- Plant cell and molecular biology
Jasmonates (JAs) are plant hormones that control both local and systemic defense response to herbivores and necrotrophic pathogens (Chapter 1). Only limited knowledge is available about the molecular mechanisms by which JAs are perceived and regulate gene expression. A genome-wide transcript profiling experiment was set up to identify novel players in the JA signalling pathway.
The transcriptome of fast-dividing suspension cultured cells of the model plant Arabidopsis was examined upon elicitation with methyl jasmonate (MeJA). This revealed a coordinated activation of genes involved in the biosynthesis of phenylpropanoids and increased levels of oligolignols in the culture medium. Simultaneously, genes related to the M-phase of the cell cycle were repressed, corresponding with an increased number of cells residing in the G2-phase. These results can be regarded as the molecular basis of a JA-mediated growth-to-defense transition (Chapter 2). When comparing the transcriptome signatures associated with JA signalling amongst different experiments, a limited overlap was observed. This suggests that the context in which JAs are perceived may be of major importance to determine the physiological response to this signal (Chapter 3). Early MeJA-responsive genes encoded predominantly JA biosynthetic enzymes and transcriptional regulators. Using high-throughput functional genomics, we examined the role of these regulators in JA signalling. This identified the transcription factors MYC2 and ORA47 as transcriptional activators of early JA-responsive genes (Chapter 4). A more detailed functional analysis was performed for the JAZ/TIFY genes. Almost all members of this uncharacterized gene family were found to be early and primary JA-responsive genes. We found that JAZ proteins are repressors of JA signalling and function by direct binding of MYC2 and inactivating its activity. JAZ1 knock-down plants had an increased sensitivity to MeJA, confirming a role as repressor. The JAZ1 protein localized to the nucleus and was rapidly degraded following JA treatment. Purification of JAZ1 protein complexes showed that JAZ1 interacts with the F-box protein COI1 in the presence of JA. These results suggest that JAZ proteins are the targets of the SCFCOI1 E3 ubiquitin ligase and that their JA-mediated destruction liberates MYC2 to activate gene expression (Chapter 5). JAZ1 protein complexes also contained a novel protein, designated Novel INteractor of JAZ (NINJA). JAZ and non-JAZ TIFY proteins interacted directly with NINJA by means of the conserved TIFY domain. Moreover, we could show that an EAR domain in NINJA was able to recruit the co-repressor TOPLESS to JAZ1. Both JAZ and NINJA are shown to act as potent transcriptional repressors, with NINJA requiring a functional EAR domain. Analysis of the dominant negative TOPLESS mutant tpl-1 underscored the role of TOPLESS as a negative regulator in JA signalling (Chapter 6).
In conclusion, systems biology approaches were successfully used to discover the central module of the JA signaling cascade. The molecular mechanism is found to be similar both not identical to that of auxin. We uncovered that the JAZ proteins are the functional equivalents of the Aux/IAA proteins and that JA, as auxin, uses the co-repressor TOPLESS as a regulator of gene expression. However, in the case of JA signalling, we discovered that an additional protein, NINJA, allows the JAZ proteins to interact with TOPLESS indirectly, possibly allowing additional fine-tuning of JAZ activity.