Roberto Solano

The JAZ family of repressors is the missing link in jasmonate signalling.

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 SCFCOI1 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 SCFCOI1 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.

ETHYLENE RESPONSE FACTOR1 Integrates Signals from Ethylene and Jasmonate Pathways in Plant Defense

Cross-talk between ethylene and jasmonate signaling pathways determines the activation of a set of defense responses against pathogens and herbivores. However, the molecular mechanisms that underlie this cross-talk are poorly understood. Here, we show that ethylene and jasmonate pathways converge in the transcriptional activation of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly by ethylene or jasmonate and can be activated synergistically by both hormones. In addition, both signaling pathways are required simultaneously to activate ERF1, because mutations that block any of them prevent ERF1 induction by any of these hormones either alone or in combination. Furthermore, 35S:ERF1 expression can rescue the defense response defects of coi1 (coronative insensitive1) and ein2 (ethylene insensitive2); therefore, it is a likely downstream component of both ethylene and jasmonate signaling pathways. Transcriptome analysis in Col;35S:ERF1 transgenic plants and ethylene/jasmonate-treated wild-type plants further supports the notion that ERF1 regulates in vivo the expression of a large number of genes responsive to both ethylene and jasmonate. These results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes.

JASMONATE-INSENSITIVE1 Encodes a MYC Transcription Factor Essential to Discriminate between Different Jasmonate-Regulated Defense Responses in Arabidopsis

In spite of the importance of jasmonates (JAs) as plant growth and stress regulators, the molecular components of their signaling pathway remain largely unknown. By means of a genetic screen that exploits the cross talk between ethylene (ET) and JAs, we describe the identification of several new loci involved in JA signaling and the characterization and positional cloning of one of them, JASMONATE-INSENSITIVE1 (JAI1/JIN1). JIN1 encodes AtMYC2, a nuclear-localized basic helix-loop-helix-leucine zipper transcription factor, whose expression is rapidly upregulated by JA, in a CORONATINE INSENSITIVE1–dependent manner. Gain-of-function experiments confirmed the relevance of AtMYC2 in the activation of JA signaling. AtMYC2 differentially regulates the expression of two groups of JA-induced genes. The first group includes genes involved in defense responses against pathogens and is repressed by AtMYC2. Consistently, jin1 mutants show increased resistance to necrotrophic pathogens. The second group, integrated by genes involved in JA-mediated systemic responses to wounding, is activated by AtMYC2. Conversely, Ethylene-Response-Factor1 (ERF1) positively regulates the expression of the first group of genes and represses the second. These results highlight the existence of two branches in the JA signaling pathway, antagonistically regulated by AtMYC2 and ERF1, that are coincident with the alternative responses activated by JA and ET to two different sets of stresses, namely pathogen attack and wounding.

Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins.

Mutations in the Arabidopsis ETHYLENE-INSENSITIVE3 (EIN3) gene severely limit a plants response to the gaseous hormone ethylene. ein3 mutants show a loss of ethylene-mediated effects including gene expression, the triple response, cell growth inhibition, and accelerated senescence. EIN3 acts downstream of the histidine kinase ethylene receptor, ETR1, and the Raf-like kinase, CTR1. The EIN3 gene encodes a novel nuclear-localized protein that shares sequence similarity, structural features, and genetic function with three EIN3-LIKE (EIL) proteins. In addition to EIN3, EIL1 orEIL2 were able to complement ein3, suggesting their participation in the ethylene signaling pathway. Overexpression of EIN3 or EIL1 in wild-type or ethylene-insensitive2 plants conferred constitutive ethylene phenotypes, indicating their sufficiency for activation of the pathway in the absence of ethylene.

ABA Is an Essential Signal for Plant Resistance to Pathogens Affecting JA Biosynthesis and the Activation of Defenses in Arabidopsis

Analyses of Arabidopsis thaliana defense response to the damping-off oomycete pathogen Pythium irregulare show that resistance to P. irregulare requires a multicomponent defense strategy. Penetration represents a first layer, as indicated by the susceptibility of pen2 mutants, followed by recognition, likely mediated by ERECTA receptor-like kinases. Subsequent signaling of inducible defenses is predominantly mediated by jasmonic acid (JA), with insensitive coi1 mutants showing extreme susceptibility. In contrast with the generally accepted roles of ethylene and salicylic acid cooperating with or antagonizing, respectively, JA in the activation of defenses against necrotrophs, both are required to prevent disease progression, although much less so than JA. Meta-analysis of transcriptome profiles confirmed the predominant role of JA in activation of P. irregulare–induced defenses and uncovered abscisic acid (ABA) as an important regulator of defense gene expression. Analysis of cis-regulatory sequences also revealed an unexpected overrepresentation of ABA response elements in promoters of P. irregulare–responsive genes. Subsequent infections of ABA-related and callose-deficient mutants confirmed the importance of ABA in defense, acting partly through an undescribed mechanism. The results support a model for ABA affecting JA biosynthesis in the activation of defenses against this oomycete.

(+)-7- iso -Jasmonoyl- L -isoleucine is the endogenous bioactive jasmonate

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.

NINJA connects the co-repressor TOPLESS to jasmonate signalling.

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose 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-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses

This work identifies two transcription factors, MYC3 and MYC4, as targets of JAZ repressors and regulators of responses to jasmonate. It finds a specificity of transcription factor activity that could be a clue to understanding the diversity of JA-regulated responses. 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 JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis

Five ethylene-insensitive loci (wei1–wei5) were identified by using a low-dose screen for “weak” ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response. wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5/EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5/eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling

Plant growth is strongly influenced by the presence of neighbors that compete for light resources. In response to vegetational shading shade-intolerant plants such as Arabidopsis display a suite of developmental responses known as the shade-avoidance syndrome (SAS). The phytochrome B (phyB) photoreceptor is the major light sensor to mediate this adaptive response. Control of the SAS occurs in part with phyB, which controls protein abundance of phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) directly. The shade-avoidance response also requires rapid biosynthesis of auxin and its transport to promote elongation growth. The identification of genome-wide PIF5-binding sites during shade avoidance revealed that this bHLH transcription factor regulates the expression of a subset of previously identified SAS genes. Moreover our study suggests that PIF4 and PIF5 regulate elongation growth by controlling directly the expression of genes that code for auxin biosynthesis and auxin signaling components.