sun Mi Hong,² Rabia Amir,¹ Zejun Yan,¹ Eunjung Kwon,¹ Alistair Elfick,³ Simon Thomlinson,⁴ Florian Halbritter,⁴ Thomas Waibel,¹ Byung-Wook Yun,¹ Gary Loake¹

¹University of Edinburgh, Institute of Molecular Plant Sciences, Edinburgh, EH9 3JR, United Kingdom, ²Unhwa Corp, 874-1, 2Ga, Wooah-Dong, Dukjin-gu, Jeonju, South Korea, ³University of Edinburgh, School of Engineering, Edinburgh, EH9 3JL, United Kingdom, ⁴University of Edinburgh, Institute of Stem Cell Research, Edinburgh, EH9 3JH, United Kingdom; [email protected]

A plethora of important, chemically diverse, natural products are derived from plants. In principle, plant cell culture offers an attractive production platform for some natural products but often is not a commercially viable strategy because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To address this problem, we have isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Utilizing a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by CMC morphology, hypersensitivity to γ-irradiation and radiomimetic drugs and the ability of these cells to differentiate at high frequency. CMCs derived from Taxus cuspidata, source of the key anticancer drug, paclitaxel, circumvented obstacles routinely associated with the commercial growth of DDCs. These cells may therefore provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products.

O7.14 BALANCING RESEARCH AND SUSTAINABILITY

Mark Perry

University of Western Ontario, Faculties of Law and Science, London, Ontario N6A 5K7, Canada; [email protected]

There is tension between the need to find new compounds and precursors to give relief from the conditions and diseases that plague humanity, with the need to ensure that sustainability of botanical sources is not compromised. An additional aspect is the desire to reward those that provide clues and knowledge on the efficacy of botanicals (traditional knowledge) that can lead to the mass production of useful compounds. This tension can be seen in the international arena with the contrasting approaches of the Agreement on Trade Related Aspects of Intellectual Property, which the vast majority (153) of nations have accepted, and the Convention on Biological Diversity, with some 193 national parties (with the notable exception of the United States of America). In 2000, the World Intellectual Property Organisation established an intergovernmental committee to provide resolution for the need for international instruments to protect the exploitation of traditional knowledge and genetic resources, and although much progress has been made, a common agreement for such instruments has yet to be met.

This paper looks at the background and outcomes of these tensions over the last decade and proposes steps that could lead to the resolution of the two basic needs, namely advancement of science and the protection of rights.

O8.1 UNRAVELLING MYSTERIES IN THE REGULATION OF PLANT SECONDARY METABOLITES AND NON-GLANDULAR TRICHOMES: NEW MECHANISMS AND CROP APPLICATIONS

Margaret Yvonne Gruber,¹ Xiang Li,³ Peng Gao,² Dejun Cui,¹ Min Yu,¹ Ali Taheri,¹ Nagabushana Nayidu,¹ Ushan Alahakoon,^1,4 Jennifer Holowachuk,¹ Sharon Regan,⁶ Abdelali Hannoufa,⁷ Zakir Hossein,⁷ Isobel Parkin,¹ S. Wei,⁵ P. Bonham-Smith,⁴ B. Yu²

¹Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, SK S7N0X2, Canada, ²Plant Biotechnology Institute, National Research Council, Saskatoon, SK S7N0W9, Canada, ³Jinlin University, College of Plant Sciences, Changchun, Jinlin 130062, China, ⁴University of Saskatchewan, Dept. of Biology, Saskatoon, SK S7N0W9, Canada, ⁵Anhui Agricultural University, School of Tea and Food Science, Hefei, Anhui 20036, China, ⁶Queen’s University, Dept. of Biology, Kingston, ON K7L3N6, Canada, ⁷Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, London, ON N5V4T3, Canada; [email protected]

Anthocyanins, proanthocyanidin, lignin, and carotenoids are important secondary metabolites which protect plants against stress and provide nutritional and health benefits to humans and livestock. Plants with modified lignin show improved cellulose (energy) availability, and trichomes (plant hairs) discourage insects and animals from foraging on plants. Some of the mystery surrounding gene regulation for these plant defence strategies is being unravelled by the new SK population of Arabidopsis enhancer lines and yellow/brown-seeded and wild Brassica systems. The presentation will highlight new tools and new regulatory mechanisms taken from the analysis of a collection of new Arabidopsis mutant lines and Brassica lines with modified flavonoid/phenolic, carotenoid, and trichome pathways and will highlight several useful applications for crop plants.

O8.2 THE ARABIDOPSIS ABCG26 TRANSPORTER: A TOOL FOR INVESTIGATING THE NATURE OF SPOROPOLLENIN

Teagen D. Quilichini, A. Lacey Samuels, Carl J. Douglas

University of British Columbia, Botany, Vancouver, BC V6T 1Z4, Canada; [email protected]

Sporopollenin, a tough biopolymer in spore and pollen walls, protects these structures from environmental stresses. From the data available, sporopollenin is a polymer of fatty acids and oxygenated aromatic compounds. Analyses of Arabidopsis male sterile mutants defective in pollen wall formation have revealed genes required for sporopollenin biosynthesis and/or deposition, including MS2, ACOS5, PKS-A/PKS-B, TKPR1 and ABCG26. Based on genetic and biochemical analysis of these genes and the corresponding enzymes, it has been proposed that an aliphatic polyketide sporopollenin monomer is synthesized in the tapetum. ABCG26, an ABC transport protein, is thought to function in sporopollenin export from tapetum cells. However, the substrate transported by ABCG26 is unknown. In the abcg26 mutant, sporopollenin precursors are predicted to accumulate in the tapetum. Through the analysis of abcg26 by two-photon microscopy, lipidic and autofluorescent compounds in tapetum cells can be visualized. No differences between wild type and abcg26 tapetum lipids were observed. Conversely, abcg26 exhibits autofluorescence in tapetum vacuoles, not observed in wild type. Transmission electron microscopy supports these findings, with enlarged, debris-filled vacuoles in the tapetum of abcg26 mutants. Identification of the autofluorescent components accumulating in abcg26 tapetum cells by biochemical methods will provide an opportunity to examine the composition of sporopollenin in planta. Using live-cell imaging and biochemical methods, we are also investigating the nature of sporopollenin precursors that accumulate in double mutants of abcg26 and acos5, pks-a/pks-b, and tkpr1.

O8.3 QUANTITATIVE ANALYSIS OF NATURAL PRODUCTS BY qNMR

Takamitsu Yoshida,¹ Keiko Hasada,^1,2 Takeshi Yamazaki,³ Naoki Sugimoto,³ Tetsuji Nishimura,³ Akito Nagatsu,² Hajime Mizukami¹

¹Nagoya City University, Graduate School of Pharmaceutical Sciences, Nagoya, 4678603, Japan, ²Kinjo Gakuin University, College of Pharmacy, Nagoya, 4638521, Japan, ³National Institute of Health Sciences, Tokyo, 1588501, Japan; [email protected]

qNMR is a powerful tool for quantitative determination of natural products since (1) it requires no standard compounds to prepare calibration lines, and (2) SI-traceable determination of the organic molecules can be attained by using a certified reference material as an internal standard. We applied qNMR to determine the purities of protoberberine alkaloid reagents obtained in the market. The purity of the reagent estimated by ¹H-NMR was, in general, lower than that claimed by the manufacturer, leading to over-estimation of the alkaloid contents of Coptis Rhizome when determined by HPLC. The present quantitative NMR method was also applicable to direct determination of protoberberine alkaloid contents in Coptidis rhizoma.

We also successfully applied the method to evaluate carthamin contents in commercial Carthamus red colourants without using carthamin as a standard compound.

O8.4 PLANT PHENOLICS ANALYSIS USING ELECTROSPRAY ION MOBILITY TIME-OF-FLIGHT MASS SPECTROMETRY

Fereshteh Zandkarimi¹, Sasidhar Nirudodhi¹, Jan F. Stevens², Claudia S. Maier¹

¹Oregon State University, Department of Chemistry, Corvallis, OR 97331, USA, ²Oregon State University, Department of Pharmaceutical Sciences and Linus Pauling Institute, Corvallis, OR 97331, USA; [email protected]

We report on the characterization of plant phenolics using an electrospray hybrid quadrupole time-of-flight mass spectrometer equipped with a traveling wave ion mobility separation device. There is increasing evidence that polyphenols have health benefits. Considering the increasing interest in plant phenolics as nutraceuticals, comprehensive profiling methods for plant extracts are highly needed. We highlight the application of advanced mass spectrometric fragmentation techniques in combination with ion mobility separation and also its current limitations for the characterization of plant extracts and plant phenolic libraries. We discuss MS and ion mobility data for the characterization of phenolics isolated from hops with focus on the prenylated flavonoids, xanthohumol (XN) and its isobaric and isomeric flavanone, isoxanthohumol (IX), and the two isobaric and structurally related flavanones, 6-prenylnaringenin (6PN) and 8-prenylnaringenin (8PN). We observed a slight difference for the arrival time distributions (ATD) for XN and IX ( 0.1msec), but identical ATDs were observed for the two prenylnaringenins. We also show the application of ion-mobility separations and time-aligned parallel fragmentation for the characterization of proanthocyanidins from hops and grapes. These analyses provide snapshots on the complexity of proanthocyanidin polymer mixtures but are highly informative. Once established these images may guide extraction processes of plant materials and serve as quality control fingerprints.

O8.5 MASS SPECTROSCOPIC FINGERPRINTING AND CHEMOMETRIC ANALYSIS FOR QUALITY ASSESSMENT OF BOTANICALS AND FOODSTUFFS

Pei Chen, Jianghao Sun, James Harnly

Agricultural Research Services, U.S. Department of Agriculture, Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Beltsville, MD 20705, USA; [email protected]

The chromatographic fingerprint technology was accepted by the WHO as a strategy for identification and quality evaluation of herbal medicines in 1991. However, the reproducibility of chromatograms is not consistent and it often takes 60 minutes or longer to generate a single chromatographic fingerprint. Flow-injection mass spectrometry (FIMS), on the other hand, can generate a mass spectrometric fingerprint in 1 minute or less. FIMS can be used to detect differences in chemical composition arising from genetic, environmental, and other factors.

Principal component analysis (PCA) and other chemometric tools can also be used to process MS fingerprints. The results are score plots that are easily interpreted visually and statistically, and can pinpoint exactly what components are responsible for the chemical difference found between different sample sets. Differentiation between Panax quinquefolius, P. ginseng, and P. notoginseng species, between Scutellaria lateriflora and the germander species, and between organic and conventionally grown grapefruits have been successfully demonstrated. The results prove that MS fingerprinting is an easy, fast, and powerful tool for quality assessment of botanicals and foodstuffs.

O8.6 STRUCTURAL CHARACTERIZATION OF PHENOLIC LIPIDS OBTAINED BY TRANSESTERIFICATION OF 3,4-DIHYDROXYPHENYLACETIC ACID AND KRILL OIL

Sarya Aziz,¹ Varoujan Yaylayan,¹ Richard St-Louis,² Marya Aziz,¹ Selim Kermasha¹

¹McGill University, Department of Food Science and Agricultural Chemistry, Ste-Anne de Bellevue, QC H9X 3V9, Canada, ²Université de Québec à Rimouski, Institut des Sciences de la Mer de Rimouski, Rimouski, QC G5L 3A1, Canada; [email protected]

The biosynthesis of novel biomolecules, phenolic lipids, rich in ω-3 polyunsaturated fatty acids (PUFAs) and phospholipids, by the enzymatic transesterification in solvent-free medium of krill oil with 3,4-dihydroxyphenylacetic acid (DHPA), using Novozym 435 was investigated. The krill oil components and its esterified phenolic lipids were separated by HPLC, using a Zorbax column (3.5 μm, packed with stable bond C₁₈-reversed-phase) as well as a solvent gradient of acetonitrile and isopropanol. The detection was performed simultaneously by ultraviolet/diode array detector (UV/DAD) at 215 and 280 nm as well as with an evaporative light scattering detector (ELSD). The experimental results indicated that ELSD was shown to be a more appropriate tool for the analysis of the krill oil components and its esterified phenolic lipids as compared to UV. Fourier transform infrared spectroscopy analysis (FTIR) confirmed the synthesis of phenolic lipids, obtained by a transesterification of phospholipids and DHPA. Liquid chromatography/mass spectrometry-electrospray ionisation/atmospheric-pressure chemical ionization (LC/MS-ESI/APCI) successfully characterized the molecular structures of the synthesized phenolic lipids.

O8.7 ANALYSIS OF PHYTOCHEMICALS BY MALDI TISSUE IMAGING MASS SPECTROMETRY

Greg Witkop

Waters Corporation, Milford, MA, USA; [email protected]

The analysis of plant tissues by Maldi Imaging mass spectrometry was investigated. The samples were prepared by making 10 μm slices and coating them with chemical matrix for analysis. The Maldi images were collected on a Synapt G2 mass spectrometer at a resolution of 20,000 FWHM. The images were then reviewed to locate specific compounds and the regions of the tissue in which they occupy.

O9.1 ANTIBACTERIAL EFFECTS OF HYDROLYZABLE TANNINS AND RELATED ARTIFICIAL TANNINS

Airi Doi,¹ Mohamed A. A. Orabi,² Shoko Taniguchi,³ Takuya Ogawa,³ Yasuo Takeuchi,³ Teruo Kuroda,³ Tomofusa Tsuchiya,⁴ Hideyuki Ito,³ Tsutomu Hatano^1,3

¹Okayama University, Faculty of Pharmaceutical Sciences, Okayama, 700–8530, Japan, ²Al-Azhar University, Faculty of Pharmacy, Assiut, 71524, Egypt, ³Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700–8530, Japan, ⁴Ritsumeikan University, College of Pharmaceutical Sciences, Kusatsu, 525–8577, Japan; [email protected]

Hydrolyzable tannins isolated from Tamarix nilotica and T. tetrandra, such as hirtellin B, showed in vitro antimicrobial effects on methicillin-resistant Staphylococcus aureus (MRSA). Artificial tannins produced on the galloylation of oligosaccharides, such as octa-O-galloyl-beta-lactose, also showed antibacterial effects on MRSA. The minimum inhibitory concentrations (MICs) of these compounds were largely different from each other depending on their structures. Inhibition of pathogenic pigment production in Pseudomonas aeruginosa was observed for almost all of the Tamarix tannins and artificial tannins.

