Functional analysis of small signaling molecules in jasmonate signaling.

Plants exhibit multitudes of defense mechanisms against different kinds of stress. Jasmonic acid (JA) is one of the identified signaling compounds mediating plant’s response to wounding, attack by herbivores or necrotrophic pathogens. Central parts of the JA signaling pathway have recently been unraveled by demonstrating that (+)-7-iso-JA-L-Ile is the most bioactive form of JA and that the SCF(COI1)-complex functions as its receptor. However, many other components of the JA signaling pathway remain unknown. This includes how and where protein kinases may be involved in JA signaling. Likewise, the mechanistic details of cross-talk between different hormone signaling pathways are unknown. Using a chemical biology approach, screening for selective compounds that can be used as tools in applications complementing genetic approaches, I aimed at identifying some of these unknown components. The advantage of this method is that it has the potential to circumvent redundancy of gene function, lethality of mutants and pleiotropic effects, problems generally encountered in genetic approaches. I developed a screening procedure for bioactive compounds that uses a transgenic Arabidopsis thaliana line, harboring the JA-responsive reporter gene LOX2p::LUC. This procedure allowed bidirectional screening for activators or inhibitors of reporter expression. Sifting through approx. 1,700 natural compounds, I identified one activator of reporter gene expression and 16 inhibitors of methyl jasmonate induced reporter expression. Critical validation of these primary hits revealed that the putative activator in fact interfered with the activity of the luciferase reporter. It presumably binds and stabilizes luciferase, thereby enhancing its apparent activity, whereas reporter gene expression was not affected. After validation and characterization of the inhibitors, one compound (12) was identified as selective inhibitor of JA signaling. Structure-activity relationship studies, using derivatives of the compound, defined parts of the molecule that where indispensable for its bioactivity. Based on this analysis, a derivatized probe was designed that harbors a ‘photoreactive’ benzophenone for establishing covalent binding and an alkyne residue to attach a detectable fluorophore using ‘click chemistry’. Importantly, this probe retained activity and was used in first affinity-based target identification experiments. In a second screen using a small, targeted library of 84 known protein kinase inhibitors, I identified three compounds that impaired JA signaling. This finding suggests the involvement of protein kinases in the JA signaling pathway that has been previously reported. Among the identified inhibitors was 5-iodotubercidin, a nucleoside antibiotic. A derivative of this compound, toyocamycin, was previously described to selectively impair auxin signaling, which is mechanistically related to JA signaling. Several structural analogs were investigated with respect to their effect on JA-dependent reporter expression or JA-independent readouts. Toyocamycin was considered to be the most specific derivative. To elucidate the role of toyocamycin in the Arabidopsis hormonal signaling network, I performed a microarray analysis after treatment with toyocamycin. The expression data showed that this compound modulates expression of JAZ genes, which are repressors of JA induced gene expression. Toyocamycin also modulated genes, which may be involved in hormonal crosstalk between e.g. auxin or salicylic acid signaling. The fact that toyocamycin caused a root growth phenotype, which is dependent on allene oxide synthase (AOS) and jasmonoyl-isoleucine synthetase (JAR1), indicated that multiple targets may exist, because inhibition of the LOX2 marker was independent of JAR1. Identification of the protein targets of toyocamycin and compound 12 may eventually lead to identification of yet unknown components in JA signaling or hormonal crosstalk