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...

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes1–5. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. Upon JA-Ile perception, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated6,7. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL)8 and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel INteractor of JAZ (NINJA). NINJA acts as a transcriptional repressor of which the activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress- and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein-protein, and protein-membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para-and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo-and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.