O9.2 COX-2 SPECIFIC INHIBITION FROM NATURAL PRODUCT (E)-HINOKIRESINOL AND A FACILE SYNTHESIS OF 3-VINYLPHENYLINDANES

Catherine P. Tunbridge,¹ Neil R. Crouch,³ Alfred E. Thumser,² Dulcie A. Mulholland¹

¹University of Surrey, Chemical Sciences, Guildford, GU2 7XH, United Kingdom, ²University of Surrey, Biological Sciences, Guildford, GU2 7XH, United Kingdom, ³South African National Biodiversity Institute, Ethnobotany Unit, Durban, 4007, South Africa; [email protected]

The total synthesis of natural product (E)-hinokiresinol had an overall yield of 20% from starting materials 4-hydroxyacetophenone and 4-hydroxybenzaldehyde. COX-1 and COX-2 assays confirmed that (E)-hinokiresinol had anti-inflammatory activity, with it showing selectivity towards COX-2 at 10 μM. During the synthesis of derivatives, an intermolecular cyclisation occured resulting in a facile route to 3-vinylphenylindanes.

O9.3 BIOACTIVE COMPOUNDS FROM THAI MARINE-DERIVED FUNGI

Prasat Kittakoop^1,2

¹Chulabhorn Research Institute, Bangkok, Thailand 10210, ²Chulabhorn Graduate Institute, Chemical Biology Program, Bangkok, 10210, Thailand; [email protected]

Marine-derived fungi are isolated from tissues of Thai marine invertebrates, and are rich sources of bioactive compounds. In our experience, these fungi could grow rapidly in salt containing media (sea water), but most of them hardly grow in media without salt supplements, and sometimes, those that do grow in non-saline media change their morphology when cultured under such conditions. We define these fungi as “marine-derived fungi” rather than marine fungi which require seawater for their growth. We have chemically explored biologically active compounds from marine-derived fungi. Results of this research will be presented.

O9.4 MULTI-DRUG RESISTANCE, VEROTOXIN PRODUCTION AND EFFICACY OF CRUDE STEM BARK EXTRACTS OF CURTISIA DENTATA AMONG ESCHERICHIA COLI (NON-O157)AND ACINETOBACTER SPP. ISOLATES OBTAINED FROM WATER AND WASTEWATER SAMPLES

James Hamuel Doughari, Patrick Alois Ndakidemi, Izanne Susan Human, Spinney Benade

Faculty of Applied Sciences, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa; [email protected]

Drug resistant diarrhea and nosocomial infections caused by verotoxic Escherichia coli and some Acinetobacter spp. has posed serious therapeutic challenges especially in developing countries. The aim of this work was to investigate multi-drug resistance, verotoxin-production and susceptibility of E. coli and Acinetobacter spp. isolated from some water samples to crude stem bark extracts of Curtisia dentata. Culture of 62 water samples on Brilliance E. coli/coliform selective medium (BECSM, Oxoid), Eosin Methylin Blue (EMB) agar, or Baumann’s enrichment medium (BEM) and Leeds Acinetobacter Medium (LAM) yielded 69 isolates of E. coli and 41 isolates of Acinetobacter spp. with 26 (53.06%) of the E. coli and 6 (14.63%) of the A. haemolyticus isolates producing verotoxins, and no A. lwoffii isolate produced the toxins. Multi-drug resistance index (MDRI) values of isolates ranged between 7–33.00% for both isolates with 12 (17.39%) of the E. coli and 10 (24.39%) of the Acinetobacter spp. resistant to 3 or more classes of the antibiotics. C. dentata stem bark extracts demonstrated low MIC values of 150–300 µg/ml for E. coli and 150–2000 µg/ml for Acinetobacter spp. The plant also contained saponins, tannins, glycosides, anthraquinones, flavonoids, steroids and phenols. The presence of verotoxic multidrug resistant E. coli and Acinetobacter spp. in the environments investigated calls for further surveillance of more water bodies and other environments. Proactive control measures need to be in place to curtail possible contamination of food and drinking water sources. Purification of C. dentata phytoconstituents, toxicological as well as in vivo studies for their antimicrobial potentials against pathogenic bacteria, should be carried out with a view to utilizing the plant in developing novel antibiotic substances.

O9.5 17-O-ACETYL, 10-HYDROXYCORYNANTHEOL, A SELECTIVE ANTIPLASMODIAL ALKALOID ISOLATED FROM STRYCHNOS USAMBARENSIS LEAVES

Martine Cao,¹ Raymond Muganga,^1,2 Monique Tits,¹ Luc Angenot,¹ Michel Frederich¹

¹University of Liege, Pharmacy, Laboratory of Pharmacognosy, CIRM, Liege, 4000, Belgium, ²National University of Rwanda, Pharmacy, Faculty of Medecine, Butare, 117, Rwanda; [email protected]

In the course of our investigations on Strychnos usambarensis leaves in order to isolate isostrychnopentamine, the main alkaloid responsible for the antiplasmodial activity of the plant, a new tertiary indolic alkaloid, has been isolated: 17-O-acetyl, 10-hydroxycorynantheol I. Its structure was determined by means of spectroscopic and spectrometric methods such as UV, IR, CD, NMR and ESI-MS. It is one of the most active monomeric indole alkaloids known to date showing an in vitro activity against Plasmodium falciparum close to 5 μM and high selectivity.

O9.6 EVALUATION OF THE ANTIMICROBIAL ACTIVITY, SUB-CHRONIC TOXICITY AND WOUND HEALING EFFECT OF CUNNINGHAMELLA SPECIES AND SOME OF ITS ISOLATED COMPOUNDS

Amani S. Awaad,¹ Derek J. Maitland,² G. A. Soliman,³ Nabilah A. Al-Jaber, Mounerah R. Al-Outhman, M. E. Zain,³ Tahani A. Alhamed

¹King Saud University, Chemistry Department, Faculty of Science (Girls Section), Riyahd, 11495, Saudi Arabia, ²Bradford University, Chemical and Forensic Sciences Department, School of Life Science, Bradford, BD7 1DP, United Kingdom,³ Al-Kharj University, Medical Laboratory Sciences Department, Faculty of Applied Medical Sciences, Al-Kharj, Saudi Arabia; [email protected]

The alcohol extracts of Cunninghamella blakesleeana, Cunninghamella elegans and Cunninghamella homothallica, as well as their successive extracts and isolated compounds, were evaluated for their antimicrobial activity against a number of microbes. The tested extracts showed significant antimicrobial activity. Total alcohol and successive extracts of C. elegans showed the highest activity against Staphylococcus aureus. Three fatty acids were isolated and identified as palmitic acid, oleic acid and stearic acid; they showed variable activities against Staphylococcus aureus. Five compounds: 2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol (adenosine), uridine, pyrimidine-2,4-dione uracil, gallic acid, and 3-(methoxycarbonyl)but-3-enoic acid were isolated from the extract of Cunninghamella elegans and identified. The results of the antibacterial activity of the isolated compounds revealed that the activity was attributed to uridine as the most active one (MIC = 20 μg mL⁻¹) followed by uracil, gallic acid and 3-(methoxycarbonyl) but-3-enoic acid (MIC = 150,130 and 210 μg mL⁻¹, respectively). The total methanol extracts of C. blakesleeana, C. elegans, and C. homothallica did not induce any signs of toxicity or mortalities in mice when administered orally at doses up to 5000 mg kg⁻¹. In a sub-chronic experiment, oral administration of the methanolic extracts of three fungi to rats in a dose of 200 mg kg⁻¹ for 35 days did not produce any significant change in their liver and kidney functions. The topical application of methanol extracts of C. elegans and uridine wound healing process was at a concentration of 5 mg mL⁻¹.

O9.7 ANTIHYPERTENSIVE EFFECT OF GENTIANA FLORIBUNDA IS MEDIATED THROUGH Ca⁺⁺ ANTAGONISTIC PATHWAY

Arif ullah Khan,^1,2 Mohamad Rais Mustafa,² Dharmani Devi Morgan²

¹Kohat University of Science and Technology, Institute of Pharmaceutical Sciences, Kohat, 26000, Pakistan, ²University of Malaya, Department of Pharmacology, Kuala Lumpur 50603, Malaysia, ³University of Karachi, International Centre for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, Karachi, 75270, Pakistan; [email protected]

The crude extract of Gentiana floribunda (Gf.Cr) caused a dose-dependent (3.0-300 mg/kg) fall in arterial blood pressure (BP) of rats under anesthesia. In rat aortic ring preparations denuded of endothelium, Gf.Cr (1.0-10 mg/Ml) relaxed high K⁺ (80 mM) and phenylephrine (PE, 1 μM)-induced contractions and shifted Ca⁺⁺ dose-response curves to the right, similar to that caused by verapamil. It also suppressed PE (1 μM) control peak responses at 0.3-1.0 mg/mL, obtained in Ca⁺⁺-free medium, like verapamil. Pre-treatment of tissues with Gf.Cr produced rightward non-parallel shift of PE-curves with decline of the maximum contractile response. The vasodilator effect of Gf.Cr was endothelial-independent, as it was not blocked by N-nitro-L-arginine methyl ester hydrochloride (0.1 mM), atropine (1 μM) or indomethacin (1 μM) in endothelium-intact aortic tissues. These data indicate that BP-lowering action of Gentiana floribunda occurred via Ca⁺⁺ antagonism (inhibition of Ca⁺⁺ ingress and release from intracellular stores) which provides a pharmacological basis to justify its effectiveness in hypertension.

O9.8 SYRBACTINS: NEW STRUCTURAL CLASS OF PROTEASOME INHIBITORS PRODUCED BY PLANT PATHOGENS

Andre S. Bachmann,^1,4 Michael Pirrung,² Robert Dudler,³ Crystal Archer,⁴ John Opoku-Ansah¹

¹College of Pharmacy, University of Hawaii at Hilo, Department of Pharmaceutical Sciences, Hilo, HI 96720, USA, ²University of California, Riverside, Department of Chemistry, Riverside, CA 92521, USA, ³University of Zurich, Institute of Plant Biology, Zurich-Basel Plant Science Center, Zurich, 8008, Switzerland, ⁴University of Hawaii at Manoa, University of Hawaii Cancer Center, Honolulu, HI 96813, USA; [email protected]

The syrbactin natural products belong to a new class of irreversible proteasome inhibitors which include syringolins and glidobactins. These small molecules are derived from the plant pathogen Pseudomonas syringae pv. syringae (Pss) and an unknown species of the order Burkholderiales, respectively. They are structurally distinct from other, well-established proteasome inhibitors (e.g., bortezomib/velcade), and bind the eukaryotic 20S proteasome by a novel mechanism (Nature, 2008, 452: 755-8). Despite the irreversible binding mode, these molecules show surprising selectivity for covalent proteasome inhibition, highlighting their potential as promising lead structures for drug discovery. Syrbactins exhibit strong anti-tumor activity in vitro against neuroblastoma, multiple myeloma, and ovarian cancer cells (Biochem. Pharmacol. 2010, 80: 170-8) and in vivo against neuroblastoma-tumor bearing mice. The chemical synthesis of these natural products has been accomplished (PNAS, 2009, 106: 6507–6512; Org. Lett. 2010, 12: 2402-5) and novel syrbactin-inspired analogues have been synthesized with improved proteasomal inhibitory activity and elevated anti-tumor potency. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

O9.9 CATEGORIZATION OF MEDICINAL PLANTS WITH IMMUNO-REGULATORY ACTIVITIES BY CYTOKINE EXPRESSION IN MOUSE BONE-MARROW DERIVED DENDRITIC CELLS

Ning-Sun Yang,^1,2,3 Wen-Chi Wei,^1,4 Sheng-Yen Lin,^1,5 Yung-Tsung Chen,^1,2 Chia-Chi Wu¹

¹Academia Sinica, Agricultural Biotechnology Research Center, Taipei, 115, Taiwan, ²National Taiwan University, Institute of Biotechnology, Taipei, 106, Taiwan, ³National Central University, Department of Life Science, Taoyuan County, 32001, Taiwan, ⁴National Sun Yat-Sen University, Department of Marine Biotechnology and Resources, Kaohsiung, 804, Taiwan, ⁵National Defense Medical Center, Graduate Institute of Life Science, Taipei, 114, Taiwan; [email protected]

Traditional medicinal plants (TMP) are increasingly recognized for use in public health care throughout the world. Numerous TMPs are reputed to confer various medicinal efficacies or effectiveness including anti-inflammation and immuno-modulatory activities. However, systematic investigation and concrete demonstration of TMPs on defined, specific and heath care-applicable immuno-regulatory activities is limited. Dendritic cells (DCs), a key type of professional antigen presenting cells (APC) are key mediators in human’s immune systems. Cytokine stimulation by DCs is known to play a crucial role in DC-mediated immuno-regulatory activities in various immune responses. Therefore, DCs are considered by many as a viable pharmacological platform or target for evaluating TMP’s immuno-regulatory activities. The objective of this study was to evaluate the regulatory activities of specific TMPs on DC maturation and activities, especially on cytokine expression. Our preliminary results show that a number of the ethanol and ethyl acetate extracts of test TMPs can exhibit an inhibitory activity on LPS-induced expression of TNF-α, IL-6 and IL-12 in mouse bone marrow derived dendritic cells (BMDCs). In addition, these test herbal extracts can be categorized into several functional groups based on their capacities to regulate cytokine. We therefore hypothesize that these key cytokines may be usefully employed as a guide or index for grouping, classifying, monitoring and manipulating the molecular and immunological specificities of different anti-inflammatory herbal extracts in key immune cell systems., These findings and future studies many have potential pharmacological application to the developmentof TMPs.

O9.10 CEMBRANOLIDES FROM CROTON GRATISSIMUS (EUPHORBIACEAE)

Dulcie Mulholland,^1,2 Moses Langat,^1,2 Neil Crouch^2,3

¹University of Surrey, Chemistry, Guildford, GU27XH, United Kingdom, ²University of KwaZulu-Natal, Chemistry, Durban, 4041, South Africa, ³South African National Biodiversity Institute, Ethnobotany Unit, Durban, 4007, South Africa; [email protected]

The phytochemical study of the leaves and stem bark of Croton gratissimis has yielded a range of novel cembranolide diterpenoids such as 1-2. Compounds 1 and 2 were screened against the PEO1 and PEO1TaxR ovarian cancer cell lines and found to have lower potency than paclitaxel (IC₅₀ values of 132 and 125 nM respectively against PEO1, cf paclitaxel 2.3 nM).

O9.11 NATURAL PRODUCT-BASED INHIBITORS OF HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1) AS CHEMICAL PROBES FOR CELLULAR SIGNALING

Yu-Dong Zhou, Fakhri Mahdi, Jun Li, Lin Du, Sandipan Datta, Dale G. Nagle

University of Mississippi, School of Pharmacy, Department of Pharmacognosy, University, MS 38677-1848, USA; [email protected]

The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates oxygen homeostasis. Decreases in oxygen tension (hypoxia) activate HIF-1, which then increases the expression of genes that promote cellular adaptation and survival under hypoxic conditions. Employing a combined approach that incorporates natural product chemistry with bioassays, we have discovered chemically diverse natural products that suppressed HIF-1 activation. Compounds (rotenone, skimmiarepins, etc.) that disrupt mitochondrial respiration selectively inhibited HIF-1 activation by hypoxia. Further mechanistic studies revealed that mitochondrial respiration inhibitors trigger a cellular stress response that stalls protein translation. In addition, mitochondrial respiration inhibitors also alter mitochondrial morphology by disrupting the balance between mitochondrial fusion and fission. Cellular signaling pathways that regulate mitochondrial morphology and protein translation may constitute part of a signaling network that transmits acute exposure-associated cellular effects exerted by natural product-based mitochondrial respiration inhibitors.

