Location: Chemistry Research2012 Annual Report
1a. Objectives (from AD-416):
To identify inducible bioactive plant metabolites that mediate resistance in key crops and other models.
1b. Approach (from AD-416):
Many plant defense chemicals are absent in non-stressed plants but accumulate locally to very high levels following attack. Therefore, by specifically targeting the identification of small molecules that accumulate after defense induction, we will enrich for plant metabolites that have biological activity in animals and microbes. To increase assay reproducibility, the plant hormones methyl jasmonate, salicylic acid, pathogens and insects will be used to elicit defense responses. We will employ a wide range of analytical skills and implement complementary mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based assays that will maximize the diversity of compounds that can be identified. We will work towards the improved annotation of novel components of the induced-defense metabolome in three well-studied plant species, Arabidopsis, maize, and rice. By targeting these species, we will benefit from sequenced genomes, well-developed genetic tools, and an extensive body of prior research in our subsequent research to identify genes that encode enzymes for the synthesis of previously unknown plant metabolites. The function of candidate genes will be confirmed through identification of natural variation, isolation of mutant plants, expression in heterologous systems, and enzyme assays. Importantly, to confirm functions of the newly identified plant metabolites, insect and microbial bioassays will be conducted with both mutant plants and purified compounds.
3. Progress Report:
This research related to inhouse project objective 1A. Isolation and identification of elicitors or biotic agents of induced plant volatile emission and other inducible plant defenses. Novel acidic sesquiterpenoid phytoalexins, termed zealexins, were discovered in Southern Leaf Blight (Cochliobolus heterostrophus) resistant maize lines that were inoculated with this fungus. Additional zealexins were found to strongly accumulate in insect and fungal damaged kernels during development on maturing maize ears. Unknown members of this phytoalexin class were highly enriched in maize lines resistant to aflatoxin accumulation compared to known susceptible lines. Work on structure elucidation and antimicrobial assays began on July 30th by grant-funded Research Associate. A time course of maize seedling scutella tissue, harboring high levels of kauralexins and zealexins, was subjected to whole transcriptome shotgun sequencing, termed RNA-seq. This defense enriched transcriptome revealed a series of kaurene synthases, cytochrome P450s and other candidate phytoalexin biosynthetic genes which will be biochemically tested for enzyme activity following purification from bacterial and yeast heterologous expression systems. A maize mutant, carrying a transposon insertion in the ent-copalyl diphosphate synthase (AN2) gene, has been identified and will be examined for the predicted loss of fungal-induced kauralexins.