Location: Natural Products Utilization Research
2021 Annual Report
Objectives
1. Discover and develop natural product-based bioherbicides with novel modes of action that are safe and effective tools for weed management. [C2, PS2A]
1.1. Discover uses of new and existing natural products for potential use as herbicides and bioherbicides for weed management.
1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues, physiological evaluations, and molecular genetics approaches.
2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals and determine the role of allelopathy in the success of invasive weeds.
2.1. Identification of transporters required for the extracellular secretion of sorgoleone in Sorghum bicolor root hair cells.
2.2. Manipulation of sorgoleone levels in vivo to generate enhanced S. bicolor germplasm.
2.3. Generation of transgenic maize, wheat and soybean plants containing the complete sorgoleone biosynthetic pathway.
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
Towards achieving Objective 1, the following research was performed in FY 2021 and is continuing in FY2022:
A series of compounds related to inhibitors of sterol 14a-demethylase (30 putatives and 2 knowns) were evaluated for phytotoxicity in a secondary bioassay against Lemna paucicostata. Six of them displayed a high level of herbicidal activity with IC50 values between 8.7 and 26 µM. Five active compounds were selected for further study and were found to inhibit the growth of a dicot model plant Arabidopsis as shown on the half strength of Murashige-Skoog culture medium containing 5 µM of a compound.
About 30 compounds/extracts from our research unit were tested in a primary bioassay and were found to have satisfactory herbicidal activity. Further testing in a secondary bioassay is in progress. Many of these include the analogs of khellin and visnagin, the lead compounds in development with INBIOAR and the subject of a pending patent application.
Determination of the mode of action of spliceostatin C (spC) is a continuation of the collaborative project with Marrone Bio Innovations, Inc. It has been shown that spC significantly inhibits the growth of Arabidopsis seedlings. To better understand the impact of spC on the protein level, proteomics approaches - two-dimensional difference gel electrophoresis (2D DIGE) technique – were employed to identify proteins for alterations in spC treated Arabidopsis seedlings. Primary results indicated that 66 proteins, as determined on 2-DIGE, were significantly affected upon treatment with spC as compared to the control. Further analysis using MALDI/TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry and Mascot software identified that 40 of them are unique proteins and 26 isoforms, and of which two proteins - extra-large guanine nucleotide-binding protein and thylakoid lumen protein - were significantly affected with the average ratio (treated seedlings vs control) of 11.36 and 8.91, respectively, the highest up-regulated proteins. Ongoing work is in progress to further elucidate the roles of these proteins in response to spC.
Mechanisms of herbicide resistance can be broadly divided into two categories, target-site resistance where an herbicide acts to disrupt a particular plant process or function (mode of action) and non-target-site resistance. It has been reported that a cytochrome P450 enzyme from cotton is responsible for the non-target site resistance against the herbicide trifloxysulfuron. To further characterize this enzyme, a binary vector containing the coding sequence of the enzyme was constructed, in which the expression of the cDNA is driven by a double 35S promoter for overexpression. The vector was transformed into Arabidopsis via Agrobacterium tumefaciens. Four homozygous lines (third generation) of transgenic plants were generated. These transgenic lines will be used for studying the possible conversion of the herbicide, as well as screening for natural products with a structure similar to trifloxysulfuron and analogs.
Streptomycetes are known for their ability to synthesize many secondary metabolites, some of which possess pesticide activity. These secondary metabolites are normally encoded by large biosynthetic gene clusters. Two regulators (pathway-specific and global) were selected for metabolic engineering to improve the production of bioactive compounds. The constructs for overexpression of these regulators were made and are being tested in two Streptomyces strains that are currently used for the extraction of active compounds in our unit.
Towards achieving Objective 2, the following research was performed in FY 2021 and is continuing in FY 2022.
One approach our group has pursued for the identification of genes associated with the extracellular transport of the allelochemical sorgoleone, is the analysis of genes differentially expressed in sorgoleone-deficient transgenic sorghum relative to wild-type sorghum, as well as null segregants derived from the same transformation events. The basis for this approach is the likelihood that within a deficient background, genes directly involved in the biosynthesis and transport of sorgoleone will exhibit expression profiles which differ from those of wild-type plants, thus providing additional clues facilitating their identification. Using a small-scale root hair isolation procedure devised by our team, root hair preparations were successfully prepared from homozygous positive and negative (null) transgenic seedlings representing multiple independent transgenic events, as well as from the nontransgenic parental Tx430 genotype. High quality total RNAs were obtained from these samples, and Illumina next-gen sequencing libraries were prepared from these RNAs. Sequence analysis experiments from this work are currently ongoing, and we anticipate that the results obtained will provide an important tool for identifying transporters and carrier proteins involved in sorgoleone rhizosecretion.
Accomplishments
Review Publications
Duke, S., Pan, Z., Bajsa Hirschel, J.N. 2020. Proving the mode of action of phytotoxic phytochemicals. Plants. https://www.doi.org/10.3390/plants9121756.
Kumarihamy, M., Rosa, L.H., Techen, N., Ferreira, D., Croom, E.M., Duke, S.O., Tekwani, B.L., Khan, S., Nanayakkara, N. 2020. Antimalarials and phytotoxins from Botryosphaeria dothidea identified from a seed of diseased Torreya taxifolia. Molecules. https://doi.org/10.3390/molecules26010059.
Pan, Z., Bajsa Hirschel, J.N., Vaughn, J.N., Rimando, A.M., Baerson, S.R., Duke, S.O. 2021. In vivo assembly of sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana. New Phytologist. 230:683-697.
Sun, Z., Li, Q., Lin, Y., Shi, Q., Baerson, S.R., Xu, C., Wang, R., Chen, L., Zeng, R., Song, Y. 2021. Olfactory perception of herbivore-induced plant volatiles elicits counter-defenses in larvae of tobacco cutworm, spodoptera litura. Functional Ecology. 2021;35:384–397. https://doi.org/10.1111/1365-2435.13716.
Hijano, N., Nepomuceno, M.P., Cantrell, C.L., Duke, S.O., Alves, P.L. 2021. Characterization of the allelopathic potential of sugarcane leaves and roots. Journal of Agricultural Chemistry and Environment. https://doi.org/10.4236/jacen.2021.103016.
Barreto, D.L., Nogueira De Azevedo, R., Carvalho, C., Ferreira, M.C., Cantrell, C.L., Duke, S.O., Rosa, L.H. 2021. Bioactive compounds produced by Neotropical endophytic fungi applied to agriculture. In: Rosa L.H. editors. Neotropical Endophytic Fungi. Springer, Cham: Springer International Publishing. p. 257-295.
Yu, C., Wang, Q., Min, L., Bajsa Hirschel, J.N., Hua, X., Cantrell, C.L., Duke, S., Liu, X. 2021. Synthesis, crystal structure, herbicidal activity, and SAR study of Novel N-(Arylmethoxy)-2-chloronicotinamides derived from nicotinic acid. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.0c07538.
Meepagala, K.M., Bracken, A.K., Fronczek, F.R., Johnson, R.D., Wedge, D.E., Duke, S.O. 2020. A novel furanocoumarin with phytotoxic activity from the leaves of Amyris elemifera (Rutaceae). Journal of Agricultural and Food Chemistry. https://www.doi.org/10.1021/acsomega.0c04778.