O9.12 RAPID AND RATIONAL IDENTIFICATION OF BIOACTIVE NATURAL PRODUCTS

Muriel Cuendet, Andreas Nievergelt, Claudia Simoes Pires, Nadine Bohni, Jean-Luc Wolfender

University of Geneva, School of Pharmaceutical Sciences, Geneva, 1211, Switzerland; [email protected]

As established by ample precedent, nature provides broad chemical diversity and compounds isolated from plants are essential in therapy. Successful hit discovery of candidates relies on rational screening strategies involving in vitro assays, compound isolation, and identification. These steps are often time consuming and require large amounts of plant material or other biological sources. Advances in analytical methods and bioassay development helped to push forward the research in this area. The development of high resolution methods related to HPLC for both chemical and biological profiling has significantly increased the efficiency of classical bioactivity-guided fractionation procedures. A comprehensive investigation becomes feasible starting with only tenth of milligram of crude extract. Enriched extracts can be fractionated in a single step by semi-preparative HPLC, and the activity of microfractions is evaluated, enabling a rapid localisation of the corresponding bioactive LC-peaks. Test samples are then analyzed by UHPLC-TOF-MS to get first information on their chemical composition. Compound identification and their approximate concentration in the original sample are feasible in the low microgram range by offline coupling with microflow-NMR equipped with automated sample injection. This discovery platform will be illustrated by a practical example of phytochemical investigation performed at the microgram level. This represents a key advantage for rapid localisation of the biological activity and subsequent identification of the compounds of interest.

O9.13 NATURALLY OCCURRING ENZYME INHIBITORS AND THEIR PHARMACEUTICAL APPLICATIONS

Athar Ata

University of Winnipeg, Department of Chemistry, Winnipeg, MB R3B 2G3, Canada; [email protected]

Enzymes are essential to human life, mediating biochemical processes including metabolism, cellular signal transduction, cell cycling, and development. Malfunction in these biochemical systems often leads to diseases that can be caused either by the dysfunction, overexpression, or hyperactivation of the enzymes involved. An understanding of diseases at the molecular level has provided several enzyme inhibitors in clinics. These enzyme inhibitors are used to treat several human health related issues. For instance, glutathione S-transferase inhibitors have applications in overcoming the drug resistance problems in cancer and parasitic chemotherapy. Fatty acid synthase inhibitors are used to discover anti-malarial, anti-parasitic, anti-TB and anti-fungal compounds. We are involved in discovering new natural products exhibiting inhibitory activities against glutathione S-transferase (GST), acetylcholinesterase (AChE), and α-glucosidase. In this presentation, structures of potent enzyme inhibitors and their structure-activity relationships will be discussed.

O9.14 NEUROTROPHIC SECO-PREZIZAANE-TYPE SESQUITERPENOIDS FROM ILLICIUM JIADIFENGPI

Yoshiyasu Fukuyama, Miwa Kubo

Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Tokushima, 770–8514, Japan; [email protected]

As part of our continuing studies on neurotrophic compounds in Illicium species, we investigated the chemical constituents of the pericarps of I. jiadifengpi, resulting in the isolation of two seco-prezizaane-type sesquiterpenoids 1 and 2 named jiadifenolide and jiadifenoxolane A. The Dess-Martin oxidation of the known sesquiterpene, neomajucin (3), gave rise to 1 in a straightforward fashion. This means that the absolute configuration of 1 can be assigned to be the same as that of 3. Jiadifenolide (1) was found not only to significantly enhance neurite outgrowth in the primary cell cultures of rat cortical neurons at concentrations ranging from 0.01 to 10 μmol L⁻¹, but also to have a potential to specifically promote differentiation of multipotent neural stem cell line MEB5 cells into neurons at 10 μmol L⁻¹.

O9.15 STEERING CLEAR OF THE DRUG DISCOVERY BLACK-HOLE

Mark O’Neil-Johnson,¹ Gary R. Eldridge,¹ Courtney Starks,¹ Russell Williams,¹ Werner Maas²

¹Sequoia Sciences, Inc., Saint Louis, MO 63114, USA, ²Bruker Instruments, Inc., Billerica, MA 01821, USA; [email protected]

There is not one single scientific innovation that you can point to and say, “this has revolutionized drug discovery.” The billions of dollars that have been invested in proteomics, genomics, metabolomics, etc. has not created the proportionate output to dollar input. Mergers and acquisitions have done nothing to invigorate the discovery process. In fact, financial data has indicated that this aggressive strategy for business growth has resulted in $1 trillion reduction of valuation over the past decade. Is this the path towards innovation as an industry? As natural product chemists, the question that we need to ask is, “what about chemical diversity?”

Sequoia Sciences identifies novel chemistry from its library of structurally diverse small molecules isolated from plants. Sequoia built this analytical process such that rapid isolation and structure elucidation of active compounds could be accomplished. Using the sensitive Bruker TCI 1.7mm MicroCryoProbe, structure elucidation of active compounds is completed on samples of limited mass.

The scientific strategy that Sequoia employs to rapidly uncover the chemical diversity contained in plant natural products will be outlined. This presentation will outline Sequoia’s unique process that is used to create a library of compounds. The MicroCryoProbe has now extended the high-throughput process to include NMR data acquisition. Sequoia’s inclusion of the MicroCryoProbe compliments its current platform technologies for high-throughput natural products research for drug discovery allowing it to uncover the chemical diversity contained in natural products.

O9.16 ENHANCING NORMAL PHASE CHROMATOGRAPHY FOR NATURAL PRODUCTS RESEARCHERS

James D. McChesney, Douglas L. Rodenburg

Ironstone Separations, Inc., Etta, MS 38627-9519, USA; [email protected]

Ironstone Separations has developed a propriety technology for the fabrication of high efficiency preparative chromatographic columns applicable to both normal and reversed phase columns, called C4 technology. Importantly, we have also perfected a technology which regenerates the efficiency of normal phase columns such that they can also be reused for hundreds of separations without adsorbent replacement. Since high quality normal phase adsorbent costs about $5,000 per kilogram, these technologies provide cost savings to users of many thousands of dollars over the lifetime of a preparative normal phase chromatographic column. Thus the advantages of normal phase preparative chromatography, increased capacity per column run and ease of purified compound recovery, are captured without the disadvantage of frequent adsorbent replacement. Ironstone fabricates and markets this technology to the synthetic chemistry and natural products research and development communities. Due to their significant economic advantages, Ironstone’s technologies are especially beneficial to academic natural product researchers.

O9.17 PHYTOAGENT DEOXYELEPHANTOPIN COTREATMENT WITH CISPLATIN SIGNIFICANTLY REDUCES NEPHROTOXICITY-INDUCED BY CISPLATIN IN B16 MELANOMA-BEARING MICE

Lie-Fen Shyur, Wen-Wan Chao, Ya-Wen Cheng, Ching-Yi Chiou

Academia Sinica, Agricultural Biotechnology Research Center, Taipei, 115, Taiwan; [email protected]

This study investigated the in vitro and in vivo efficacy of deoxyelephantopin (DET), a major sesquiterpene lactone constituent of Elephantopus scaber L. (Asteraceae), against B16 melanoma. Isobologram analysis revealed the synergism of DET and chemotherapeutic drug cisplatin (CP) against B16 cell proliferation. A series of drug treatment protocols were thus designed in this study, i.e., DET or CP treatment alone, DET co-treated with CP (DET+CP), and sequential treatment with CP then DET (CP-DET). A stable B16 melanoma cell clone carrying COX-2 promoter driven-luciferase reporter gene was established to monitor the lung metastasis of melanoma in syngeneic mice. Our results showed that Pre-DET10 and CP-2 have a similar profound effect on inhibiting lung metastasis of B16 melanoma and increase of median survival rate in tested mice. CP treatment, however, resulted in renal damage and haematological toxicity in mice that was not detected in DET or DET and CP cotreatment groups. The metabolomic results obtained by UPLC-QTOF MS showed that CP-induced nephrotoxicity in mice kidney can be reflected in the levels of specific metabolites involved in primary metabolism or urea cycle in animals. Mechanistic study indicated that DET could induce cell cycle arrest at G2/M phase, as well as apoptosis in B16 melanoma cells. Our findings may prove useful for the future application of combinational DET and CP treatment against metastatic melanoma.

O9.18 ANTIVIRAL PROPERTIES OF SILYMARIN AND PURIFIED FLAVONOLIGNANS

Stephen J. Polyak,¹ Jessica Wagoner,¹ Jan McClure,¹ Erica Lovelace,¹ Tyler Graf,² Nicholas Oberlies²

¹University of Washington, Laboratory Medicine, Seattle, WA 98104, USA, ²University of North Carolina at Greensboro, Chemistry and Biochemistry, Greensboro, NC 27412, USA; [email protected]

Silymarin, an extract of milk thistle seeds [Silybum marianum (L.) Gaertn. (Asteraceae)], prevents liver injury and disease progression in animal models. To maximize the clinical value of this natural product, we are pursuing the molecular mechanisms by which silymarin protects the liver. We have previously shown that silymarin and silymarin-derived purified flavonolignans block hepatitis C virus (HCV) infection in liver cells and have immunomodulatory and anti-inflammatory effects on T cells. We now report that SIL suppresses human immunodeficiency virus (HIV) infection in peripheral blood mononuclear cells (PBMC) in vitro, the results of which have been validated in 5 different donor PBMC preparations. Furthermore, SIL inhibits 2 replication-competent viruses and 4 pseudoviruses in TZM-Bl cells. Thus, SIL inhibits Clade A, B, and C HIVs. Cumulatively, the data show that silymarin-derived compounds have antiviral effects against HCV and HIV in their natural cellular contexts: liver and T cells. We hypothesize that these antiviral effects against divergent viruses in vastly different cell types arise through the interaction of silymarin-derived compounds will cellular biomolecules that regulate virus infection. Current work is focused on identifying the cellular targets of silymarin flavonolignans using biochemical, genetic, and systems biology approaches. These studies may lead to novel cell-targeted antiviral therapies, identification of biomarkers of silymarin treatment and efficacy, and refinements in silymarin-based treatments for liver disease in HCV and HCV/HIV infected patients. This research is partially supported by NCCAM and the University of Washington Virology Division Pilot Award.

S10.1 REALISING THE POTENTIAL OF PLANT METABOLOMICS

Michael H. Beale, Jane L. Ward

Rothamsted Research, Plant Sciences, Harpenden, AL5 2JQ, United Kingdom; [email protected]

Plant metabolomics technology has now developed to an extent where large-scale screening programmes are possible. A key feature of untargeted metabolite profiling is that both primary and secondary metabolites are observed in the same spectra. This enables the study of how plants switch metabolic flow from primary to secondary metabolism in response to stress. We describe a sample preparation protocol, streamlined for robotic operation, that is used to generate NMR-MS fingerprint data from many thousands of samples. The data, because they originate from multiparallel analysis of single solvent extracts, can be interpreted together to identify discriminatory metabolites. Furthermore, structural information on those metabolites can be extracted from the fingerprints, in a way that is analogous to the classical structure determination of isolated, pure natural products. Use of this technology for the screening of recombinant inbred lines of Arabidopsis revealed that metabolic variation could be ascribed to relatively few areas of the genome. The metabolome is dynamic and studies must take into account the natural diurnal rhythms, tissue specificity and developmental programming. The presentation will describe examples of natural product biomarkers detected in Arabidopsis, either locally or systemically in response to microbe infection. A very striking example of the power of the technique has emerged from recent work on nutrient deprived Arabidopsis plants, where novel hemiterpenoids, directly associated with leaf N depletion, are induced by metabolic re-programming that is co-ordinated with synthesis of root phenylpropanoids.

S10.2 SPECTROSCOPIC METABOLITE PROFILING OF LASER-MICRODISSECTED PLANT CELLS

Bernd Schneider

Max Planck Institute for Chemical Ecology, Biosynthesis/NMR, Jena, 07745, Germany; [email protected]

The analysis of single plant cells and special plant cell populations is of considerable interest in natural product chemistry, chemical ecology and other disciplines of plant science. Laser-microdissection has become an established method for harvesting cells from plant tissue for RNA isolation and amplification. Identification of metabolites from single cells and microscopic tissue pieces so far has been reported by mass spectrometry. However, miniaturization of NMR and the enhanced sensitivity of cryogenically cooled probes enable NMR-based metabolic profiling of secondary metabolites specifically accumulating in special plant cells. ¹H NMR, 2D COSY and HSQC spectra were recorded at 500 MHz from extracts obtained from a limited number of cells and the metabolites were identified in the mixture. Using ¹H NMR, the relative proportions of metabolites in the samples were determined by integration and/or quantified by means of added standards. The method and results from plant species accumulating metabolites in specialized cells will be discussed.

S10.3 EXPLOITING METABOLIC DIVERSITY THROUGH INTEGRATED METABOLOMICS FOR THE DISCOVERY AND ELUCIDATION OF SAPONIN BIOSYNTHETIC GENES IN MEDICAGO TRUNCATULA

Dong Sik Yang, John H. Snyder, David V. Huhman, Vered Tzin, Stacy Allen, Yuhong Tang, and Lloyd W. Sumner

Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA; [email protected]

Triterpene saponins are a class of structurally diverse plant natural products with a wide range of demonstrated bioactivities including allelopathic, antifungal, antibacterial, anti-insect, anticancer, and antinutritive activities. The antinutritive properties of triterpene saponins in legume forages such as alfalfa and soybean are of particular and substantial economic importance. However, the genes and proteins responsible for the biosynthesis of legume saponins are mostly unknown. Thus, we are using cutting-edge metabolomics, correlated gene expression profiling, and eventually genome wide association mapping to identify, prioritize and characterize gene candidates related to triterpene saponin biosynthesis in M. truncatula. This presentation will provide a specific example whereby large-scale metabolite profiling (i.e. metabolomics) was used to survey a diverse collection of M. truncatula germplasm for the identification of hyper- (high) and hypo- (low) saponin accumulating lines. Comparative gene expression analyses were then performed on the hyper and hypo saponin accumulating lines, and correlation analyses performed to identify and prioritize genes candidates likely involved in triterpene saponin biosynthesis and regulation. This presentation will focus on a specific cytochrome P450 that was characterized as a multi-functional oxidase in saponin biosynthesis.

