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United States Department of Agriculture

Agricultural Research Service


Location: Natural Products Utilization Research

2009 Annual Report

1a.Objectives (from AD-416)
To discover phytotoxins and allelochemicals for use in pest management and to provide the fundamental information on mode of action, structure-activity relationships, natural resistance mechanisms, and biosynthesis of natural phytotoxins that will be required for development medicinal/nutraceutical crops. To discover, characterize, manipulate and utilize genes involved in the production of natural products and in chemical defense and resistance mechanisms against allelochemicals and environmental phytotoxins.

1b.Approach (from AD-416)
Conduct bioassays in collaboration with research chemists during bioassay-directed isolation of new phytotoxins. Molecular sites of action will be determined with genomic and biochemical approaches. Biosynthetic pathways of toxins from plants will be investigated. Genes controlling synthesis of useful plant secondary products and plant defenses to phytotoxins will be identified, cloned, and manipulated.

3.Progress Report
Constituents of camphorweed, Heterotheca subaxillaris, were found to be highly phytotoxic. The strongest plant growth inhibitor against Agrostis stolonifera and Lactuca sativa seedlings, as well as duckweed (Lemna pausicostata), was 2-methoxy-calamenene-14-carboxylic acid. This level of activity is in the range of commercial herbcides evaluated with the same bioassay in our laboratory. We investigated the physiological responses of calamenene-type sesquiterpenes isolated from camphorweed on plant growth inhibition. Acetylation of calamenene molecule was found to have a dramatic effect on the biological activity of these natural phytotoxins.

Databases of transcriptome signatures for compounds with known modes of action can serve as important resources for obtaining clues concerning the mode of action of unknown bioactive compounds. We have been using Arabidopsis thaliana as a model for generating whole-genome transcriptome responses, using conditions emulating standard field application procedures. Our data set has recently been expanded to include post-emergence herbicides targeting the enzymes protoporphyrinogen oxidase as well as 4-hydroxyphenyl-pyruvate-dioxygenase, which participate in the biosynthesis of protoporphyrin and carotenoids, respectively.

We found the blister beetle-derived compound, cantharidin, to be a good natural phytotoxin with activity as good as the herbicide endothall. Camtharidin is a known protein phosphatase inhibitor. The responses of Arabidopsis to it at the transcriptional and translational levels were complex and in keeping with its activity as a protein phosphatase inhibitors. This is a novel mode of action for a phytotoxin.

Catechin was reported to be a potent phytotoxin responsible for the success spotted knapweed, a major invasive species. We found both isomers of catechin to be weakly phytotoxic in liquid culture and to have essentially no activity in soil. Furthermore, was confirmed to be an antioxidant, not a compound causing oxidative stress, as reported by those claiming it to be a potent phytotoxin. Numerous compounds from plants and microbes were evaluated for phytotoxicity, and several were found to have activity of interest. At this time, the identity of the more phytotoxic compounds cannot be revealed for proprietary reasons. Constructs for identifying root-hair-specific promoters were designed and made. Four genes (DES3, Gama-tocopheral methyltransferase, GST and RCc3 (code for hypothetical protein)) were selected for promoter analysis. Analysis of these genes using computer programs and sorghum expressed sequence tag (EST) databases had shown that they are exclusively expressed in root hair tissue. Promoters and terminators of these genes were cloned and fused with GUSPlus, an expression marker, to form expression cassettes (promoter-GUSPlus-terminator). These constructs are being transformed into rice and tobacco.

