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Research Project: New Weed Management Tools from Natural Product-Based Discoveries

Location: Natural Products Utilization Research

2018 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
A series of compounds related to a natural phytotoxin were evaluated for potential as herbicides. One of these had unusually high phytotoxicity. An invention disclosure was filed. Results of these studies are confidential until a patent is filed. 2-Benzoxazolinone (BOA) is a phytotoxic compound that induces strong effects on plant metabolism. BOA effects include increased membrane permeability, degradation of proteins and pigments, increased lipid peroxidation, oxidative stress and senescence induction. In this work, the effects of amino acid supplementation on BOA action were analyzed. Roles of histidine, tryptophan and cysteine in the mode of action of BOA action were proposed from their protective effects. The mode of action of the terpenoid phytotoxin citral was probed in Arabidopsis thaliana with RNA-Seq transcriptome analysis. The concentration of citral that inhibited growth by 50% (202 µM) down regulated transcription of 9156 and 5541 genes in roots and shoots, respectively, at the p = 0.001 level after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the roots, these effects dissipated after 3 and 6 h, but similar effects began again at 12 and continued until 24 h. In the shoots, the down regulation increased at 3 h (6239 genes downregulated, vs. 66 upregulated) with the effect decreasing at from 6 to 24 h, with only 1 gene downregulated at 24 h. Of all genes affected in roots at 1 h and shoots at 3 h (times of greatest effect), 6.5 and 9.2 %, respectively, of affected genes were for DNA and RNA binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly down downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at 12, and 24 h. Citral docking calculations for WHY1, WHY2, and WHY3 crystal structures and the SSBP homology model predicted strong binding in the hydrophobic pockets of these proteins. Such an effect could account for the profound and unusual effects on downregulation of gene transcription. New cassane-like diterpenes from Eragrostis plana were found to be moderately phytotoxic. An invention disclosure was filed. A series of triazole derivatives containing a pyrazole moiety were synthesized and characterized by 1H nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Most of the tested compounds were moderately herbicidal to lettuce and bentgrass. Indirect evidence was via transcriptome and biochemical methods to link the mode of action of the natural phytotoxin t-tchalcone to inhibition of tyrosine metabolism. Hymnoclea salsola, a weed species that is found in the arid regions of Utah, Texas and Arizona was investigated in search of phytotoxic compounds. Confertin and several analogs of confertin and two novel chalcones were found to be phytotoxic. These compounds are being evaluated further. Bioassay guided isolation of an ethyl acetate extract from an Amryis species led to isolation and identification of a novel phytotoxic furanocoumarin. Palmitoleoyl-CoA is found in virtually every plant cell type due to its central role in plant fatty acid and lipid metabolism. Therefore in principle, the transfer of the sorgoleone biosynthetic pathway genes DES2, DES3, ARS1/2, OMT3, and CYP71AM1 to any plant cell could result in the accumulation of dihydrosorgoleone, which upon exposure to oxygen will spontaneously form the benzoquinone sorgoleone. Furthermore, the expression of these genes specifically in root hair cells is likely prerequisite to the successful deployment of sorgoleone as a bioherbicide in other crop plants. Toward this end, our research unit previously identified and characterized highly specific gene promoters to direct the root hair-specific expression of transgenes in plants, and as mentioned we have also isolated all of the genes required for the biosynthesis of the potent bioherbicide sorgoleone starting from palmitoleoyl-CoA. Recently, we have constructed five transgene cassettes containing the complete open reading frames of S. bicolor DES2, DES3, ARS1, OMT3, and CYP71AM1, which also feature the root-hair specific gene promoters our research unit has identified. These five transgene cassettes were all positioned within a single binary transformation vector, which was then sent to the Iowa State University Plant Transformation Facility and used for the generation of transgenic rice events. R1 seed were produced in our research unit's greenhouse from the primary transformants generated by Iowa State University, and multiple tranformant lines are currently being evaluated by our research unit to determine if a functional sorgoleone biosynthetic pathway has been successfully transferred to rice. A positive outcome from these efforts will represent a major breakthrough in the plant-produced biopesticide field, and the generation of novel rice germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests as well.

4. Accomplishments
1. Completion of biochemical and genetic characterization of the sorgoleone biosynthetic pathway. ARS researchers at Oxford, Mississippi, goal of identifying and characterizing the genes and enzymes required for the biosynthesis of the potent bioherbicide sorgoleone is now completed with the characterization of a root-hair specific cytochrome P450 from S. bicolor genotype BTx623. The cytochrome P450 protein was determined to belong to the CYP71 enzyme subfamily and has been formally designated S. bicolor CYP71AM1. Biochemical characterization of recombinant CYP71AM1 using both yeast (S. cerevisiae) and a Nicotiana benthamiana transient expression system demonstrated that CYP71AM1 is capable of performing dihydroxylation on the proposed physiological substrate 3-methyl-5-pentadecatrienyl resorcinol, yielding dihydrosorgoleone. These results clearly demonstrate a role for CYP71AM1 in the biosynthesis of sorgoleone in Sorghum bicolor, and represent the first example of a cytochrome P450 enzyme capable of catalyzing a dihydroxylation reaction occurring on a resorcinolic ring structure. Previously, our research team successfully identified the fatty acid desaturases, alkylresorcinol synthases, and an O-methyltransferase participating in the sorgoleone biosynthetic pathway. The characterization of CYP71AM1 represents a highly significant milestone, as our research unit has now characterized the genes and enzymes required for all of the biosynthetic steps leading to the formation of dihydrosorgoleone, beginning with palmitoleoyl-CoA. With this accomplishment we now have the tools in hand to engineer the production of sorgoleone in other crop species, which could potentially lead to the generation of novel germplasm possessing enhanced resistance to weed competition, and potentially against other agronomic pests as well.

