Jasmonates are essential phytohormones for plant development and survival. However, the molecular details of their signalling pathway remain largely unknown. The identification more than a decade ago of COI1 as an F-box protein suggested the existence of a repressor of jasmonate responses that is targeted by the SCF(COI1) complex for proteasome degradation in response to jasmonate. Here we report the identification of JASMONATE-INSENSITIVE 3 (JAI3) and a family of related proteins named JAZ (jasmonate ZIM-domain), in Arabidopsis thaliana. Our results demonstrate that JAI3 and other JAZs are direct targets of the SCF(COI1) E3 ubiquitin ligase and jasmonate treatment induces their proteasome degradation. Moreover, JAI3 negatively regulates the key transcriptional activator of jasmonate responses, MYC2. The JAZ family therefore represents the molecular link between the two previously known steps in the jasmonate pathway. Furthermore, we demonstrate the existence of a regulatory feed-back loop involving MYC2 and JAZ proteins, which provides a mechanistic explanation for the pulsed response to jasmonate and the subsequent desensitization of the cell.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo-and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JAregulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response. INTRODUCTIONThe plant hormones jasmonates (JAs) are fatty acid-derived oxylipins required for the regulation of multiple physiological aspects of plant growth, development, and defense (Wasternack, 2007;Kazan and Manners, 2008;Browse, 2009;Pauwels et al., 2009). Thus, JAs are widely recognized as regulators of plant responses to environmental stresses such as pathogen and pest attack, wounding, ozone exposure, and water deficit (Devoto et al., 2005;Browse and Howe, 2008). They are also important regulators of growth and developmental programs such as gamete development, the cell cycle, root growth, tendril coiling, and senescence in many plant species (Pauwels et al., 2008;Zhang and Turner, 2008;Reinbothe et al., 2009;Yoshida et al., 2009). JAs are being recognized as important integrators of developmental and stress signals to modulate the allocation of resources to grow or to defend (Moreno et al., 2009;Robson et al., 2010).Transcription is a major regulatory step in the activation of these responses, and JAs trigger an important transcriptional reprogramming of the cells to switch the basal developmental programs into the necessary stress response program (Reymond et al., 2004;Devoto et al., 2005;Mandaokar et al., 2006;Pauwels et al., 2008). The signaling events that lead to transcriptional reprogramming are starting to be elucidated. Upon elicitation by exogenous or endogenous signals, the hormone (+)-7-iso-jasmonoyl-L-isoleucine [also known as (3R,7S)-jasmonoyl-L-isoleucine or JA-Ile] is synthesized by JAR1 (Fonseca et al., 2009b;Suza et al., 2010;Wasternack and Kombrink, 2010). JA-Ile is perceived by a receptor complex formed by the protein COI1 and the JAZ repressors (Xie et al., 1998;Thines et...

Hormone-triggered activation of the jasmonate signaling pathway in Arabidopsis thaliana requires SCF(COI1)-mediated proteasome degradation of JAZ repressors. (-)-JA-L-Ile is the proposed bioactive hormone, and SCF(COI1) is its likely receptor. We found that the biological activity of (-)-JA-L-Ile is unexpectedly low compared to coronatine and the synthetic isomer (+)-JA-L-Ile, which suggests that the stereochemical orientation of the cyclopentanone-ring side chains greatly affects receptor binding. Detailed GC-MS and HPLC analyses showed that the (-)-JA-L-Ile preparations currently used in ligand binding studies contain small amounts of the C7 epimer (+)-7-iso-JA-L-Ile. Purification of each of these molecules demonstrated that pure (-)-JA-L-Ile is inactive and that the active hormone is (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine. In addition, we show that pH changes promote conversion of (+)-7-iso-JA-L-Ile to the inactive (-)-JA-L-Ile form, thus providing a simple mechanism that can regulate hormone activity through epimerization.

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