S10.4 ARTHUR NEISH YOUNG INVESTIGATOR AWARDLECTURE

EFFECTS OF EXOGENOUSLY APPLIED BRASSINOSTEROID IN SECONDARY XYLEM OF YELLOW POPLAR

Hyunjung Jin,² Wook Kim,² Mi Kwon^1,2

¹Institute of Life Science and Natural Resources, Korea University, Seoul, South Korea 136–701, ²Biotechnology, Korea University, Seoul, 136–701, South Korea; [email protected]

Brassinosteroids (BRs) are a group of plant steroidal hormones with various physiological roles including control of cell elongation and cell division, tracheary element differentiation, and resistance against biotic and abiotic stresses in plants. Recently, roles of BRs in vascular cambium development were proposed based on the analysis of Arabidopsis thaliana BR mutants as well as suspension cells of Zinnia elegans. However, the effects of BRs in the vascular development have not been demonstrated in woody species which possesses a well-developed vascular system. Thus, this study was designed to uncover the roles of brassinosteroids during secondary xylem formation in woody species. When 24-epi-brassinolide (BL) had been applied to the vascular cambium of the vertical stem of two-year-old yellow poplar, the growth promotion of tracheary elements were clearly visualized. Statistical analysis of cell length and cell diameter indicate that the length of both types of cells, fiber and vessels, was significantly increased upon BL application. Since anatomical and histochemical analysis implied changes in cell wall structure and chemistry, an expression analysis was performed for the genes involved in cell wall biosynthesis at the transcriptional level. In the case of lignin, almost all lignin biosynthetic genes were significantly down-regulated in the stem where BR was applied exogenously, as compared to the control stem. On the other hand, cellulose synthase (CesA) was significantly up-regulated, indicating an active involvement of BR in secondary xylem formation in woody plants.

S10.5 PLANT METABOLOMICS FOR PHYTOCHEMICAL GENOMICS

Kazuki Saito

RIKEN, Plant Science Center, Yokohama, 230-0045, Japan, and Chiba University, Graduate School of Pharmaceutical Sciences, Chiba, 263–8522, Japan; [email protected]

Metabolomics plays a major role in plant functional genomics and biotechnology. We have established an excellent analytical platform of plant metabolomes based on the combination of multiple mass spectrometers. An integrated analysis of metabolome and transcriptome data led to the prediction of gene-to-metabolite relations with a model plant, Arabidopsis thaliana. Holistic identification of genes involved in biosynthesis and modification of flavonoids in Arabidopsis has been carried out by combination of a transcriptome co-expression network. Metabolomics developed in Arabidopsis is further applicable to crops to decipher their gene functions and to improve their traits by biotechnology. An excellent coverage of chemical diversity of our analytical platform was suitably applied to the assessment of objective substantial equivalence of genetically-modified tomatoes over-expressing the taste-modifying protein miraculin. Application to a study in rice leading to the prediction of the rice agronomical and food traits was made by regression analysis of the metabolome for the World Rice Core Collection (WRC). Metabolome QTL analysis has been also performed to figure out the overview of metabolic genomics in rice. In this presentation, the crucial roles of metabolomics in plant functional genomics and crop biotechnology will be discussed.

S10.6 DEVELOPING MINT AS AN EXPERIMENTAL MODEL SYSTEM FOR UNDERSTANDING AND MANIPULATING TERPENOID ESSENTIAL OIL BIOSYNTHESIS

Mark Lange

Washington State University, Institute of Biological Chemistry, Pullman, WA 99164-6340, USA; [email protected]

Mints have been used and valued as aromatic herbs and sources of essential oils for thousands of years. The biosynthesis of the terpenoid essential oils in mint is confined to modified epidermal hairs called glandular trichomes. These structures contain highly specialized, non-photosynthetic, cells - termed secretory cells - that are solely responsible for the synthesis of essential oils. An EST sequencing effort with isolated secretory cells, which began in 1997, provided sequences of cDNAs with potential roles in terpenoid essential oil biosynthesis. By 2004 all of the structural genes with direct involvement in mint essential oil biosynthesis had been cloned and characterized, thus demonstrating the utility of working with specialized plant cell types for gene discovery. A metabolic engineering effort aimed at manipulating essential oil yield and composition commenced in 1998 and has continued to generate a large number of transgenic mint lines with purposefully altered oils. More recently (since 2005), mathematical modeling has provided fascinating novel insights into the regulatory control of this process, which has enabled further metabolic engineering advances. The genome of a diploid mint was sequenced using Illumina technology in 2011, which has opened up new opportunities for using integrative approaches, based on metabolic engineering and/or molecular breeding, for the sustainable agricultural production of high quality essential oils at a competitive cost.

S10.7 ARTHUR NEISH YOUNG INVESTIGATOR AWARD LECTURE

IMPROVING THE QUANTITY AND QUALITY OF LC- AND GC-MS DATA FOR PLANT METABOLOMICS

Paul G. Boswell¹, Will I. Menzel¹, Mikel R. Roe¹, Jerry D. Cohen¹, Adrian D. Hegeman^1,2

¹University of Minnesota-Twin Cities, Department of Horticultural Science and the Microbial and Plant Genomics Institute, Saint Paul, MN 55108,USA, ²University of Minnesota-Twin Cities, Plant Biology, Saint Paul, MN 55108, USA; [email protected]

Current LC- and GC- mass spectrometry-based metabolomics approaches are capable of providing thousands of chromatographic “features” from typical plant extracts. While accurate mass alone is insufficient to allow annotation of most of those observed peaks, orthogonal information, in the form of chromatographic retention, is usually captured but not under suitably controlled conditions to be useful for metabolite identification. Some success in using retention information in this way has come in the field of GC-MS (Kovats indices etc.), where the theoretical basis for compound retention is relatively simple and instrumentation is more easily standardized. In LC-MS, the higher degree of complexity in analyte retention and operational inconsistencies from run to run and greater differences between instruments have decreased the usefulness of retention information for metabolite identification. Here we describe a new approach that allows us to harness LC retention data for metabolite identification by carefully measuring multi-variable retention properties of compounds on several widely used C18-reversed phase media and by precisely accounting for variation across time and instrument platforms using a new methodology in which we “back-calculate” all significant instrument-related factors controlling retention from the gradient retention times of a small set of standard compounds. In addition, we will briefly discuss our recent efforts in constructing two environmental chambers for stable isotopic labeling of plants using [¹³C]-carbon dioxide for both absolute and relative quantification and for measurement of metabolic flux.

S10.8 CHEMICAL DEFENCE OF CONIFERS AND BIOENERGY APPLICATIONS

Jörg Bohlmann

University of British Columbia, Michael Smith Laboratories, Vancouver, BC V6T1Z4, Canada; [email protected]

Conifer trees produce large volumes of terpenoid oleoresin defenses for their protection against insects (e.g. bark beetles and weevils) and insect-associated fungal pathogens (e.g., ophiostomaoid fungi). Using a combination of genomics, transcriptomics, proteomics, and biochemical approaches, we have functionally characterized large gene families of terpenoid synthases (TPS-d family) and cytochrome P450 dependent monoxygenases (CYP720B family) of conifer oleoresin biosynthesis. The TPS-d and CYP720B gene families are critical for the plasticity and diversity of secondary metabolism in conifer defense and the successful evolution of long-lived conifer trees, which often survive for several hundred years in the same location defeating many generations of faster evolving insect pests and pathogens. In parallel, using genome sequencing of fungi and bark beetles, we have discovered new genes that allow fungal pathogens to overcome the toxic defenses of conifer hosts. The gene space of conifer defense against insects revealed genes for the improved production of biofuels and bioproducts.

S10.9 ELUCIDATION OF BIOSYNTHETIC PATHWAY OF CAMPTOTHECIN BY METABOLOMICS

Mami Yamazaki,^1,2 Takashi Asano,^1,2 Ko Aoki,^2,3 Kazuki Saito^1,4

¹Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263–8522, Japan, ²CREST, JST, Tokyo, 102-0075, Japan, ³Kazusa DNA Institute, Kisarazu, 292-0818, Japan, ⁴RIKEN Plant Science Center, Yokohama, 230-0045, Japan; [email protected]

Camptothecin (CPT), a monoterpenoid indole alkaloid, is a natural toxin that binds topoisomerase I, and CPT derivatives are clinically used as anticancer drugs. The plant species producing CPT, such as Camptotheca acuminata, Nothapodytes foetida and Ophiorrhiza pumila posses CPT-resistant topoisomerase I to survive CPT produced by themselves. The catalytic reactions and intermediates are still unclear in the late steps in CPT biosynthesis. To find out intermediate compounds and figure out the whole pathway of CPT biosynthesis, we conducted metabolome analysis of genetically modified hairy roots of Ophiorrhiza pumila. The CPT production was observed in the hairy roots induced by Agrobacterium rhizogenes, however not in the dedifferentiated cell suspension culture. The amount of CPT accumulation was correlated with gene expression level of both tryptophan decarboxylase (TDC) and secologanin synthase (SLS) in hairy roots in which corresponding genes were knocked down by RNAi technique. The general metabolic change in these tissues was analyzed by using infusion FT-MS, LC/FT-MS and LC/MS. Among the specific mass ion peaks detected in hairy root but not in cell suspension culture, several peaks exhibited positive or negative correlation with gene expression levels of TDC and SLS in RNAi hairy roots as well as CPT peak. The pathway mining using these data will be discussed.

S10.10 FLAVONOID-SPECIFIC PRENYLTRANSFERASES, A MEMBRANE-BOUND ENZYME FAMILY RESPONSIBLE FOR POLYPHENOL DIVERSITY

Kazufumi Yazaki, Kanako Sasaki, Tomoyoshi Akashi, Hirobumi Yamamoto, Shin-ichi Ayabe

Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan; Department of Applied Biological Sciences, Nihon University, Fujisawa 252-0880, Japan; Faculty of Life Science, Toyo University, Gunma 374-0193, Japan; [email protected]

Prenylation largely contributes to the diversification of aromatic natural products, such as the flavonoids, coumarins, and phenylpropanoids, via differences in the prenylation position on the aromatic rings, various lengths of prenyl chains, and further modifications of the prenyl moiety, e.g. cyclization and hydroxylation. The entry steps represents the crucial coupling process of the shikimate and polyketide pathways providing an aromatic moiety and the isoprenoid pathway derived from either mevalonate or MEP (methyl erythritol phosphate) pathways, which provides the prenyl (isoprenoid) chain. Recently, membrane-bound prenyltransferases have been reported as being responsible for these prenylation reactions. An updated understanding about this enzyme family is discussed.

O10.1 UNEXPECTED HEMITERPENOIDS IN ARABIDOPSIS, REVEALED BY METABOLOMIC FINGERPRINTING, GIVE NEW INSIGHTS INTO C/N METABOLIC BALANCING

Jane L. Ward, John M. Baker, Aimee M. LLewellyn, Nathaniel D. Hawkins, Michael H. Beale

Rothamsted Research, National Centre for Plant and Microbial Metabolomics, Harpenden, AL5 2JQ, United Kingdom; [email protected]

Unlike many other species, the model plant Arabidopsis thaliana contains relatively low levels of terpenoids, consisting mainly of sesquiterpene volatiles produced in flowers. However, metabolomic analysis of polar solvent extracts, using combined NMR-MS, has revealed the presence of two novel (to Arabidopsis) hemiterpenoid glycosides (HTGs) that accumulate in leaves, to quite high levels (Ward et al., PNAS, 2011,108,10762–10767). The structure of the compounds was determined by 2D-NMR and confirmed by synthesis. Using a hydroponic growth system we applied a series of stresses to roots and could follow the formation of these compounds in leaves. The formation of the HTGs was induced specifically in leaves by nitrate deficiency, and some other, but not all, root applied stresses such as oxidative stress. Replacement of growth media nitrate with ammonia failed to suppress the formation of the HTGs indicating that nitrate ion sensing was a key factor in signalling their formation. The formation of the HTGs in leaves was strongly correlated with the induction of phenylpropanoid secondary metabolites (coniferin and scopolin) in roots of the same plants. Feeding of MEP pathway intermediates to detached leaves of control and nitrate deficient plants was used to delineate the pathway to the hemiterpenoids and investigate the regulatory processes behind the induction. The shunts of photosynthetic carbon flow to HTGs will be discussed in terms of safety valve/overflow mechanisms that are involved in the balancing leaf photosynthetic carbon flow against nitrogen availability.

O10.2 MEASURING AND COMPARING THE MAGNITUDES OF METABOLOMIC CHANGE

Steven C. Halls, Jay M. Harrison, George G. Harrigan, Angela Hendrickson Culler, Marie A. Coffin

Monsanto Company, St. Louis, MO 63167, USA; [email protected]

Metabolomic analyses allow examination of numerous biochemical pathways networks and complex interactions. Several statistical tools have been developed to help identify potential metabolic effects related to a particular treatment. When metabolomic analyses are combined with multiple experimental design factors, it becomes challenging to understand the relative magnitude of the overall metabolic effect from each factor because of the complexity of the data. Developing tools such as Principal Variance Component Analysis (PVCA), oPLS-DA Eigen values and Stochastic Drift Ratios allow an overall comparison to be made between the relative magnitudes of change for each experimental factor (genotype, location, treatment, etc.)

O10.3 EMBEDDED SECRETORY CAVITIES: NATURAL PRODUCT BIOFACTORIES

Jason Q.D. Goodger, Allison M. Heskes, Ian E. Woodrow

University of Melbourne, School of Botany, Parkville, Victoria 3010, Australia; [email protected]

Many plants possess specialised extracellular secretory structures, such as glandular trichomes and embedded secretory cavities, which produce a range of high-value natural products. There have been rapid advances in research on the biosynthesis of natural products from trichomes because, unlike embedded cavities, these structures can be readily isolated from leaf surfaces and purified in a functional state. We have recently developed a method to isolate functional embedded cavities from within leaves and make use of this to study unique aspects of the structure, biosynthetic function and metabolome of embedded secretory cavities. In particular, we have shown that Eucalyptus secretory cavities house an array of natural products, in addition to the well known terpene essential oils, including chromanones, flavanones, flavonol glycosides and a large number of monoterpenoid glucose esters.

O10.4 EXPLORING THE ORGANIZATION AND FUNCTION OF BELOWGROUND TERPENE SPECIALIZED METABOLISM IN ARABIDOPSIS ROOTS

Martha Vaughan,² Qiang Wang,¹ Jung-Hyun Huh,¹ Reza Sohrabi,¹ Jim Tokuhisa,¹Dorothea Tholl¹

¹Virginia Tech, Biological Sciences, Blacksburg, VA 24061, USA, ²USDA-ARS, Center for Medical, Agricultural & Veterinary Entomology, Gainesville, FL 32608, USA; [email protected]

Understanding plant metabolism as a whole requires its analysis in both aboveground and belowground tissues. Specifically, how specialized metabolism is maintained in plant roots at cell and subcellular levels is not well understood. We investigate the organization of terpene specialized metabolic pathways and their function in Arabidopsis roots. We have found that constitutively expressed terpene biosynthetic enzymes and modules are largely restricted to specific cell types generating layers or gradients of terpene metabolites in the root tissue. As an example, we have identified a novel diterpene olefin (rhizathalene), which is exclusively produced by the terpene synthase TPS08 from MEP pathway precursors in leucoplasts of the root vascular tissue. Diffusion of the diterpene compound from the root stele into the surrounding cell layers has an anti-feeding effect on root herbivores. As a second example, we have characterized two (E)-β-farnesene synthases (TPS22, TPS25) with complimenting expression patterns in different root growth zones and surprising subcellular compartmentation in mitochondria. In addition to the constitutive formation of terpenes, we characterize biosynthetic modules involved in pathogen-induced volatile homoterpene formation. An unusual biosynthetic route in the formation of the homoterpene DMNT from the triterpene arabidiol and its potential function will be discussed.

