Hoo Sun Chung

Regulation and Function of Arabidopsis JASMONATE ZIM-Domain Genes in Response to Wounding and Herbivory

Jasmonate (JA) and its amino acid conjugate, jasmonoyl-isoleucine (JA-Ile), play important roles in regulating plant defense responses to insect herbivores. Recent studies indicate that JA-Ile promotes the degradation of JASMONATE ZIM-domain (JAZ) transcriptional repressors through the activity of the E3 ubiquitin-ligase SCFCOI1. Here, we investigated the regulation and function of JAZ genes during the interaction of Arabidopsis (Arabidopsis thaliana) with the generalist herbivore Spodoptera exigua. Most members of the JAZ gene family were highly expressed in response to S. exigua feeding and mechanical wounding. JAZ transcript levels increased within 5 min of mechanical tissue damage, coincident with a large (approximately 25-fold) rise in JA and JA-Ile levels. Wound-induced expression of JAZ and other CORONATINE-INSENSITIVE1 (COI1)-dependent genes was not impaired in the jar1-1 mutant that is partially deficient in the conversion of JA to JA-Ile. Experiments performed with the protein synthesis inhibitor cycloheximide provided evidence that JAZs, MYC2, and genes encoding several JA biosynthetic enzymes are primary response genes whose expression is derepressed upon COI1-dependent turnover of a labile repressor protein(s). We also show that overexpression of a modified form of JAZ1 (JAZ1Δ3A) that is stable in the presence of JA compromises host resistance to feeding by S. exigua larvae. These findings establish a role for JAZ proteins in the regulation of plant anti-insect defense, and support the hypothesis that JA-Ile and perhaps other JA derivatives activate COI1-dependent wound responses in Arabidopsis. Our results also indicate that the timing of JA-induced transcription in response to wounding is more rapid than previously realized.

A Critical Role for the TIFY Motif in Repression of Jasmonate Signaling by a Stabilized Splice Variant of the JASMONATE ZIM-Domain Protein JAZ10 in Arabidopsis

JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form (JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block JAZ10.4–JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further suggest that the dominant-negative action of this splice variant involves protein–protein interaction through the ZIM/TIFY domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex directly represses the activity of transcription factors that promote expression of JA response genes.

Jasmonate signaling: a conserved mechanism of hormone sensing.

The lipid-derived hormone jasmonate (JA) regulates diverse aspects of plant immunity and development. Among the central components of the JA signaling cascade are the E3 ubiquitin ligase SCFCOI1 and Jasmonate ZIM-domain (JAZ) proteins that repress transcription of JA-responsive genes. Recent studies provide evidence that amino acid-conjugated forms of JA initiate signal transduction upon formation of a coronatine-insensitive1 (COI1)-JA-JAZ ternary complex in which JAZs are ubiquitinated and subsequently degraded. Coronatine, a virulence factor produced by the plant pathogen Pseudomonas syringae, is a potent agonist of this hormone receptor system. Coronatine-induced targeting of JAZs to COI1 obstructs host immune responses to P. syrinage, providing a striking example of how pathogens exploit hormone signaling pathways in the host to promote disease. These findings, together with homology between COI1 and the auxin receptor, TIR1, extend the paradigm of F-box proteins as intracellular sensors of small molecules, and suggest a common evolutionary origin of the auxin and JA response pathways.

A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine‐ and jasmonoyl isoleucine‐dependent interactions with the COI1 F‐box protein

SUMMARY Coronatine is an important virulence factor produced by several pathovars of the bacterial pathogen Pseudomonas syringae. The structure of coronatine is similar to that of a class of plant hormones called jasmonates (JAs). An important step in JA signaling is the SCF(COI1) E3 ubiquitin ligase-dependent degradation of JAZ repressor proteins. We have recently shown that jasmonoyl isoleucine (JA-Ile) promotes physical interaction between Arabidopsis JAZ1 and COI1 (the F-box component of SCF(COI1)) proteins, and that the JA-Ile-dependent COI1-JAZ1 interaction could be reconstituted in yeast cells (i.e. in the absence of other plant proteins). Here we show that coronatine, but not its two biosynthetic precursors, also promotes interaction between Arabidopsis COI1 and multiple JAZ proteins. The C-terminal Jas motif, but not the N-terminal (NT) domain or central ZIM domain of JAZ proteins, is critical for JA-Ile/coronatine-dependent interaction with COI1. Two positively charged amino acid residues in the Jas domain were identified as essential for coronatine-dependent COI1-JAZ interactions. Mutations of these two residues did not affect the ability of JAZ1 and JAZ9 to interact with the transcription factor AtMYC2. Importantly, transgenic Arabidopsis plants expressing JAZ1 carrying these two mutations exhibited JA-insensitive phenotypes, including male sterility and enhanced resistance to P. syringae infection. These results not only suggest that coronatine and JA-Ile target the physical interaction between COI1 and the Jas domain of JAZ repressors, but also illustrate the critical role of positively charged amino acids in the Jas domain in mediating the JA-Ile/coronatine-dependent JAZ interaction with COI1.

Identification of a Peroxisomal Acyl-activating Enzyme Involved in the Biosynthesis of Jasmonic Acid in Arabidopsis

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of β-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.

Top hits in contemporary JAZ: an update on jasmonate signaling.

The phytohormone jasmonate (JA) regulates a wide range of growth, developmental, and defense-related processes during the plant life cycle. Identification of the JAZ family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid-derived hormone controls gene expression. Recent analysis of JAZ proteins has provided insight into the nature of the JA receptor, the chemical specificity of signal perception, and cross-talk between JA and other hormone response pathways. Functional diversification of JAZ proteins by alternative splicing, together with the ability of JAZ proteins to homo- and heterodimerize, provide mechanisms to enhance combinatorial diversity and versatility in gene regulation by JA.

Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling

Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-l-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCF(COI1) ) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates the JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and seven C-terminal (X(5) PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X(5) PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre-mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

This work uses a facile protein-based screen to systematically examine the efficacy and action mechanism of amiRNAs, thereby allowing selection of optimal amiRNAs for gene silencing from hundreds of computationally predicted candidates. Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5′ coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot–root coordination and developmental plasticity in shaping organ biomass and architecture.

Negative Feedback Control of Jasmonate Signaling by an Alternative Splice Variant of JAZ10

A transcriptional regulator generated by alternative splicing uses a cryptic binding site to negatively regulate bHLH-type transcription factors that promote jasmonate responses. The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.