Final Activity Report Summary - JASMONATE SIGNALING (Analysis of the ORA47 transcription factor involved in jasmonic acid signal transduction in Arabidopsis thaliana)
Jasmonic acid (JA) and derivatives are plant defence hormones involved in biotic or abiotic stress responses in plants. Stress induces JA biosynthesis via the so-called octadecanoid pathway. All JA biosynthesis genes are induced by JA indicating that JA initiates an auto-stimulatory loop. In the host lab the AP2-domain transcription factor ORA47 from Arabidopsis thaliana has been identified as a regulator of the JA-responsive expression of several JA biosynthesis genes, including the AOC2 gene. The expression of ORA47 gene itself is transiently induced by JA. It has been shown that the protein synthesis inhibitor cycloheximide did not inhibit JA-responsive expression of the AOC2 gene, indicating that de novo synthesis of ORA47 protein is not necessary. The working hypothesis is that JA does not induce JA biosynthesis gene expression by increasing ORA47 protein abundance, but activates pre-existing ORA47 protein. The main aim of the project was thus to identify proteins that could interact with and / or activate ORA47 in the context of JA signalling.
Two approaches were used to identify ORA47 interactors, the TAP-tag method and the yeast-two hybrid screening technique. The first approach was to express ORA47 tagged with a TAP-tag in Arabidopsis plants. Despite a correct expression of the tagged ORA47, the TAP tag used did not allow efficient recovery of the tagged protein. Thus this technique was unsuccessful. However, using yeast-two hybrid screening, two potential ORA47 interactors were identified. Interaction was confirmed using an in vitro interaction assay. Relevance and function of these interactions in vivo are still under investigation. Recently a new family of proteins called JAZ were discovered which regulate JA-responsive expression of certain genes.
These proteins regulate the JA-responsive activity of the transcription factor AtMYC2 at the protein level. Interaction of ORA47 with JAZ proteins was tested using yeast two-hybrid assays. None of the 12 JAZ protein family members interacted with ORA47 in this assay. During this project, two new interactors of ORA47 were identified, whereas JAZ proteins, newly identified key regulators of a certain branch of the JA response pathway, are not interacting with ORA47. Further experimentation is needed to elucidate the involvement of these new interactors in regulation of ORA47 activity.
Two approaches were used to identify ORA47 interactors, the TAP-tag method and the yeast-two hybrid screening technique. The first approach was to express ORA47 tagged with a TAP-tag in Arabidopsis plants. Despite a correct expression of the tagged ORA47, the TAP tag used did not allow efficient recovery of the tagged protein. Thus this technique was unsuccessful. However, using yeast-two hybrid screening, two potential ORA47 interactors were identified. Interaction was confirmed using an in vitro interaction assay. Relevance and function of these interactions in vivo are still under investigation. Recently a new family of proteins called JAZ were discovered which regulate JA-responsive expression of certain genes.
These proteins regulate the JA-responsive activity of the transcription factor AtMYC2 at the protein level. Interaction of ORA47 with JAZ proteins was tested using yeast two-hybrid assays. None of the 12 JAZ protein family members interacted with ORA47 in this assay. During this project, two new interactors of ORA47 were identified, whereas JAZ proteins, newly identified key regulators of a certain branch of the JA response pathway, are not interacting with ORA47. Further experimentation is needed to elucidate the involvement of these new interactors in regulation of ORA47 activity.