O10.5 TRACING GLUCOSINOLATE METABOLISM AND DETOXIFICATION IN SMALL HERBIVORES

Daniel G. Vassão, Katharina Schramm, Michael Reichelt, Kimberly L. Falk, Jonathan Gershenzon

Max Planck Institute for Chemical Ecology, Biochemistry Department, Jena, TH 07745, Germany;[email protected]

The glucosinolates present in Brassicales plants are phytoanticipins that, upon activation, result in the so-called “mustard oilbomb”. This “bomb” comprises a number of toxic glucosinolate-hydrolysis products, most prominently their isothiocyanatederivatives, but also nitriles and thiocyanates. While their biosyntheses from amino acids and their activation steps arewell-studied, we know only little regarding their mode(s) of action and ecological effects. Even less is understood about the biochemical means employed by some small herbivores to disarm or safely “detonate” this bomb, a necessary strategy to permit successful herbivory and development.

We are now producing and utilizing isotope-labeled glucosinolates to detect, elucidate and quantify these differentbiochemical strategies within generalist herbivores. More specifically, we are characterizing the metabolic fates of the major A. thaliana methionine-derived alkylglucosinolate (4-methylsulfinylbutyl glucosinolate, glucoraphanin) in several insectherbivores, as well as in the mollusk pest Arion lusitanicus. We have found these processes to primarily consist of conjugationto amino acids and derivatives. Such results are now allowing us to correlate these metabolic strategies to the apparent herbivory success of these herbivores, i.e. associating their growth and development to their management of intaken toxicglucosinolate products.

O10.6 PHENOLIC ACIDS IN CATHARANTHUS ROSEUS ANALYZED BY A TARGETED APPROACH OF METABOLOMICS

Marcos Soto-Hernandez,^1,2 Young Hae Choi,¹ Robert Verpoorte¹

¹Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, 2300RA, The Netherlands, ²Colegio de Postgraduados, Campus Montecillo, Botanica, Texcoco, 56230, México; [email protected]

Phenolic acids containing leaves of intact plants from Catharanthus roseus responded to treatment with an elicitor from the oomicete Pythium aphanidermathum by shifting their phenol metabolism towards wall-bound phenylpropanoids derivatives: ferulic and p-coumaric acids.

The regulation of these metabolic changes, the integration of the phenylpropanoic acids and the signal transduction after elicitation were studied.

O10.7 SOYBEAN 14-3-3 PROTEINS: ARE THEY INVOLVED IN THE REGULATION OF ISOFLAVONOID BIOSYNTHESIS IN SOYBEAN?

Xuyan Li,¹ Sangeeta Dhaubhadel^1,2

¹Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, London, ON N5V 4T3, Canada, ²University of Western Ontario, Department of Biology, London, ON N6A 5B7, Canada; [email protected]

Isoflavonoids are legume specific plant natural compounds that play important roles in nitrogen fixation as well as biotic and abiotic stresses. Many clinical studies have suggested a role for isoflavonoids in human health and nutrition. Therefore, understanding the regulation of isoflavonoid biosynthesis is critical to develop soybean cultivars with altered levels of isoflavonoids. Recently, we have identified an R1 MYB transcription factor, GmMYB176, which regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean. Further, we identified the requirement of a 14-3-3 protein binding site within GmMYB176 for its cytoplasmic localization. Plant 14-3-3 proteins have been found to regulate a variety of biological processes such as metabolic, growth and developmental or signaling pathways via interactions with their target proteins. We identified 18 14-3-3 family members in soybean genome, of which 16 are transcribed. Tissue specific expression pattern of soybean 14-3-3 genes in various soybean tissues indicated that all 16 14-3-3s were expressed in embryos during the development suggesting that 14-3-3 proteins may play an important role in seed development. All of the 14-3-3s expressed in soybean were able to interact with GmMYB176 both in in vivo and in vitro condition. The detailed analysis of 14-3-3 binding sites within GmMYB176 identified a critical motif for 14-3-3 protein-GmMYB176 interaction where Ser29 located within the motif is potentially phosphorylated. Our results demonstrate that soybean consists of the largest members of 14-3-3 gene family identified to date and that 14-3-3 regulate the intracellular localization of GmMYB176 and control turnover of GmMYB176 thereby affecting isoflavonoid biosynthesis in soybean. The role of 14-3-3s in isoflavonoid biosynthesis is demonstrated by altering its expression in soybean hairy roots and using virus induced gene silencing in soybean and monitoring the impact on isoflavonoids levels.

O10.8 THE STUDY OF NORTH AMERICAN GINSENG METABOLISM IN RATS BY LC-MS/MS

Edmund M.K. Lui,¹ Takeo Sakuma,² Suma Ramagiri,² Tom Moy,² Carmai Seto²

¹University of Western Ontario, Physiology and Pharmacology, Ontario Ginseng Innovation and Research Consortium, London, ON N6A 5C1, Canada, ²AB-Sciex, Concord, ON L4K 4V8, Canada; [email protected]

The metabolism of ginseng in Zucker rats was studied using high resolution accurate mass spectrometry (HRMS) techniques coupled with UPLC. Fast and generic single injection HRMS methods allow for simultaneous qualitative and quantitative analysis of complicated ginsenosides in biological samples. Plasma and intestinal content were collected after chronic oral treatment with aqueous and alcoholic extracts. LC-MS and MS/MS technology and software programs have allowed for rapid analysis of complex mixtures of ginsenosides and their metabolites as well as metabolic pathways involved. The data showed that the primary ginsenoside, Rb1, was extensively metabolized in the intestine with the formation of oxidative metabolites and its aglycones. In contrast, the other less abundant ginsenosides, Re, Rd and Rc were detected the plasma but not in the colon. This indicated their intestinal absorption and systemic bioavailability after oral administration (supported by MRI: RE02-049).

Table

Ginsenoside	R1	R2	R3	MW
Rb1	–Glc–Glc	H	–Glc–Glc	1108
Rb2	–Glc–Glc	H	–Glc–Arap	1078
Rc	–Glc–Glc	H	–Glc–Araf	1078
Rd	–Glc–Glc	H	–Glc	946
Rf	H	–Glc–Glc	H	800
Rg1	H	–Glc	–Glc	800

O10.9 THE METABOLISM OF LIGNANS FROM FRUCTUS SCHISANDRA

Jun Yin,¹ Feng Yun Cao,^1,2 Ling Yang²

¹Shenyang Pharmaceutical University, School of Traditional Chinese Medicines, Shenyang, 110016, China, ²Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Laboratory of Pharmaceutical Resource Discovery, Dalian, 116023, China; [email protected]

After metabolites from dibenzocyclooctadiene lignans of Schisandra chinensis incubated with human liver microsomes were isolated by HPLC and their structures were identified, the study combined correlation analysis, chemical inhibition studies, assays with recombinant CYPs and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform. Three metabolites were identified and the results showed little difference among the mentioned species. Deoxyschizandrin undergo hydroxylation in HLMs, and schizandrin was the corresponding metabolite specially mediated by CYP3A4. The high affinity and high turnover make deoxyschizandrin hydroxylation an excellent probe drug for CYP3A4 activity in vitro. The metabolic pathway of schisantherin A validated the inhibitory mechanism against CYP3A4 undergoes demethylenenation in the human liver microsomes. IC₅₀ drift and dynamics test indicate that schisantherin A is inhibitor which shows dependency to NADP⁻ and time, which illustrates that the methylene of schisantherin A produced active intermediate during the catalyzed reaction, thereby presents forceful inhibitory effect against CYP3A4. In mice liver microsomes, schisantherin A, schizandrol B and schizandrin B showed strong inhibition against metablism schizandrin and deoxschizandrin. Our research results indicate that why deoxyschizandrin presented forceful anti-multidrug resistance in vitro but reduced or vanished in vivo, and the extract of Fructus Schisandra containing deoxyschizandrin presents forceful anti-multidrug resistance in vivo.

O10.10 SELF-POLLINATED ARTEMISIA ANNUA PLANTS FORM A NEW PLATFORM TO UNDERSTAND ARTEMISININ BIOSYNTHESIS

De-Yu Xie, Fatima Alejos-Gonzalez, Guosheng Qu

North Carolina State University, Plant Biology, Raleigh, NC 27695, USA; [email protected]

Artemisinin is the currently most effective medicine for human being to fight against malaria disease. However, its low production in Artemisia annua, the only natural resource, limits its sufficient supply for the treatment of malarial victims. The past endeavors have made certain great progresses in understanding the biochemical pathway of artemisinin formation; however, little is known in the genetics of its biosynthesis, which leads to difficulties in metabolic engineering of this medicine. In this presentation, we report the development of self-pollinated A. annua plants in growth chambers. The F5 progeny plants were developmentally and morphologically uniform and were predicted to be homozygous. LC-MS based metabolic profiling showed that except for roots, seedlings after the removal of roots, leaves, and capitula produced artemisinin. ADS and CYP71AV1 genes, two pathway genes involved in artemisinic acid formation, were expressed in young leaves and flowers. It was interesting that CYP71AV1 was expressed in roots of seedling although this tissue did not produce artemisinin. Self-pollinated plants form a new genetic resource to understand the biosynthesis of artemisinin.

S11.1 LIVERWORTS-POTENTIAL SOURCE OF MEDICINAL COMPOUNDS

Yoshinori Asakawa

Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Tokushima, 770–8514, Japan; [email protected]

The present paper concerns chemical constituents from liverworts (Marchantiophyta) and their biological activity. The liverworts are taxonomically placed between algae and pteridophytes (ferns) and there are 6000 species. Almost all liverworts possess beautiful cellular oil bodies. Over several hundred new compounds (terpenoids, phenolic compounds and acetogenins) have been isolated from liverworts[1-3]. Many of them show characteristic scents, pungency and bitterness, and some can give allergenic contact dermatitis. Many also have cytotoxic, anti-HIV inhibitory, antimicrobial and antifungal, insect antifeedant and mortality, nematocidal, superoxide anion radical release and NO production inhibitory, neurotrophic, piscicidal, muscle relaxing, antiobesity and vasorelaxant effect and antiinfluenza activities [1-5]. The chemical structures of the active compounds and some biological activities including fragrant and tasty components will be discussed.

[1]	Asakawa, Y. Prog. Chem. Org. Nat. Prod. 42, 1–285 (1982).
[2]	Asakawa, Y. Prog. Chem. Org. Nat. Prod. 65, 1–618 (1995).
[3]	Asakawa, Y. Ludwiczuk, A. Nagashima, F. Prog. Chem. Org. Nat. Prod. 93. (in preparation).
[4]	Asakawa, Y. Curr. Pharm. Design. 14, 3067–3088 (2008).
[5]	Kuzuhara, T. & Asakawa, Y. et al. PLoS One 6(5), e19825.

S11.2 TRACING SECONDARY METABOLITES ON BRAZILIAN BIODIVERSITY: HOW TO DO IT USEFULLY TO FIND NEW BIOLOGICALLY ACTIVE COMPOUNDS?

Vanderlan da S. Bolzani,¹ Alberto J. Carvalheiro,¹ Ian Castro-Gamboa,¹ Marília Valli,¹ Meri E. Pinto,¹ Marcos Pivatto,¹ Adriano D. Andricopulo,² Claudia Pessoa,³ Celia R. Garcia⁴

¹Universidade Estadual Paulista, Instituto de Química, Araraquara, SP14800-900, Brazil, ²Universidade de São Paulo, Instituto de Física, São Carlos, SP 13560-590, Brazil, ³Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Fortaleza, CE 60.430-270, Brazil, ⁴Universidade de São Paulo, Instituto de Biociências, São Paulo, SP 05508-900, Brazil; [email protected]

Natural products represent a vast and complex structural diversity that is not matched by any other sources of small molecules, as well as providing not only a source of raw material, but an inspiration for the discovery of new molecular targets. Brazilian biodiversity holds a tremendous resource of secondary metabolites, which has still hardly been explored. During the past ten years, we have isolated and published ca. 834 compounds from Cerrado and Atlantic Forest species that constitute a valuable set of leads, useful for further medicinal chemistry studies. Some casearin diterpenes, piperidine alkaloids and small cyclic peptides have been used as starting materials for synthetic and semi-synthetic derivatives, aiming at SAR studies for optimization of pharmacological properties. [Biota-FAPESP, CNPq, FINEP].

S11.3 BIFLAVONOID BIOSYNTHESIS

Lydia Fumiko Yamaguchi,¹ Andre Luis Wendt dos Santos,² Eny Iochevet Segal Floh,² Massuo Jorge Kato¹

¹Chemistry Institute, University of São Paulo, Chemistry, São Paulo, SP 05508000, Brazil, ²Biosciences Institute, University of São Paulo, Botany, São Paulo, SP 05508-090, Brazil.

Flavonoids are ubiquitous in the plant kingdom but their dimers, the biflavonoids, are restricted to some Gymnospermae families such as Ginkgoacea and Araucariaceae and a few Angiospermae species. The biosynthesis of these compounds is controversial, but their formation could involve the oxidative coupling of two apigenin or two chalcones moieties possibily mediated by a peroxidase. However, this important biosynthetic step has not been examined in terms of precursor or enzymes involved. Phytochemical studies carried out on Araucaria angustifolia leaves demonstrated the presence of amentoflavone-type biflavonoids and lignans. On the other hand, its callus culture produced only p-coumaroyl/feruloyl esters but no flavonoids. Nevertheless, when apigenin is provided to the cell cultures, a methoxylated amentoflavone-type biflavonoid, isoginkgetin is formed. Then, the biosynthesis of biflavonoids in A. angustifolia proceeds through a long sequence of steps involving the apigenin production followed by the action of peroxidase and dirigent protein to mediate oxidative coupling forming the 3′-8′′ linkage, and methylation reaction by O-methyltransferase. These enzymes and the dirigent protein were detected and characterized in cell cultures.

