Location: Natural Products Utilization Research
2020 Annual Report
Objectives
The overall goal of this project is to discover and develop natural product-based weed management solutions. The research is divided into discovery and development of biochemical bioherbicides and creation of crops that are resistant to weeds by transgenically imparting or improving plant/plant allelopathy. This research should provide new avenues for the development of affordable and effective, yet sustainable, weed control strategies.
1. Discover and develop natural product-based bioherbicides that provide environmentally safe and toxicologically benign tools for weed management, with novel modes of action, to address current problems associated with herbicide resistant weeds.
1.1. Discover new and existing natural products for potential use as herbicides and bioherbicides.
1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations.
1.3. Develop natural products as new weed management tools.
2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals.
2.1. Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway.
2.2. The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone.
2.3. Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis.
2.4. Engineering de novo sorgoleone biosynthesis in non-producing host plants.
Approach
Bioassay-directed isolation of phytotoxin will be followed by their evaluation of their potential as bioherbicides and determination of their modes of action. Genes of the sorgoleone synthesis pathway with root hair-specific promoters will be inserted into plants with the intent to impart or improve allelopathic capacity for enhanced weed management.
Progress Report
This is the final report for this project; therefore, it includes highlights from the entire 5 year period as well as information for the current year. This project will be replaced with a new project, "Natural Product-Based Bioherbicides" pending completion of the National Program 304 research review.
Significant progress on discoveries of natural products as potential herbicides were made over the past five years. A large number of natural products from plants, insects, and fungi were characterized. Many of these compounds are new compounds or are known natural compounds that had not been previously determined to be phytotoxic. The major natural compounds or derivatives discovered to have significant phytotoxicity include khellin, visnagin, curvularin, alfa,beta-dehydrocurvularin, saponins, pyrichalasin, cleistanthane terpenoids, triterpene novel lignans, new cassane diterpenoids, cytochalasins, confertin, etc. Khellin and visnagin are the subject of a patent filed on the use of these compounds as natural herbicides.
Omics methods were used to probe the mode of action of serval phytotoxins. For example, proteomics was used to study the mode of action of the potent insect-derived phytotoxin cantharidin. RNA-seq was used to probe the mode of action of t-chalcone and citral. In silico binding studies were also used to determine that citral inhibits single-stranded DNA binding proteins. The mode of action of romidepsin and spliceostatin C were determined to be inhibition of histone deacetylase and spliceosome function. The roles of three amino acids (histidine, tryptophan, and cysteine) were implicated in the mode of action of the allelochemical 2-benzoxazolinone. Several contributions to the understanding of plant/plant allelopathy were made. For example, the chemistry and biological activity of triketone allelochemicals were further explored. The transcriptional responses to chemical cues from a weed (barnyard grass) of genes of the gene cluster for synthesis of the allelochemical momilactone B in rice were characterized. Three genes in the cluster were upregulated in the presence of barnyard grass.
With the successful characterization and identification of a cytochrome P450 enzyme (CYP71AM1) from sorghum, all of the genes required for sorgoleone biosynthesis beginning with the C16:1 fatty acid now been identified by ARS researchers in Oxford, Mississippi. Thus, the primary tools required to engineer the production of sorgoleone in transgenic crops are now in hand for creating the potential to generate novel varieties with reduced reliance on synthetic herbicides. As a proof of concept, a multi-gene vector was constructed which contains the complete open reading frames for desaturases (DES2, DES3), alkylresorcinol synthase (ARS2), O-methyltransferase (OMT3), and cytochrome P450 enzyme (CYP71AM1) driven by the constitutive promoters to direct the production of sorgoleone. When tested in a Nicotiana benthamiana transient assay system using this vector, sorgoleone was detected on the leaves expressing these genes, indicating that the expression of these sorghum genes simultaneously is capable of producing sorgoleone. One of the major goals has been the development of technologies that could confer the ability to conduct de novo biosynthesis of sorgoleone to root hair cells of non-producing crop species. Toward this aim, four different promoters with 3’-flanking region combinations capable of directing high-level root hair-specific expression were successfully characterized, with the potential to deliver recombinant gene products to root hair cells at significantly higher levels. The tissue specificity and activity of these transcriptional elements were confirmed by extensive promoter:reporter quantitative fluorimetric and histochemical analyses of beta-glucuronidase. These root hair-specific promoter/3’-flanking region combinations were subsequently used for the assembly of second-generation transgene cassettes for high-level expression of sorgoleone biosynthetic enzymes in root hairs. Preliminary experiments have also been recently initiated to analyze the ability of these root hair-specific transgene cassettes to direct the synthesis of sorgoleone in stably transformed rice plants, and these experiments are ongoing and will be continued in the new project cycle.
