Location: Chemistry Research2013 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 work relates to sub-objective 1a. Isolation and identification of elicitors or biotic agents of induced plant volatile emission and other inducible plant defenses. This agreement was put into place to establish guidelines for determining intellectual property in the context of the collaboration with Japan. To date no significant issues have arisen. Metabolomics: Identification of inducible bioactive plant metabolites: Two novel acidic sesquiterpenoid phytoalexins, termed zealexin A4 and zealexin B3, have been discovered in maize. Zealexin A4 exhibits significant antimicrobial activity against a range a fungal pathogens including Southern Leaf Blight (Cochliobolus heterostrophus) and Aspergillus flavus. A transposon insertion mutant in the gene AntherEar2, encoding an ent-copalyl diphosphate synthase, was identified in the maize W22 background. Maize plants homozygous for this mutation completely lack production of all kauralexin diterpenoid phytoalexins following insect and pathogen attack. Propagated seeds are currently being used in greenhouse and field experiments to examine altered resistance phenotypes with naturally occurring fungal and insect pests. Whole transcriptome shotgun sequencing, termed RNA-seq, of seedling scutella tissue revealed candidate a type II kaurene synthase for the kauralexin biosynthetic pathway. Preliminary analysis of this transposon insertion mutant supports the role of this enzyme as a second step in the pathway. Further functional characterization of this inducible maize kaurene synthase is currently underway.