S11.4 MODERNIZATION OF TRADITIONAL CHINESE MEDICINE: CHALLENGES AND OPPORTUNITIES

De-an Guo

Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China; [email protected]

Traditional Chinese Medicine (TCM) has over 3000 years of history and played important role in the peoples’ health and social development in Chinese history. In the past 50 years, Chinese scientists have made great effort to modernize TCM to make it an evidence-based medicine from the experience-based origin. The current review has summarized the recent advances in the modern research on the various respects of TCM including the resources investigation, GAP cultivation, phytochemistry, quality assessment, safety issues, metabolic investigation, pharmacology, systems biology etc. It pointed out that GAP for Chinese herbs is the first and key step in the quality control cycle of traditional Chinese medicine. In addition, the research on TCM active principles and quality control methods were also summarized. Over 12,000 chemical constituents have been isolated from over 600 species of traditional Chinese medicines, among which over 3000 new compounds were discovered, which laid a solid foundation for clarifying the material basis of action and providing reference substances for the quality control of TCM. Systems biology approach is now being actively practiced for the action mechanism studies of TCMs and their active principles, which provided a valuable approach for complex TCM systems. Currently, genomics, proteomics and metabolomics have obtained good application in TCM studies. Finally, the progress of TCM new drug research and development has been outlined. Several famous new drugs from TCM or based on TCM have been successfully marketed. Examples are artemisinin, artemether, huperzine A, etc. Other types of new drugs include Fufang Danshen Dripping Pill, Diao Xinxuekang, etc.

O11.1 COMPOSITION AND CHEMICAL STABILITY OF IRIDOIDS OCCURRING IN MORINDA CITRIFOLIA L. (NONI)

Johannes Westendorf, Simla Basar

Institute of Experimental and Clinical Pharmacology and Toxicology, University Clinic Hamburg Eppendorf, Toxicology, Hamburg, D-20246, Germany; [email protected]

Morinda citrifolia L. (noni) is among the most important medicinal plants used by ancient Polynesian people. Pharmacological activities, such as anti-inflammatory, immunostimulting and anti-oxidative properties have been confirmed by modern research. Although the chemistry of the noni plant has been widely investigated, the compounds that are responsible for the pharmacological profile, which is best explained as adaptogenic are not known. Possible candidates for the adaptogenic properties are a group of structurally related iridoids. We investigated a variety of noni fruit samples from different tropical areas with respect to their iridoid content. Local differences in the quantitative composition of iridoids could be observed, however, the qualitative composition was almost the same. Most prominent were the structurally related iridoids deacetyl asperulosidic acid (DAA) and asperulosidic acid (AA). Both compounds are stable under acidic conditions of the stomach (pH 1-2). DAA is stable at room temperature and pH-values between 1-12, whereas AA decomposes to DAA at pH >10. The hydrophilic glycosides do not enter the bloodstream after oral ingestion. DAA and AA are hydrolyzed by β-glycosidases, present in the human intestinal wall and in fecal bacteria. The resulting aglycones are highly reactive and have a short half life in biological fluids. Our findings make it unlikely that DAA and AA or their aglycones are responsible for the pharmacological activity of noni, which might nevertheless be due to reaction products formed by the reactive aglycones and biological molecules occurring in the body.

O11.2 LIMONOIDS FROM MANGROVE PLANTS OF THE XYLOCARPUS GENUS AND THEIR BIOACTIVITIES

Minyi Li, Jun Wu

South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, P. R. China; [email protected]

Mangrove plants are a large group of different salt tolerant plants growing in tropical and subtropical intertidal estuarine zones. We will introduce mangrove resources worldwide, and then focus on new limonoids from mangrove plants of the genus Xylocarpus. Limonoids, derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton, are tetranortriterpenoids with a β-furyl ring moiety. During the last 10 years, we have identified more than 100 new limonoids from Xylocarpus plants collected in south China and India. The structures of these compounds were elucidated by NMR spectroscopic method combined with single-crystal X-ray diffraction techniques. The absolute configurations of limonoids with a new carbon skeleton were established by circular dichroism quantum chemical calculations. Antifeedant, insecticidal, and antitumor activities of some limonoids are reported.

O11.3 MULTIDISCIPLINARY ASSESSMENT OF WIKSTROEMIA ENDEMIC SPECIES (THYMELAEACEAE) OF EASTERN POLYNESIA

Nicolas Ingert,^1,3 Isabelle Bombarda,^1,2 Gaetan Herbette,² Robert Faure,² Christian Moretti,³ Phila Raharivelomanana¹

¹Université de la Polynésie Française, Laboratoire BIOTEM, Faa’a, 98702, French Polynesia, ²Université Paul Cézanne, Marseille, 13397, France, ³Institut de Recherche pour le Développement, Papeete, 98713, French Polynesia; [email protected]

Wikstroemia genus (Thymelaeaceae family), used in traditional medicine in Asia and Pacific areas, is well known to possess various interesting therapeutic properties (such as cytotoxicity, anti-mitotic, anti-inflammatory, anti-oxidant, antiviral, antifungal, antimalarial...) and also to contain many bioactive components.

Three endemic species of Wikstroemia are found in French Polynesia: W. coriacea (endemic of Eastern Polynesia), W. raiateensis (endemic of Raiatea Island) and W. johnplewsii (endemic of Hiva Oa Island). We report herein a first investigation to establish their chemical composition and pattern. Eleven constituents belonging to different metabolite classes (sesquiterpenoids, lignans, triterpenoids, biflavonoids, coumarin and phenyl derivatives) were identified including the new natural compounds, oleodaphnoic acid, 2-hydroxy-1,5-diphenylpentan-1-one and 3-hydroxy-1,5-diphenylpentan-1-one. Comparison of the chemical profile of these species put in evidence the chemodiversity of these three endemic species which could be also discriminated by some morphological traits and genetic patterns. These findings will be helpful to solve remaining taxonomic confusion among Wikstroemia species from French Polynesia.

O11.4 THE OXIDATIVE IN VITRO METABOLISM OF LAPACHOL, CHARACTERIZED BY BIOMIMETIC MODELS

Michael Niehues,¹ Valéria Priscila Barros,¹ Marilda das Dores Assis,² Norberto Peporine Lopes¹

¹FCFRP/USP, Departamento de Física e Química, Ribeirão Preto, SP 14040-903, Brazil, ²FFCLRP/USP, Departamento de Química, Ribeirão Preto, SP 14040-901, Brazil; [email protected]

Lapachol, a natural naphthoquinone, has been demonstrated in the past to have a great number of biological properties, such as activity against enterovirus and Trypanosoma cruzi. Envisioning future therapeutic applications, twelve potential oxidation metabolites were therefore generated in vitro by means of different oxidizing agents with the Jacobsen catalyst or metalloporphyrins. Thereafter, characterized derivatives were compared to lapachol with isolated rat liver microsomes.

Acknowledgements: To FAPESP, CAPES and CNPq for the financial support.

S12.2 GENOMICS APPROACHES FOR BIOCHEMICAL PATHWAY DISCOVERY IN MEDICINAL PLANT SPECIES

Elsa Gongora, Kevin Childs, John Hamilton, Brieanne Vaillancourt, C. Robin Buell

Michigan State University, Department of Plant Biology, East Lansing, MI 48824,USA; [email protected]

Medicinal plants produce a wide range of compounds of pharmaceutical interest. The great structural diversity and biological activities of plant-derived compounds suggest that additional relevant compounds remain to be discovered. However, the secondary metabolic pathways that produce such compounds are poorly understood. To address this problem, whole transcriptome sequencing using next generation sequencing methods was used by the Medicinal Plant Consortium (http://medicinalplantgenomics.msu.edu/) to generate the transcriptomes of 14 medicinal plant species for which reference genomes are not available. Our genomics and bioinformatics approach integrates transcript abundance data with functional annotation for these species to identify genes in key biochemical pathways associated with the synthesis, transport and accumulation of target compounds. We have sequenced an array of cDNA libraries and assembled the transcriptome using the de novo short read assembler Velvet/Oases. Pseudo-reference sequences were constructed from unigenes that represent the transcriptome. To estimate the transcript abundance, single end reads were mapped to the pseudo-reference sequence and expression levels were quantified. Functional annotation of the unigenes, coupled with downstream analyses of “expression matrices” for each species in conjunction with metabolomics data, has enabled the identification genes in biochemical pathways related to the medicinal compounds of interest.

S12.3 DE NOVO ASSEMBLY OF EXPRESSED TRANSCRIPTS AND CONSTRUCTION OF A TRANSCRIPTOME DATABASE OF PHALAENOPSIS APHRODITE

Chun-lin Su,¹ Ya-Ting Chao,² Yao-Chien Alex Chang,³ Wan-Chieh Chen,¹ Chun-Yi Chen,¹ Ann-Ying Lee,¹ Tuan Hwa Kee,¹ Ming-Che Shih¹

¹Academia Sinica, Agricultural Biotechnology Research Center, Taipei, 11529, Taiwan, ²Yuan Ze University, Department of Computer Science and Engineering, Chungli, 32003, Taiwan, ³National Taiwan University, Department of Horticulture, Taipei, 10617, Taiwan; [email protected]

Orchids are one of the largest families in Angiosperm with more than 25,000 species, displaying a wide diversity of unique and interesting biological features. Molecular and genetic research studies of orchids are hindered due to limited genomic information available in current databases. Recent advances in high throughput DNA sequencing technology make it economically feasible to generate orchid genomic information. We have designed an effective workflow to fast accumulate genomic information for these non-model organisms in order to facilitate their genome research. We used 454 GS FLX Titanium and Illumina Genome Analyzer IIx platforms to sequence cDNAs prepared from different organs of Phalaenopsis aphrodite. After quality trimming and assembly pipeline, 246,242 contigs were obtained, with 43,358 annotated to protein coding genes. Designed into the workflow is to achieve both gene discovery and expression profiling with the same sequencing efforts of Solexa reads. The expressed genes were blasted to database and their functional identities found in Pfam, Gene Ontology and KEGG. All datasets and analysis results were organized in a user-friendly manner in the Orchidstra website (URL: http://orchidstra.abrc.sinica.edu.tw).

S12.4 TRANSCRIPTOME PROFILING OF PODOPHYLLUM HEXANDRUM TISSUES FOR GENES IN PODOPHYLLOTOXIN BIOSYNTHESIS

Joaquim Vogt Marques,¹ Kye Won Kim,¹ Choonseok Lee,¹ Kerry C. Roby,¹ Michael A. Costa,¹ Gregory D. May,² John A. Crow,² Laurence B. Davin,¹ Norman G. Lewis¹

¹Washington State University, Institute of Biological Chemistry, Pullman, WA 99164-6340, USA, ²National Center for Genomic Resources, Santa Fe, NM 87505, USA; [email protected]

The roots and rhizomes of Podophyllum hexandrum have been used for decades as a source of the aryltetralin lignan podophyllotoxin, precursor to drugs used to treat several types of cancer. Yet in spite of its extensive use in medicine, much of the biosynthetic pathway to podophyllotoxin is mostly speculative. In an effort to further our understanding of its biosynthetic pathway, we have taken advantage of recent developments in gene sequencing technologies, as part of a broader program to study several medicinally important plant species. (To date we have examined 24 important medicinal plant species, see http://uic.edu/pharmacy/MedPlTranscriptome/index.html for transcriptome/metabolome data). Using Illumina sequencing approaches and various gene assembly construction strategies, we have, for example, generated a transcriptome database using different Podophyllum tissues and are mining these to identify the candidates responsible for the putative steps in the podophyllotoxin biosynthetic pathway. This approach has been initially successful first in identifying (and confirming assembly protocols for) all potential gene family members in this species for known biochemical steps, as well as for identification of putative unknown upstream enzymes in the phenylpropanoid pathway. Progress in identifying the missing steps (methylenedioxy bridge formation, etc.) in podophyllotoxin biosynthesis is described. This work is supported by 5 RC2 GM092561-02

S12.5 ARTHUR NEISH YOUNG INVESTIGATOR AWARD

INSIGHTS INTO STORAGE OIL BIOSYNTHESIS: COMPARATIVE TRANSCRIPTOMICS OF SEED AND NON-SEED TISSUES

Aruna Kilaru,^1,2 John Ohlrogge²

¹East Tennessee State University, Department of Biological Sciences, Johnson City, TN 37614, USA, ²Michigan State University, Department of Plant Biology, East Lansing, MI 48823, USA; [email protected]

Storage oils in the form of triacylglycerols (TAGs) in seeds serve as a high-energy carbon resource for post-germinative growth whereas they provide a source of food for seed dispersers when they occur outside the seed such as mesocarp. Using high-throughput deep transcriptional profiling tools, we generated 10 million ESTs for various developing seed and non-seed tissues. I will present insights from analysis of the similarities and differences in lipid gene expression and regulation in developing seed (rapeseed and castor) and non-seed (mesocarp of oil palm and avocado) tissues.

S12.6 FUNCTIONAL GENOMICS USING NON-MODEL PLANTS AND SYNTHETIC BIOSYSTEMS FOR GENE DISCOVERY IN SPECIALIZED METABOLISM

Peter J. Facchini,¹ Jillian M. Hagel,¹ Isabel Desgagné-Penix,¹ Eun-Jeong Lee,¹ Andrew Ekins,² Elena Fossati,² Jean-François Lauzon,² Vincent Martin²

¹University of Calgary, Department of Biological Sciences, Calgary, AB, T2N 1N4, Canada, ²Concordia University, Department of Biology, Montréal, QC, H4B 1R6, Canada; [email protected]

Among the vast catalogue of plant natural products are ~2500 benzylisoquinoline alkaloids (BIAs) that include codeine and morphine produced in opium poppy. cDNAs corresponding to most of the enzymes involved in morphine biosynthesis and several functioning in other branch pathways have been identified. The first committed step in BIA metabolism, norcoclaurine synthase, is catalyzed by a unique enzyme. All other known enzymes belong to a limited number of families including cytochromes P450, O- and N-methyltransferases, FAD oxidoreductases, dioxygenases, acyltransferases and three different types of reductases. Tapping into the enzyme variants responsible for the immense diversity of BIAs requires the generation of resources for a variety of plant species and the development of tools that provide a common functional genomics platform. The PhytoMetaSyn Project (www.phytometasyn.com) represents a consortium of Canadian researchers advancing the application of genomics and synthetic biology to the discovery of novel natural product biosynthetic genes and the reconstitution of pathways in microbes. The tools generated are allowing the discovery of novel BIA biosynthetic genes using plug-and-play synthetic biology based on candidate genes in our deep transcriptome databases, and complement our plant functional genomics tools such as virus-induced gene silencing.

O12.1 TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF REED (PHRAGMITES AUSTRALIS) RHIZOMES

Ruifeng He,¹ Min-Jeong Kim,¹ William Nelson,² Tiago Balbuena,³ Jay Thelen,³ Carol Soderlund,² David R. Gang¹

¹Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA, ²BIO5 Institute, University of Arizona, Tucson, AZ 85721, USA, ³Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211,USA; [email protected]

The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. To identify candidate genes and proteins involved in rhizome growth, development and metabolism, we employed next-generation sequencing and quantitative proteomics technologies to characterize the reed rhizome transcriptome and proteome. Combining 336,514 Roche 454 Titanium reads and 103,350,802 Illumina paired-end reads in a hybrid assembly yielded 124,450 unique transcripts with an average length of 549 bp, including 31,675 contigs (average length 1,283 bp) and 92,775 singletons, of which 54,202 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome tips (apical meristematic region) and rhizome elongation zones. A total of 1,280 non-redundant proteins were identified by label-free proteomics coupled to GeLC-MS/MS, of which 174 and 77 proteins were up regulated in the rhizome tip and elongation zone tissues, respectively. Identification and characterization of specific genes with potential roles in rhizome differentiation, development and function will be discussed.

