Location: Natural Products Utilization Research2012 Annual Report
1a. Objectives (from AD-416):
Objective 1: Discover natural product-based materials and technologies for weed and other pest management that would be accepted by organic farmers and/or farmers who desire more environmentally and toxicologically benign weed and other pest management tools. Subobjective 1.1: Discover uses of new and existing natural products for weed management in conventional and organic farming. Subobjective 1.2: Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. Subobjective 1.3: Discovery of the mechanisms of action for newly discovered phytotoxins using a genetics approach with resistant mutants. Subobjective 1.4: Discovery of the mechanisms of action for newly discovered phytotoxins using transcriptome analysis. Objective 2: Identify and characterize the biochemical pathways of phenolic lipid-type allelochemicals and fungicides from cereals, and manipulate these pathways to produce enhanced allelopathic and disease-resistant crops with reduced requirements for synthetic herbicides and/or fungicides. Subobjective 2.1: Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. Subobjective 2.2: Functional analysis of putative sorgoleone pathway enzymes by genetically engineering sorghum to either increase or reduce expression of the corresponding genes, and the use of these transformants to investigate the ecological role of sorgoleone. Subobjective 2.3: Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. Subobjective 2.4: Manipulation of phenolic lipid biosynthesis using rice and Arabidopsis models.
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. Genes controlling synthesis of useful plant secondary products involved in plant defenses to pests will be identified, cloned, and manipulated.
3. Progress Report:
The natural herbicide leptospermone was found to be stable in agriculturally relevant soils. Its bioavailability was increased when applied as a mixture in triketone-rich manuka oil, indicating that this natural compound has potential as a commercial herbicide. Soil composition and pH had strong effects on the longevity and bioavailability of leptospermone. Rapid leaf disc bioassays were developed to measure the effect of natural and synthetic toxins on photosynthesis and membrane stability. This work provides assays to test large number of natural herbicides for their effect on two key plant physiological functions. Tabanone, a phytotoxin from the invasive weed cogongrass was isolated and its effect on various physiological functions was established. Additionally, phytotoxins from horseweed were identified. This work serves to develop environmentally compatible strategies for weed control. The mode of herbicide action of the natural product cantharidin and its synthetic analog endothall as inhibitors of serine-threonine protein phosphatase was validated. Genetic identification of a key enzyme involved in allelochemical detoxification was made. Manipulation of allelochemical synthesis in transgenic plants requires the ability of the producing plant to tolerate exposure to the allelochemical. Identification of genes associated with allelochemical detoxification pathways therefore represents a key component of our project. Previously, we performed gene expression profiling experiments using arabipdopsis as a model plant to examine transcriptional responses to the allelochemical benzoxazolin-2(3H)-one (BOA), and also identified BOA-6-OH and BOA-O-glucoside as breakdown products of BOA produced by arabidopsis. To identify cytochrome P450 and UDP-glucosyltransferases associated with this detoxification pathway, genetic screens were performed using T-DNA insertional mutants of arabidopsis. The genes mutated in the arabidopsis lines corresponded to P450 and glucosyltransferase genes previously shown to be up-regulated in response to BOA exposure. The mutant lines and wild-type plants were screened against BOA. While several mutant lines had reduced sensitivity to BOA compared to wild-type seedlings, one glucosyltransferase mutant had more than a 50-fold increase in sensitivity to BOA, with no growth abnormalities in the absence of BOA. These results strongly suggest involvement of this gene in BOA detoxification. Two P450 enzymes from sorghum root hairs were characterized as having the function of hydroxylation of methylated resorcinolic intermediate, a strong indicator that they are involved in biosynthesis of the allelochemical sorgoleone. Because of the extreme difficulty in doing test tube enzyme assays with these enzymes because both substrates and products have extremely low water solubility, a system expressing genes for both enzyme was engineered in yeast to make substrates for the cloned P450s. In this system, both enzymes consecutively convert endogenous fatty acids into methoxy-alkylresorcinol that serves as a substrate for the P450s. The expected methoxy-dihydroresorcinol product was observed.
1. Identification of gene promoters for directing root hair-specific expression in monocotyledonous plants. Transgenic manipulation of the allelochemical sorgoleone, which is synthesized in root hair cells and then released into the rhizosphere, represents a key component of this project at the USDA, ARS, Natural Products Utilization Research Unit (NPURU) located in Oxford, MS, to produce weed-fighting crops. The identification of highly-active, root hair-specific gene promoters is therefore essential to achieving our technical goals. Previously, scientists at the USDA, ARS, NPURU have generated a database comprised of sequences expressed in root hair cells of sorghum (Sorghum bicolor), and the most highly expressed sequences in this data set were further compared against all publicly-available sorghum expressed sequence data sets. A number of highly expressed root hair sequences were only found within this data set, and the corresponding promoter and 3’ flanking regions were identified. These constructs were transformed into both rice and Arabidopsis to examine their performance in both a monocotyledonous and dicotyledonous host. Histochemical analyses were performed on multiple tramsformed individuals representing a minimum of 8 rice and 8 Arabidopsis events for each construct to confirm that these constructs directed highly root hair-specific expression patterns. Given that these regulatory sequences were derived from the most highly expressed genes in sorghum root hair cells, it is likely that they will serve as very powerful tools for the cell-specific manipulation of sorgoleone biosynthesis. Additionally, these promoters could have other important biotechnological applications such as in influencing rhizosphere-associated microbial communities, enhancement of plant nutrient acquisition, as well as bioremediation of contaminated soils.
Bajsa, J.N., Pan, Z., Duke, S.O. 2011. Transcriptional responses to cantharidin a protein phosphatase inhibitor in Arabidopsis thaliana reveal the involvement of multiple signal transduction pathways. Physiologia Plantarum. 143:188-205.