Towards achieving Objective 1 during FY 2020, 130 of extracts and pure compounds were tested. Diplopyrone is a phytotoxin originated from the fungus Diplodia mutila. The herbicidal activity of diplopyrone analogues derived from a pyranopyran core was evaluated using a primary ranking bioassay, a commonly used method for preemergence test, against bentgrass and lettuce. Two compounds (pyranopyran nitrile and pyranopyran alkyne) demonstrated significant phytotoxic potential against lettuce. All tested pyranopyrans have a wide range of activity against monocot species. Although pyranopyran nitrile is less active than pyranopyran alkyne as a preemergence agent against the monocot bentgrass, its herbicidal activity is greater than pyranopyran alkyne using postemergence bioassay on the monocot duckweed. Among the tested compounds, the phytotoxicity of pyranopyran amide, desmethyldiplopyrone and desmethyldiplopyrone acetate is insignificant, similar to the previously reported results on diplopyrone derivatives. However, pyranopyran alkyne analog showed potent activity against bentgrass with IC50 = 38.1 µM, which offers a promising potential for further development.
To evaluate the diplopyrone analog pyranopyran nitrile further, experiments in greenhouse setting were carried out. Four plant species, velvetleaf, field bindweed, crabgrass, and barnyardgrass were selected for spray experiments. Leaf tissues of both monocot species (crabgrass and barnyardgrass) developed signs of injures within 24 hours after application (1 day after treatment - DAT), which then turned into necrotic brown lesions at 4 DAT. Leaves of dicot species (velvetleaf and field bindweed) showed little or no signs of damage. However, necrosis spots were observed, possibly a result of direct contact of plant tissue with droplets of sprayed solution. This finding suggests that pyranopyran nitrile may act in a similar manner as a contact herbicide.
Azoles are a group of five-membered heterocyclic compounds wildly used in agriculture and medicine. Thirty compounds that belong to the class of azoles were tested and almost all of them exhibited very satisfying herbicidal activity by inhibiting the germination of bentgrass (Agrostis stolonifera) and lettuce (Lactuca sativa). As a highly effective group of fungicides, azoles target a class of cytochrome P450 enzymes (CYP51), for example, lanosterol 14a-demethylase. It has been reported that azoles also inhibit plant CYP51 enzymes leading to the reduction of plant sterol hormones brassinosteroids (BR). Seeds of transgenic Arabidopsis overexpressing CYP51 from Fragaria vesca were obtained for further studying the mode of action of azole compounds.
For Objective 2, characterization of transformed rice plants expressing the sorgoleone biosynthetic pathway was conducted. These transgenic rice plants were generated previously using a multi-gene vector constructed in our unit via agrobacterium-mediated plant transformation performed at the Iowa State University transformation laboratory in Ames, Iowa. In collaboration with ARS researchers in Oxford, Mississippi, an high-performance liquid chromatography-ultraviolet (HPLC-UV) based detection method was developed for the detection of sorgoleone in transgenic seedling root samples, and in addition, a novel soil-free in vitro propagation system was developed and successfully utilized which optimized root hair production in seedling root tissues. In addition to the transformation work performed at Iowa State, a second multi-gene vector was designed and is currently being utilized by the University of Wisconsin (UW) Crop Innovation Center for the generation of transgenic corn, wheat, and soybean plants. The UW Crop Innovation center is also actively working on the generation of transgenic sorghum plants with enhanced sorgoleone content, utilizing vectors containing root hair-specific gene promoters driving the expression of ARS1, a rate-limiting enzyme which catalyzes the formation of the 5-pentadecatrienyl resorcinol sorgoleone pathway intermediate. The root hair-specific gene promoters used for the above-described transformation experiments were isolated from S. bicolor by our research unit and were mentioned in a previous annual report.
The transgenic plant screens performed to date have utilized total root tissues from seedlings grown in vitro under conditions which optimize root hair production, as the direct analysis of isolated root hairs is not amenable to high-throughput approaches, and the number of seeds obtained from individual R0 transformants is generally not sufficient for performing established methods for root hair isolation from sorghum. To address this problem, we have recently developed a novel small-scale root hair isolation procedure which we have successfully tested with both sorghum and rice 10-day old seedlings grown in vitro. The new method enables biochemical and molecular analyses of highly-purified root hair tissues, starting with as few as 100 R1 transgenic seedlings. This small-scale root hair isolation procedure will dramatically improve the sensitivity of our assays, and greatly enhances our ability to detect sorgoleone accumulation specifically occurring in root hairs of transgenic seedlings. We anticipate that positive outcomes from these efforts will represent major breakthroughs in the plant-produced biopesticide field, resulting in the generation of novel germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests.