O12.2 DISCOVERY OF DITERPENE BIOSYNTHETIC PATHWAYS USING TARGETED TRANSCRIPTOME ANALYSIS AND FUNCTIONAL CHARACTERIZATION OF GENES AND ENZYMES FOR METABOLIC ENGINEERING

Philipp Zerbe,¹ Angela Chiang,¹ Mack Yuen,¹ Björn Hamberger,² Britta Hamberger,² Jörg Bohlmann¹

¹University of British Columbia, Michael Smith Laboratories, Vancouver, BC V6T 1Z4, Canada, ²University of Copenhagen, Department of Plant Biology and Biotechnology, Copenhagen, 1871, Denmark; [email protected]

Plant diterpenoids are well characterized for their diverse physiological functions and many specialized (i.e., secondary) diterpenoids are also of substantial value as pharmaceuticals and other industrial products. Therefore, plants are perhaps the best known renewable resource for diterpene natural products, including compounds for the fragrance industry (e.g., cis-abienol, sclareol), precursors for industrial resins and coatings (e.g., conifer diterpene resin acids), and a plethora of pharmaceuticals with a wide range of applications as anti-cancer drugs (e.g., taxol), anti-inflammatory agents (e.g., marrubiin), or antimicrobials/antifungals (pseudolaric acids). The goal of our research is the discovery of enzymes for the production of a suite of diterpenoid compounds in metabolically engineered microbial host systems. For a set of ten different plant species we characterized the diterpenoid metabolite profiles, followed by tissue-specific 454- and Illumina transcriptome sequencing. Sequence databases were established for the discovery and functional characterization of diterpene biosynthetic pathway genes, targeting new diterpene synthases and cytochrome P450 monooxygenases. We will present the overall strategy for the rapid and successful genomics-based gene discovery of various diterpenoid biosyntheses and highlight specific examples of the functional characterization of enzymes, and details of reactions mechanisms gleaned from structural analyses.

O12.3 INVESTIGATING ESSENTIAL OIL METABOLISM IN LAVANDULA BY TRANSCRIPT PROFILING

Soheil Mahmoud

University of British Columbia, Okanagan, Biology, Kelowna, BC V1V 1V7, Canada; [email protected]

Several members of the genus Lavandula (lavenders) produce valuable essential oils (EO), which are extensively used in cosmetics, hygiene products and medicines. These oils are predominantly constituted of monoterpenes, the C₁₀ class of the isoprenoids. Although numerous genes encoding monoterpene synthase enzymes have been described from a wide range of plants including gymnosperms and angiosperms, regulation of production and secretion of EO constituents is still poorly understood. We are investigating EO metabolism in lavender glandular trichomes, and have generated extensive genomics resources for these plants. We obtained over 22,000 expressed sequence tags (ESTs) from flowers, leaves and glandular trichomes, and assessed their expression pattern in various tissues of three lavender species by microarrays. Our investigation led to the cloning and functional characterization of two EO biosynthetic enzymes. We also identified putative genes that control trafficking of EO constituents in glandular trichomes. Further, our data confirmed that the biosynthesis of EO constituents is regulated at multiple levels, including transcriptional control of the terpene synthase genes.

O12.4 TRANSCRIPTOME AND METABOLITE ANALYSIS OF POLYUNSATURATED FATTY ACID-RICH SEA BUCKTHORN (HIPPOPHAE RHAMNOIDES) SEED

Tahira Fatima,¹ Crystal L. Snyder,² Randall J. Weselake,² Priti Krishna¹

¹University of Western Ontario, Department of Biology, London, ON N6A 5B7, Canada, ²University of Alberta, Department of Agricultural, Food & Nutritional Science, Edmonton, AB T6G 2P5, Canada; [email protected]

Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit producing, plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its seed and pulp oil. Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the seed transcriptome using 454 sequencing. GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33–36% and 30–36%, respectively, while the pulp oil contained palmitoleic acid at 32–42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds identified sequences related to fatty acid biosynthesis. A subset of these was examined for transcript expression at four developing stages of the berry. This study provides the first comprehensive genome sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.

O12.5 METABOLITE AND GENE EXPRESSION STUDIES IN ENDOPHYTE INFECTED AND UNINFECTED TALL FESCUE UNDER WATER DEFICIT STRESS

Padmaja Nagabhyru,¹ Randy D. Dinkins,² Charles W. Bacon,³ Christopher L. Schardl¹

¹University of Kentucky, Plant Pathology, Lexington, KY 40546, USA, ²University of Kentucky, USDA, Forage-Animal ProductionResearch Unit, Lexington, KY 40516, USA, ³University of Georgia, Toxicology and Mycotoxin Research Unit, USDA, Athens, GA 30605, USA; [email protected]

Tall fescue plants symbiotic with the endophytic fungus, Neotyphodium coenophialum (E+), have better survivability and persistence under stressful conditions, especially under drought stress, than plants lacking the endophyte (E-). To understand more about the grass-endophyte interactions, how endophyte affects the host plant physiology and geneexpression especially when the plants are subjected to water deficit stress conditions, we conducted a time course water deficit stress experiment using 3 clone pairs of tall fescue. Upon rewatering, survival and retillering were significantly greater for E+ than E- plants starting from day 2 or 3 of the treatment. We observed higher accumulation of the free sugars like glucose, fructose, trehalose, and amino acid proline in E+ plants at early days of onset of stress compared to E- plants. Loline alkaloids and mannitol, which are fungal metabolites, also increased with water deficit stress. Thus endophyte aids in survival and recovery of plants from drought, and may act in part by inducing rapid accumulation of these compatible solutes, soon after imposition of stress. Illumina mRNA sequencing of these E+ and E- clones shown 125 unigenes were differentially expressed two-fold or more between them. Sequencing of these E+ and E- stressed as well as watered controls tissues, is in progress to see any specific effects of endophyte on plant gene expression especially under water deficit stress.

O12.6 A GENOMICS APPROACH TO GENE DISCOVERY RELATED TO BIOSYNTHESIS OF THUJONE IN WESTERN RED CEDAR (THUJA PLICATA)

Adam Foster,¹ Dawn Hall,² Shelley Abercromby,¹ Regine Gries,¹ Gerhard Gries,¹ Jörg Bohlmann,² John Russell,³ Jim Mattsson¹

¹Simon Fraser University, Biological Sciences, Burnaby, BC V5A1S6, Canada, ²University of British Columbia, Michael Smith Laboratories, Vancouver, BC V6T1Z4, Canada; ³British Columbia Ministry of Forests, Burnaby, BC V0R2N0, Canada; [email protected]

Reforestation with T. plicata is severely hampered by extensive ungulate browsing of plantlets. High foliar monoterpenoid content correlates with reduced browsing, providing a target for resistance breeding. The most abundant monoterpenoids in T. plicata foliage are α- and β-thujone, both of which strongly deter ungulate browsing. We found that these compounds are stored in foliar resin glands. Thereafter, we used next generation DNA sequencing technology to compare transcript profiles of foliage with and without glands to identify >600 genes whose expression associated with these structures. The differential expression was confirmed for the majority of tested genes by RT-Q-PCR. One of the most differentially expressed genes encodes a putative monoterpene synthase. In situ RNA hybridization showed that this gene is expressed in the epithelium of foliar resin glands. Furthermore, in vitro enzyme assays showed that the corresponding protein converts geranyl pyrophosphate almost entirely into sabinene, a known precursor of thujone synthesis. We are currently assessing whether candidate genes for the conversion of sabinene into thujones, primarily cytochrome P450s and reductases, are also expressed in gland epithelium.

O12.7 EST ANALYSIS OF TRANS-RUBBER PRODUCING PLANT, EUCOMMIA ULMOIDES OLIVER AND IDENTIFICATION OF CANDIDATE GENES IN TRANS-1,4-POLYISOPRENE PRODUCTION

Nobuaki Suzuki,^1,3Shinya Takeno,^2,3Hirotaka Uefuji,^1,3 Takashi Nishikawa,^1,3 Takeshi Bamba,^1,3 Ei-ichiro Fukusaki,^1,3 Akio Kobayashi,^1,3 Yoshiyuki Ogata, Daisuke Shibata, Yoshihisa Nakazawa^1,2,3

¹Osaka University, Department of Biotechnology, Graduate School of Engineering, Suita, 5650871, Japan, ²Technical Research Institute, Hitachi Zosen Corporation, Osaka, 5510022, Japan; [email protected]

Eucommia ulmoides Oliver is one of the few woody plants producing abundant trans-polyisoprene rubber in leaf, bark and seed coat. Two cDNA libraries derived from the bark and inner tissue in the stem were constructed and 27752 expressed sequence tags (ESTs) including 10520 unigenes consisting of 4302 contigs and 6218 singlets were generated. Homologues of major latex protein (MLP) and rubber particle membrane proteins (RPMPs) which function for effective synthesis of high molecular poly-isoprene in latex were isolated and shared high proportions in the ESTs, indicating abundant expression for trans-polyisoprene rubber biosynthesis. The six MVA pathway genes involved in synthesis of isopentenyl diphosphate (IPP), a starting material for poly‐isoprene biosynthesis, were isolated and shown to be involved in IPP biosynthesis. Five full length trans-isoprenyl diphosphate synthase were also isolated and two of them functioned to synthesize farnesyl diphosphate which is assumed as an intermediate of rubber biosynthesis.

O12.8 COMPARATIVE PROTEOMIC STUDY REVEALS THE BIOSYNTHESIS OF COUMARINS IN LEAVES OF CLEMATIS TERNIFLORA UPON UV RADIATION

Runze Chen,¹Wen Zheng,² Lei Cui,¹ Yanjuan Guo,² Mingyao Sun,¹ Wei Ning,² Lin Zhang,² Jingkui Tian¹

¹Education Ministry Key Laboratory for Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou, 310027, China, ²College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou, 310027, China; [email protected]

Phytochemical studies have been carried out to reveal the effects of UV radiation on leaves of Clematis terniflora DC. Three coumarins were prominently induced by UV-B radiation and could not be detected in control and UV-A treatment groups. Harmful effects of UV-B radiation were found on the surface of leaves. A comparative proteomic research method has been used to study the different expression levels of proteins under high intensity (120.8 μw/cm²) UV-A and UV-B radiation. Seventy three differentially expressed proteins were identified by MALDI-TOF/TOF MS. Functions of the successfully identified sixty three proteins that mainly focus on photosynthesis and respiration, transporting, amino acid biosynthesis, secondary metabolism, defence and stress responses, carbohydrate metabolism, energy metabolism and other categories. Results showed that proteins which were involved in the secondary metabolic pathway of phenylpropanoid biosynthesis were significantly stimulated. These proteins are very important in the UV-B induced production of coumarins. This study helps to increase our understanding of the comprehensive functional network Clematis terniflora uses to adapt to UV-A and UV-B stress. More importantly, this opens up new areas for the exploration of the changes in plant secondary metabolic pathways under UV radiation.

S13.1 BIOPOLYMERS, BIOPRODUCTS AND BIOFUELS IN ALTERNATE RUBBER-PRODUCING SPECIES

Katrina Cornish

Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA; [email protected]

Efforts are underway to move sustainable biorefineries, powered with renewable fuels, from a subsidized concept to commercial reality. The production of rubber, resins, bioproducts and bio-fuels from Parthenium argentatum, or rubber, inulin and biofuels from Taraxacum kok-saghyz, are both attractive to a true biorefinery capable of producing a wide range of phytochemicals. A combination of products and fuels, from single feedstocks, is needed for profitability. However, as both are new industrial crops, scale-up issues are extremely complex, and species-specific chemistry and biochemistry must be understood and capitalized upon. The expansion of acreage must be intimately tied to expansion of processing capacity and to specific markets.

S13.2 ADVANCES IN UNDERSTANDING PLANT CELL WALL PECTIN SYNTHESIS AND STRUCTURE AND IMPACT ON THE BIOFUEL INDUSTRY

Debra Mohnen, Melani Atmodjo, Ajaya Kumar Biswal, Kimberly Hunt, Sushree Sangita Mohanty, Ivana Gelineo-Albersheim, Sivakumar Pattathil, Michael G. Hahn, Robert Amos, Zhangying Hao, Li Tan

Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA; [email protected]

Plant cell walls are comprised of cellulose, so-called matrix polysaccharides (pectin and hemicellulose), cell wall proteins, and in many secondary walls, lignin. Among these, pectin is the most structurally complex polysaccharide and is often considered as mainly associated with primary cell walls, although recent results indicate an important role in secondary walls as well. Studies in our group on pectin structure and synthesis have centered on the potential function of pectin in the recalcitrance of plant biomass to deconstruction for biofuel production and on the role of a family of glycosyltransferases known as GAUTs in pectin synthesis. The results suggest that pectin may be more realistically viewed as a polysaccharide domain within a complex glycoconjugate matrix. The role of GAUTs in the synthesis of pectic domains and implications for engineering plants for enhanced biofuel production will be discussed.

The work was supported by USDA AFRI 2010–65115-20396; National Science Foundation NSF-MCB 0646109, DOE center grant DOE DE-FG02-09ER20097 and BioEnergy Science Center grant DE-PS02-06ER64304. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.

S13.3 PECTIN MODIFICATION IMPROVES UTILIZATION OF PLANT BIOMASSES TO BIOFUEL CONVERSION

Simone Ferrari, Vincenzo Lionetti, Fedra Francocci, Manuel Benedetti, Daniela Pontiggia, Susanna Tomassetti, Daniela Bellincampi, Giulia De Lorenzo, Felice Cervone.

