Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.

Role and functioning of bHLH transcription factors in jasmonate signalling.

Plant growth, development and interaction with the environment involve the action of multiple phytohormones. Transcription factors (TFs) of diverse families play essential roles in the signalling cascades triggered by the perception of a particular hormone. TFs may act alone or in a combinatorial fashion with other TFs, and may act specifically in a single hormonal signalling cascade or as signalling hubs for multiple hormones. In the signalling cascades triggered by the phytohormone jasmonate (JA), which modulates a diverse, but specific, range of aspects of plant growth, development and defence, the TFs of the basic helix-loop-helix (bHLH) family play an essential and often conserved role in the plant kingdom. Here, we first discuss the bHLH TFs involved in all kinds of JA-modulated processes in the model plant Arabidopsis thaliana. Secondly, we elaborate on the identity and role of bHLH TFs in the conserved JA-mediated elicitation of specialized metabolism of medicinal and crop species. Finally, we discuss which directions future fundamental research on the functioning of bHLH TFs in JA signalling may head for and how this research can be translated from model plants into crop and medicinal plant species to engineer traits of agronomical and industrial interest.

Jasmonates: signal transduction components and their roles in environmental stress responses.

Jasmonates, oxylipin-type plant hormones, are implicated in diverse aspects of plant growth development and interaction with the environment. Following diverse developmental and environmental cues, jasmonate is produced, conjugated to the amino acid isoleucine and perceived by a co-receptor complex composed of the Jasmonate ZIM-domain (JAZ) repressor proteins and an E3 ubiquitin ligase complex containing the F-box CORONATINE INSENSITIVE 1 (COI1). This event triggers the degradation of the JAZ proteins and the release of numerous transcription factors, including MYC2 and its homologues, which are otherwise bound and inhibited by the JAZ repressors. Here, we will review the role of the COI1, JAZ and MYC2 proteins in the interaction of the plant with its environment, illustrating the significance of jasmonate signalling, and of the proteins involved, for responses to both biotic stresses caused by insects and numerous microbial pathogens and abiotic stresses caused by adverse climatic conditions. It has also become evident that crosstalk with other hormone signals, as well as light and clock signals, plays an important role in the control and fine-tuning of these stress responses. Finally, we will discuss how several pathogens exploit the jasmonate perception and early signalling machinery to decoy the plants defence systems.

The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability.

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCF(COI1)) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCF(COI1) components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability.

Change of a conserved amino acid in the MYC2 and MYC3 transcription factors leads to release of JAZ repression and increased activity.

The bHLH transcription factor MYC2, together with its paralogues MYC3 and MYC4, is a master regulator of the response to the jasmonate (JA) hormone in Arabidopsis (Arabidopsis thaliana). In the absence of JA, JASMONATE ZIM (JAZ) proteins interact with the MYC proteins to block their activity. Understanding of the mechanism and specificity of this interaction is key to unravel JA signalling. We generated mutant MYC proteins and assessed their activity and the specificity of their interaction with the 12 Arabidopsis JAZ proteins. We show that the D94N mutation present in the atr2D allele of MYC3 abolishes the interaction between MYC3 and most JAZ proteins. The same effect is observed when the corresponding conserved Asp (D105) was mutated in MYC2. Accordingly, MYC2(D105N) activated target genes in the presence of JAZ proteins, in contrast to wild-type MYC2. JAZ1 and JAZ10 were the only JAZ proteins still showing interaction with the mutant MYC proteins, due to a second MYC interaction domain, besides the classical Jas domain. Our results visualize the divergence among JAZ proteins in their interaction with MYC proteins. Ultimately, the transferability of the Asp-to-Asn amino acid change might facilitate the design of hyperactive transcription factors for plant engineering.

The Molecular Basis of JAZ-MYC Coupling, a Protein-Protein Interface Essential for Plant Response to Stressors.

The jasmonic acid (JA) signaling pathway is one of the primary mechanisms that allow plants to respond to a variety of biotic and abiotic stressors. Within this pathway, the JAZ repressor proteins and the basic helix-loop-helix (bHLH) transcription factor MYC3 play a critical role. JA is a volatile organic compound with an essential role in plant immunity. The increase in the concentration of JA leads to the decoupling of the JAZ repressor proteins and the bHLH transcription factor MYC3 causing the induction of genes of interest. The primary goal of this study was to identify the molecular basis of JAZ-MYC coupling. For this purpose, we modeled and validated 12 JAZ-MYC3 3D in silico structures and developed a molecular dynamics/machine learning pipeline to obtain two outcomes. First, we calculated the average free binding energy of JAZ-MYC3 complexes, which was predicted to be -10.94 +/-2.67 kJ/mol. Second, we predicted which ones should be the interface residues that make the predominant contribution to the free energy of binding (molecular hotspots). The predicted protein hotspots matched a conserved linear motif SL••FL•••R, which may have a crucial role during MYC3 recognition of JAZ proteins. As a proof of concept, we tested, both in silico and in vitro, the importance of this motif on PEAPOD (PPD) proteins, which also belong to the TIFY protein family, like the JAZ proteins, but cannot bind to MYC3. By mutating these proteins to match the SL••FL•••R motif, we could force PPDs to bind the MYC3 transcription factor. Taken together, modeling protein-protein interactions and using machine learning will help to find essential motifs and molecular mechanisms in the JA pathway.

A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.