Accomplishments
Review Publications
Duke, S.O. 2019. Enhanced metabolic degradation: The last evolved Glyphosate resistance mechanism of weeds? Plant Physiology. 181:1401-1403. https://doi.org/10.1104/pp.19.01245.
Yu, W., Zhai, Z., Min, L., Wedge, D.E., Duke, S.O., Wu, H., Weng, J., Tan, C., Zhang, Y., Liu, X. 2019. Synthesis and biological activity of novel 1,3,4-oxadiazole derivatives containing a pyrazole moiety. Research on Chemical Intermediates. 45:5989-6001. https://doi.org/10.1007/s11164-019-04015-8.
Gu, C., Xia, X., Lv, J., Tan, J., Baerson, S.R., Pan, Z., Song, Y., Zeng, R. 2019. Diterpenoids with herbicidal and antifungal activities from hulls of rice (Oryza sativa). Fitoterapia. 136:104183. https://doi.org/10.1016/j.fitote.2019.104183.
Duke, S.O. 2020. Glyphosate: Environmental fate and impact. Weed Science. 68(3):201-207. https://doi.org/10.1017/wsc.2019.28.
Dayan, F.E., Duke, S.O. 2020. Discovery for new herbicide sites of action by quantification of plant primary metabolite and enzyme pools. Engineering. 6:509-514. https://doi.org/10.1016/j.eng.2020.03.004.
Meepagala, K.M., Clausen, B., Johnson, R.D., Wedge, D.E., Duke, S.O. 2019. A phytotoxic and antifungal metabolite (Pyrichalasin H) from a fungus infecting Brachiaria eruciformis (Signal Grass). Journal of Agricultural Chemistry and Environment. 8(3):115-128. https://doi.org/10.4236/jacen.2019.83010.
Grana, E., Diaz-Tielas, C., Sanchez-Moreiras, A.M., Reigosa, M.J., Celeirao, M., Abagyan, R., Teijeira, M., Duke, M.V., Clerk, T., Pan, Z., Duke, S.O. 2019. Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins. Physiologia Plantarum. 169(1):99-109. https://doi.org/10.1111/ppl.13055.
Bajsa Hirschel, J.N., Pan, Z., Duke, S.O. 2020. Rice momilactone gene cluster: Transcriptional response to Barnyard grass (Echinochloa crus-galli). Molecular Biology Reports. 47:1507-1512. https://doi.org/10.1007/s11033-019-05205-8.
Owens, D.K., Bajsa Hirschel, J.N., Duke, S.O., Carbonari, C.A., Gomes, G.L., Asolkar, R., Boddy, L., Dayan, F.E. 2020. The contribution of Romidepsin to the herbicidal activity of Burkholderia rinojensis biopesticide. Journal of Natural Products. 83(4):843-851. https://doi.org/10.1021/acs.jnatprod.9b00405.
Perera, W.H., Meepagala, K.M., Wedge, D.E., Duke, S.O. 2020. Sesquiterpenoids from culture of the fungus Stereum complicatum (Steraceae): Structural diversity, antifungal, and phytotoxic activities. Phytochemistry Letters. 37:51-58. https://doi.org/10.1016/j.phytol.2020.03.012.
Nocera, P., Bajsa Hirschel, J.N., Masi, M., Ross, S.A., Cantrell, C.L., Duke, S.O., Surico, G., Evidente, A. 2020. Secondary metabolites of Thymelaea hirsuta, a plant collected from the Sicilian Island of Lampedusa. Natural Product Research. https://doi.org/10.1080/14786419.2020.1752212.
Liu, X., Yu, W., Min, L., Wedge, D., Tan, C., Weng, J., Wu, H., Cantrell, C.L., Bajsa Hirschel, J.N., Hua, X., Duke, S.O. 2020. Synthesis and pesticidal activities of new Quinoxalines. Journal of Agricultural and Food Chemistry. 68:7324-7332. https://doi.org/10.1021/acs.jafc.0c01042.
Cerveira, W.R., Santos Da Costa, Y.K., Carbonari, C., Duke, S.O., Aguiar Alves, P., Bianco De Carvalho, L. 2020. Growth, morphological, metabolic, and photosynthetic responses of clones of eucalyptus to glyphosate. Forest Ecology and Management. https://doi.org/10.1016/j.foreco.2020.118218.
Kepler, R., Epp Schmidt, D.J., Yarwood, S.A., Cavigelli, M.A., Buyer, J.S., Duke, S.O., Reddy, K.N., Williams, M., Bradley, C.A., Maul, J.E. 2020. Soil microbial communities in diverse agroecosystems exposed to glyphosate. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.01744-19.