Review Publications
Heap, I., Duke, S.O. 2017. Overview of glyphosate-resistant weeds worldwide. Pest Management Science. 74:1040-1049. doi 10.1002/ps.4760.

Pan, Z., Baerson, S.R., Wang, M., Bajsa Hirschel, J.N., Rimando, A.M., Wang, X., Nanayakkara, N., Noonan, B.P., Fromm, M.E., Dayan, F.E., Khan, I., Duke, S.O. 2018. A cytochrome P450 CYP71 enzyme expressed in S. bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone. New Phytologist. doi: 10.1111/nph.15037.

Belz, R.G., Duke, S.O. 2018. Predicting hormesis in mixtures of herbicidal compounds – where are we and how far can we go? Julius Kühn Archiv. 458:161-167.

Labruzzo, A., Cantrell, C.L., Carrubba, A., Ali, A., Wedge, D.E., Duke, S.O. 2018. Phytotoxic Lignans from Artemisia arborescens. Natural Product Communications. 13(3):237-240.

Lu, X., Zhang, J., Xu, M., Li, R., Liu, B., Rodriguez-Romero, J., Luo, D., Pan, Z., Baerson, S.R., Li, Z., Sesma, A., Yang, B., Peters, R.J. 2018. Inferring roles in defense from metabolic allocation of rice diterpenoids. The Plant Cell. 30:1119-1131.

Maroli, A.S., Nandula, V.K., Duke, S.O., Gerard, P., Tharayil, N. 2017. Comparative metabolomic analyses of two Ipomoea lacunosa biotypes with contrasting glyphosate tolerance elucidates glyphosate-induced differential perturbations in cellular physiology. Journal of Agricultural and Food Chemistry. (66):2027-2039.

Meepagala, K.M., Briscoe, W.E., Techen, N., Johnson, R.D., Clausen, B.M., Duke, S.O. 2017. Isolation of a phytotoxic isocoumarin from Diaporthe eres-infected Hedera helix (English Ivy) and synthesis of its phytotoxic analogs. Pest Management Science. 74:37-45.

Morimoto, M., Cantrell, C.L., Khan, S., Tekwani, B.L., Duke, S.O. 2017. Antimalarial and antileishmanial activities of phytophenolics and their synthetic analogues. Chemistry and Biodiversity. DOI: 10.1002/cbdv.201700324.

Sanchez-Moreiras, A.M., Pedrol, N., Duke, S.O. 2018. Influence of amino acids on the phytotoxicity of 2-benzoxazolinone on Lemna paucicostata. Journal of Allelochemical Interactions. 4(1):33-39.

Wang, R., Liu, S., Baerson, S.R., Qin, Z., Ma, Z., Su, Y., Zhang, J. 2018. Identification and functional analysis of a novel cytochrome P450 gene CYP9A105 associated with pyrethroid detoxification in Spondoptera exigua Hubner. International Journal of Molecular Sciences. 19:737.

Westwood, J.H., Charudattan, R., Duke, S.O., Finnimore, S.A., Marrone, P., Slaughter, D.C., Swanton, C., Zollinger, R. 2018. Weed management in 2050: Perspective on the future of weed science. Weed Science. 66:275-285.

Carvalho, L.B., Duke, S.O., Alves, P. 2018. Physiological responses of Eucalyptus x urograndis to glyphosate are dependent on the genotype. Scientia Forestalis. 46(118): 177-187.

Duke, S.O. 2018. Interaction of chemical pesticides and their formulation ingredients with microbes associated with plants and plant pests. Journal of Agricultural and Food Chemistry. 66:7753-7561. DOI: 10.1021/acs.jafc.8b02316.

Duke, S.O., Pan, Z., Bajsa Hirschel, J.N., Sanchez-Moreiras, A.M., Vaughn, J.N. 2018. Use of omics methods to determine the mode of action of natural phytotoxins. American Chemical Society Symposium Series. 1294:33-46.

Seiber, J.N., Coats, J., Duke, S.O., Gross, A.D. 2018. Pest management with biopesticides. Frontiers of Agricultural Science and Engineering. 5(3):205-300.

Beck, J.J., Duke, S.O., Rering, C.C. 2018. Roles of natural products for biorational pesticides in agriculture. ACS Symposium Series. 1294:1-4. doi:10.1021/bk-2018-1294.ch001.