Sapienza Università di Roma, Dipartimento di Biologia e Biotecnologie “Charles Darwin”, 00185 Rome, Italy; [email protected]

Cell wall recalcitrance to enzymatic hydrolysis is the main bottleneck for the industrial scale-up of biomass processing and bioconversion to fermentable sugars. We aimed to overcome the difficulties of converting plant biomass into usable products by genetically engineering or selecting plants with altered expression of proteins that help either break down the components of the cell wall or prevent the cell wall polysaccharides from forming crosslinks. Pectin acts as a glue by affecting cell wall stiffening through homogalacturonan (HGA) calcium-mediated cross-links. Saccharification efficiency of dicot and monocot biomass can be improved by reducing the amount of acidic HGA domains through the constitutive expression of a fungal polygalacturonase (PG) or the overexpression of an inhibitor of pectin methylesterase (PMEI). We are also exploring the possibility to improve cell wall degradation without causing growth defects through the controlled expression of pectin-degrading enzymes. Saccharification is also improved in Arabidopsis mutants with a lower content of de-methylated stretches of HGA as compared to the wild type, indicating that the level of unesterified HGA is a useful parameter to isolate natural variants with improved saccharification efficiency. Understanding the biochemical and genetic determinants of cell wall degradability will be useful to identify markers for the breeding of new varieties suited for the dual food and bioenergy production

S13.4 ENGINEERING IMPROVED CELLULASES FOR BIOFUEL PRODUCTION

Michael E. Himmel, Gregg T. Beckham, Christina M. Payne, Lintao Bu, James F. Matthews, Stephen R. Decker, John O. Baker, Larry Taylor II, Arjun Singh, Qi Xu, Michael F. Crowley

Biosciences Center, NREL, Golden, CO 80401, USA; [email protected]

The Trichoderma reesei Families 6 and 7 cellulases (Cel6A and Cel7A) are key industrial enzymes used for the production of biofuels from lignocellulosic biomass. These enzymes are multi-modular, with a Family 1 carbohydrate-binding module linked to a large catalytic domain via a flexible O-glycosylated linker. We have used simulation to elucidate new functions for these three sub-domains in general, and have demonstrated a new route to increase the activity of Cel7A. These findings include new roles for glycosylation, which we have shown can be used to tune the binding affinity. We have also examined the structure of the catalytically-active complex of Cel7A and its non-processive counterpart, Cel7B, engaged on cellulose, which suggests allosteric mechanisms may be involved in chain binding when these cellulases are complexed on cellulose. Our computational results also suggest that product inhibition varies significantly between Cel7A and Cel7B, and we offer a molecular-level explanation for this observation. These results highlight new considerations in protein engineering for processive and non-processive cellulases.

S13.5 ENGINEERING PLANT CELL WALLS FOR SECOND GENERATION BIOFUEL PRODUCTION

Charis Cook, Paul G. Bolwell, Alessandra Devoto

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, United Kingdom; [email protected]

The substrate for second generation biofuels is lignocellulosic material obtained from plant cell walls. Genetic modification of the cell wall has the potential to improve cellulose accessibility and hydrolysis, therefore decreasing the cost and energy input in biofuel production. This study aims to improve understanding of cell wall biosynthesis and organisation to increase cellulose content and extractability by genetic modification and pretreatment with white rot fungus Phanerochaete chrysosporium.

Enzymatic saccharification assays have shown differences in soluble sugars released from transgenic tobacco lines down-regulated in both lignin and xylan. Significantly, TOBACCO PEROXIDASE 60 down-regulated line 1074 shows 30% increase in glucose release as compared to the wildtype. Xylan down-regulation by suppression of UDP-GLUCURONATE DECARBOXYLASE, which synthesises the xylan precursor xylose, also caused improvement in saccharification. Treatment of the cell wall modified lines with P. chrysosporium, a white rot fungus that naturally hydrolyses and metabolises lignin further improved saccharification after pretreatment.

We also show that lignin biosynthesis pathway is down-regulated at the transcriptional level in lignin modified lines, while the polysaccharide biosynthesis response differs depending on the position of disruption in lignin biosynthesis.

S13.6 COMMON THEMES IN LIGNIN BIOSYNTHESIS AND LIGNIN BIODEGRADATION

Yi-ru Chen, Simo Sarkanen, Yun-Yan Wang

University of Minnesota, Bioproducts and Biosystems Engineering, St. Paul, MN 55108, USA; [email protected]

The primary structures of lignins may be replicated through a direct template polymerization mechanism during the final step of lignin biosynthesis. The process is governed by powerful forces arising from electron correlation between precursor aromatic rings and the pre-existing substructures about to undergo replication. Correspondingly, the enzymatic degradation of lignin macromolecules requires two consecutive steps. After enzyme-catalyzed cleavage, dissociation between individual lignin fragments and components may be facilitated by other proteins that can compete with the strong noncovalent interactions remaining between the oligomers and polymeric lignin chains.

S13.7 LIGNIN: THE NEW PARADIGM IN BIOFUELS

Art J. Ragauskas

BioEnergy Science Center, Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA; [email protected]

The biological conversion of biomass to biofuels is a multi-faceted technical challenge that has focused on the basic structure of the plant cell wall and its chemical components. Although the native recalcitrance of biomass is attributed to many factors including cellulose crystallinity/degree of polymerization, lignin/hemicellulose structure, lignin-carbohydrate complexes, cell wall structure and accessibility, recent advances in transgenic plants have highlighted the special role that lignin holds on recalcitrance. By down-regulating select monolignol pathways it has been possible to significantly decrease recalcitrance and improve the overall efficiency of cellulosic ethanol by +20%. Research studies in lignin structure for transgenic plants have shown that the observed reduction in recalcitrance for lignin-lite transgenic plants is due to reductions in lignin content and structure. Furthermore, pretreatment a key technology in 2^nd and 3^rd generation biofuels significantly alters the structure of lignin and facilitates biological deconstruction of plant polysaccharides. This presentation will integrate our investigations into the structure of lignin pre‐ and post‐pretreatment for native and transgenic biomass with a special emphasis on the role of β-O-aryl ethers, condensed and non-condensed lignin structures as determined by 1D and 2D NMR. Finally, lignin to date remains an under-utilized biofuels resource in itself especially from cellulosic ethanol technologies. Understanding its structure after pretreatment has facilitated its utilization as a feedstock resource for heterotrophic oleaginous microorganism leading to the generation of triacylglycerols which opens new avenues for securing biodiesel from lignocellulosics.

S13.8 METABOLIC VERSUS TRANSCRIPTIONAL CONTROL TARGETS FOR LIGNIN MODIFICATION

Richard A. Dixon, Fang Chen, Lina Gallego-Giraldo, Huanzhong Wang, Hui Shen, Xian-Zhi He, Qiao Zhao

Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK 73401, USA; [email protected]

Lignocellulosic biomass is recalcitrant to saccharification, and this is, at least in part, due to the presence of the cell wall polymer lignin. Analysis of alfalfa (Medicago sativa) plants in which lignin content and composition had been modified through independently down-regulating each of eight enzymes in the lignin pathway revealed that reduction in lignin content progressively increased saccharification efficiency. However, the gains in fermentable sugar production are partially offset by reductions in plant yield in some, but not all, of these transgenic lines. Levels of the stress hormone salicylic acid (SA) inversely mirror lignin levels in a series of transgenic alfalfa plants, and genetic experiments in Arabidopsis point to SA production as being responsible for most of the reduced growth phenotypes of lignin down-regulated plants. Targeting transcriptional regulators of the lignin or whole secondary cell wall pathways provides an alternative strategy for lignin down-regulation that potentially avoids metabolic spillover effects. Forward genetic screening of a transposon mutagenized population of barrel medic (Medicago truncatula) has led to the identification of NAC and WRKY master switches that work as positive and negative regulators of lignification, respectively. Loss of function of WRKY12 in Arabidopsis leads to a 50% increase in stem biomass density. Manipulation of lignin biosynthetic and regulatory genes has potential to deliver bioenergy feedstocks or forages combining increased cell wall density with improved digestibility.

S13.9 CHARACTERIZATION OF LIGNIN AND RELATED COMPOUNDS OF ERIANTHUS RAVENNAE

Masaomi Yamamura,¹ Yuichiro Otake,¹ Soichiro Noda,¹ Takefumi Hattori,^1,2 Keiji Takabe,³ Shiro Suzuki,^1,2 Nozomu Sakurai,⁴ Hideyuki Suzuki,⁴ Masakazu Ike,⁶ Ken Tokuyasu,⁶ Jun Kikuchi,⁵ Daisuke Shibata,^1,4 Toshiaki Umezawa^1,2

¹Kyoto University, Research Institute for Sustainable Humanosphere, Uji, Kyoto 611-0011, Japan, ²Kyoto University, Institute of Sustainability Science, Uji, Kyoto, 611-0011 Japan, ³Kyoto University, Graduate School of Agriculture, Kyoto 606–8052, Japan, ⁴Kazusa DNA Research Institute, Kisaradu, Chiba 292-0818, Japan, ⁵RIKEN, Plant Science Center, Yokohama, Kanagawa 230-0045, Japan, ⁶National Food Research Institute, Tsukuba, Ibaraki 305–8642, Japan; [email protected]

Recently, plant biomass such as Erianthus, switchgrass, Miscanthus, Jatropha and oil-producing algae have been receiving a lot more interest as chemical and biofuel feedstocks. Liquid fuels from the plant biomass or lignocellulosic materials offer an attractive alternative to fossil fuels. Lignocellulosic biomass is composed of cellulose, hemicelluloses and lignins. The three components constitute a suprastructure, where lignin encrusts cellulose microfibrils and confers mechanical strength and imperviousness to the cell wall. At the same time, lignins are obstacles in the enzymatic conversion of plant cell wall polysaccharides into biofuels. However, little is known about lignins of energy-producing plants, especially Erianthus ravennae. Recently, we established a microscale, high-throughput, and highly reproducible nitrobenzene oxidation method. We have successfully applied the method as well as a number of analytical methods to the analysis of E. ravennae, thereby lignins, related compounds, and enzymatic saccharification efficiency of the plant have been characterized.

S13.10 NORTHWEST ADVANCED RENEWABLE ALLIANCE (NARA) AND THE QUEST FOR BIOFUELS/PETROCHEMICAL SUBSTITUTES

Norman G. Lewis

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA; [email protected]

Vascular plants have evolved remarkable phytochemical armouries, not only from a lignocellulosic recalcitrance perspective but also through specialized metabolism leading to a variety of bioactive/defense molecules. Many countries now wish to explore the potential of sustainably using these renewable (woody) lignocellulosic materials as “feedstocks” for liquid fuels and petrochemical substitutes at the scale and cost needed.

The Northwest Advanced Renewables Alliance has been created to establish the feasibility of using various woody biomass (Douglas-fir, western hemlock, poplar and red alder) for such applications and uses; this presentation focuses upon the progress made thus far in genetically modifying model plant (Arabidopsis) and poplar in efforts to overcome lignocellulosic recalcitrance through gene downregulation/transcription factor modulation/introduction of new biochemical pathways.

O13.1 BIOPOLYMER PRODUCTION IN TRANSGENIC POPLAR

David Dalton,¹ Caiping Ma,² Shreya Shrestha,¹ Peter Kitin,² Steven Strauss²

¹Reed College, Biology, Portland, OR USA 97202, ²Oregon State University, Forest Ecosystems and Society, Corvallis, OR 97331, USA; [email protected]

Poplar is an attractive model species for the production of commercially promising biopolymers such as polyhydroxybutyrate (PHB). A PHB-producing system was developed in poplar based on the introduction of three bacterial transgenes under the control of an ecdysone-inducible promoter. The negative impacts of PHB production on plant health were minimized by targeting of PHB synthesis to the chloroplast and delaying production until application of an inducing chemical - the licensed insecticide Intrepid. PHB concentrations up to 1-2 % (dry weight) were detected in leaves of 18 separate events after 6-8 weeks of application of the inducing chemical. There was no direct toxicity due to the inducing agent, but growth (mass and height) was reduced substantially in plants in which the concentration of PHB exceeded 1%. PHB granules were visualized within chloroplasts using confocal fluorescence microscopy and found to measure 3.6 μm in length. Ongoing efforts to develop new senescence-associated promoters that might enable enhanced PHB yields will be discussed.

O13.2 NAC DOMAIN TRANSCRIPTION FACTORS AND SECONDARY WALL FORMATION CONTROL IN POPLAR (POPULUS TRICHOCARPA)

Hong Yang, Chanyoung Ki, Claudia L. Cardenas, Kye-won Kim, Laurence B. Davin, Norman G. Lewis

Institute of Biological Chemistry (IBC) and Northwest Advanced Renewables Alliance (NARA), Washington State University, Pullman, WA 99164-6340, USA; [email protected]

The use of woody plants, such as poplar, as bioenergy/bioproduct plantation feedstock is limited, due in part to lignocellulosic recalcitrance that impedes facile fermentation/saccharification into products suitable as scalable petrochemical replacement/intermediate commodity chemicals. To begin to consider new ways to overcome this form of recalcitrance, it was recently discovered that NST/SND and VND subgroups of NAC domain transcription factors can function as “master switches” for fiber and vessel secondary wall formation in the model plant, Arabidopsis. This raised the possibility of engineering woody plants more amenable to lignocellulosic processing. Accordingly, genes encoding poplar NAC TFs (PtNAC1, PtNAC6, PtNAC7, PtNAC13 and PtNAC17) were initially studied for their abilities to restore secondary wall formation in the Arabidopsis SND1/NST1-RNAi double mutant. Overexpression of all five PtNACs in the double mutant rescued secondary wall formation in the fiber cells and restored lignin contents to near WT levels as well, as determined by comprehensive histochemical and chemical analyses. These findings suggest that all five PtNAC are functional (potentially) redundant TFs involved in poplar secondary wall formation and lignin biosynthesis. In-depth functional analyses using overexpression and silencing of five poplar PtNAC TFs are also currently underway with transgenic poplars.

O13.3 INTERACTIONS BETWEEN THE CO₂ CONCENTRATING MECHANISM AND LIPID PRODUCTION OF TWO SPECIES OF ALGAE: CHLAMYDOMONAS REINHARDTII AND NANNOCHLOROPSIS SALINA

Samuel Lopez-Nieves,¹ Howland Jones,² Omar Garcia,² Aaron Collins,² Jerilyn Timlin,² David Hanson¹

¹University of New Mexico, Biology, Albuquerque, NM 87131, USA, ²Sandia National Laboratories, Albuquerque, NM 87185, USA; [email protected]

The demand for energy is rapidly depleting available resources and creating a need for renewable energy. Wind and solar power are valuable sources of electricity, but they do not generate energy dense compounds needed for long distance uses such as long-haul trucking and air transportation. Algal lipid production may help to solve these needs without impacting food sources. However, many fundamental questions remain unanswered in algal biology and this is impairing development of viable algal lipid production systems. We have chosen to examine the basic research question of how algal CO₂ concentrating mechanism (CCM) function affects lipid production. CCM function is often induced at low CO₂ and it increases the efficiency of CO₂ capture, though the allocation of carbon under these conditions is not well understood. We analyzed lipid content and CCM function at low CO₂ (CCM expressed) and high CO₂ (CCM suppressed) to determine if CCM expression increases or decreases lipid storage. Our measurements of online carbon isotope discrimination showed that high CO₂ suppressed the CCM in both C. reinhardtii and N. salina. Our preliminary discrimination data also provided evidence for an unusual HCO₃⁻ pump-CO₂ leak style CCM in N. salina that was first characterized in other species of Nannochloropsis. In addition, our preliminary data using a hyperspectral imaging suggest lipid content increases in both species after exposure to high CO₂. However, the effect is smaller in C. reinhardtii and much of the lipid accumulation occurred in the eyespot.