1. Arabidopsis and Tobacco Transformed for the Production of Pterostilbene, a High-potency Resveratrol Analogue with Antifungal Activity. Stilbenoid phytoalexins, such as resveratrol, play an important defense role in several plant species against pathogens such as Botrytis cinerea. In addition to their antimicrobial properties, resveratrol and related stilbenes have also generated considerable interest as nutraceuticals, due to their potential role in promoting cardiovascular health, inhibiting tumor formation, and increasing cell longevity. Several studies have demonstrated that pterostilbene, a methyl ether derivative of resveratrol, possesses similar potent antioxidant and anticancer properties, and in addition could provide host plants with more broad-spectrum resistance to fungal pathogens. A dual- transgene cassette approach was therefore employed using a resveratrol synthase sequence from peanut in conjunction with an O-methyltransferase (OMT) from Sorghum bicolor (patent filed by this project) capable of catalyzing the conversion of resveratrol to pterostilbene, both expressed behind the CaMV 35S promoter in transgenic Arabidopsis as well as tobacco. The OMT is the root hair-specific enzyme involved in synthesis of sorgoleone (see FY07 progress report). Significant levels of pterostilbene accumulated in transformants of both species, demonstrating the potential utility of this strategy for the development of novel crop germplasm with enhanced nutraceutical value and disease resistance.

Review Publications
Duke, S.O., Blair, A.C., Dayan, F.E., Johnson, R.D., Meepagala, K.M., Cook, D., Bajsa, J.N. 2009. Is (-)-Catechin a "Novel Weapon" of Spotted Knapweed (Centaurea stoebe)? Journal of Chemical Ecology. 35:141-153.

Bajsa, J.N., Duke, S.O., Tekwani, B.L. 2008. Plasmodium falciparum Serine/Threonine Phosphoprotein Phosphatases (PPP): From Housekeeper to 'Holy Grail'. Current Drug Targets. 9:997-1012.

Chang, Z., Li, L., Pan, Z., Wang, X. 2008. Crystallization and Preliminary X-ray Analysis of Allene Oxide Synthase, Cytochrome P450 CYP74A2, from Parthenium argentatum. Acta Crystallographica Section F. F64:668-670.

Dayan, F.E., Cantrell, C.L., Duke, S.O. 2009. Natural Products in Crop Protection. Bioorganic and Medicinal Chemistry. 17:4022-4034.

Dayan, F.E., Howell, J., Weidenhamer, J. 2009. Dynamic Root Exudation of Sorgoleone and Its in Planta Mechanism of Action. Journal of Experimental Botany. 60(7):2107-2117.

Dayan, F.E., Singh, N., Mccurdy, C., Godfrey, C., Larsen, L., Weavers, R.T., Van Klink, J., Perry, N. 2009. B-Triketone Inhibitors of Plant p-Hydroxyphenylpyruvate Dioxygenase: Modeling and Comparative Molecular Field Analysis of Their Interactions. Journal of Agricultural and Food Chemistry. 57:5194-5200.

Duke, S.O., Dayan, F.E., Bajsa, J.N., Meepagala, K.M., Hufbauer, R.A., Blair, A.C. 2009. The Case Against (-)-Catechin Involvement in Allelopathy in Centaurea stoebe (spotted knapweed). Plant Signaling and Behavior. 4(5):422-424.

Kini, S.G., Bhat, A.R., Bryant, B., Williamson, J.S., Dayan, F.E. 2009. Synthesis, Antitubercular Activity and Docking Study of Novel Cyclic Azole Substituted Diphenyl Ether Derivatives. European Journal of Medicinal Chemistry. 44(2):492-500.

Morimoto, M., Cantrell, C.L., Libous Bailey, L.M., Duke, S.O. 2009. Phytotoxicity of Constituents of Glandular Trichomes and the Leaf Surface of Camphorweed, Heterotheca subaxillaris. Phytochemistry. 70:69-74.

Techen, N., Pan, Z., Scheffler, B.E., Kahn, I.A. 2009. Detection of Illicium anisatum as Adulterant of Illicium verum. Planta Medica. 75:392–395.

Dayan, F.E., Duke, S.O. 2009. Biological Activity of Allelochemicals. Book Chapter. In: Plant-Derived Natural Products. A.E. Osbourn and V. Lanzotti (eds.). Springer Science, New York, New York, pp. 361-384.

Gimsing, A., Baelum, J., Dayan, F.E., Locke, M.A., Sejero, L.H., Jacobsen, C. 2009. Mineralization of the Allelochemical Sorgoleone in Soil. Chemosphere. 76:1041-1047.

Last Modified: 4/19/2014
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