Abraham J.K. Koo

Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database.

The genome of Arabidopsis has been searched for sequences of genes involved in acyl lipid metabolism. Over 600 encoded proteins have been identified, cataloged, and classified according to predicted function, subcellular location, and alternative splicing. At least one-third of these proteins were previously annotated as “unknown function” or with functions unrelated to acyl lipid metabolism; therefore, this study has improved the annotation of over 200 genes. In particular, annotation of the lipolytic enzyme group (at least 110 members total) has been improved by the critical examination of the biochemical literature and the sequences of the numerous proteins annotated as “lipases.” In addition, expressed sequence tag (EST) data have been surveyed, and more than 3,700 ESTs associated with the genes were cataloged. Statistical analysis of the number of ESTs associated with specific cDNA libraries has allowed calculation of probabilities of differential expression between different organs. More than 130 genes have been identified with a statistical probability > 0.95 of preferential expression in seed, leaf, root, or flower. All the data are available as a Web-based database, the Arabidopsis Lipid Gene database (http://www.plantbiology.msu.edu/lipids/genesurvey/index.htm). The combination of the data of the Lipid Gene Catalog and the EST analysis can be used to gain insights into differential expression of gene family members and sets of pathway-specific genes, which in turn will guide studies to understand specific functions of individual genes.

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 rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis

Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F-box protein COI1 triggers the SCF(COI1)/ubiquitin-dependent degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the expression of JA-response genes. JA is required for many wound-inducible systemic defense responses, but little is known about the role of the hormone in long-distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA-Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound-induced systemic production of JA-Ile required the JA biosynthetic enzyme 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA-conjugating enzyme JAR1. Interestingly, the wound-induced synthesis of JA/JA-Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA-Ile, activates the expression of early response genes by the SCF(COI1)/JAZ pathway.

The wound hormone jasmonate

Plant tissues are highly vulnerable to injury by herbivores, pathogens, mechanical stress, and other environmental insults. Optimal plant fitness in the face of these threats relies on complex signal transduction networks that link damage-associated signals to appropriate changes in metabolism, growth, and development. Many of these wound-induced adaptive responses are triggered by de novo synthesis of the plant hormone jasmonate (JA). Recent studies provide evidence that JA mediates systemic wound responses through distinct cell autonomous and non-autonomous pathways. In both pathways, bioactive JAs are recognized by an F-box protein-based receptor system that couples hormone binding to ubiquitin-dependent degradation of transcriptional repressor proteins. These results provide a framework for understanding how plants recognize and respond to tissue injury.

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.

Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers

Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases essential for cutin biosynthesis, glycerol-3-phosphate acyltransferase (GPAT) 4 and GPAT8. Double knockouts gpat4/gpat8 were strongly reduced in cutin and were less resistant to desiccation and to infection by the fungus Alternaria brassicicola. They also showed striking defects in stomata structure including a lack of cuticular ledges between guard cells, highlighting the importance of cutin in stomatal biology. Overexpression of GPAT4 or GPAT8 in Arabidopsis increased the content of C16 and C18 cutin monomers in leaves and stems by 80%. In order to modify cutin composition, the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis, were overexpressed. When both enzymes were overexpressed together the epidermal polyesters accumulated new C20 and C22 ω-hydroxyacids and α,ω-diacids typical of suberin, and the fine structure and water-barrier function of the cuticle were altered. These results identify GPATs as partners of fatty acyl oxidases in lipid polyester synthesis and indicate that their cooverexpression provides a strategy to probe the role of cutin composition and quantity in the function of plant cuticles.

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.

Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) signals through the COI1-JAZ coreceptor complex to control key aspects of plant growth, development, and immune function. Despite detailed knowledge of the JA-Ile biosynthetic pathway, little is known about the genetic basis of JA-Ile catabolism and inactivation. Here, we report the identification of a wound- and jasmonate-responsive gene from Arabidopsis that encodes a cytochrome P450 (CYP94B3) involved in JA-Ile turnover. Metabolite analysis of wounded leaves showed that loss of CYP94B3 function in cyp94b3 mutants causes hyperaccumulation of JA-Ile and concomitant reduction in 12-hydroxy-JA-Ile (12OH-JA-Ile) content, whereas overexpression of this enzyme results in severe depletion of JA-Ile and corresponding changes in 12OH-JA-Ile levels. In vitro studies showed that heterologously expressed CYP94B3 converts JA-Ile to 12OH-JA-Ile, and that 12OH-JA-Ile is less effective than JA-Ile in promoting the formation of COI1-JAZ receptor complexes. CYP94B3-overexpressing plants displayed phenotypes indicative of JA-Ile deficiency, including defects in male fertility, resistance to jasmonate-induced growth inhibition, and susceptibility to insect attack. Increased accumulation of JA-Ile in wounded cyp94b3 leaves was associated with enhanced expression of jasmonate-responsive genes. These results demonstrate that CYP94B3 exerts negative feedback control on JA-Ile levels and performs a key role in attenuation of jasmonate responses.

Functional Diversification of Acyl-Coenzyme A Oxidases in Jasmonic Acid Biosynthesis and Action

The biosynthesis of jasmonic acid (JA) in plant peroxisomes requires the action of acyl-coenzyme A oxidase (ACX). Among the five expressed members (ACX1–5) of the ACX gene family in Arabidopsis (Arabidopsis thaliana), only ACX1 is known to serve a role in JA production. Here, we used transgenic promoter-reporter lines to show that ACX1 is highly expressed in mature and germinating pollen, stem epidermal cells, and other tissues in which jasmonate-signaled processes occur. Wound-induced JA accumulation was reduced in a mutant that is defective in ACX1 and was abolished in a mutant that is impaired in both ACX1 and its closely related paralog, ACX5. The severe JA deficiency in acx1/5 double mutants was accompanied by decreased resistance to the leaf-eating insect Trichoplusia ni. The double mutant also showed reduced pollen viability and fecundity. Treatment of acx1/5 plants with JA restored both protection against T. ni larvae and normal seed set. Unexpectedly, acx1/5 plants accumulated JA in response to infection by the necrotrophic fungal pathogen Alternaria brassicicola. In contrast to mutants that are impaired in jasmonate perception or early steps of the JA biosynthetic pathway, acx1/5 plants maintained resistance to A. brassicicola infection. These results indicate that ACX1/5-mediated JA synthesis is essential for resistance to chewing insects and male reproductive function and further suggest that other ACX isozymes contribute to JA production in response to A. brassicicola challenge. Thus, different types of biotic stress may induce JA synthesis via distinct enzymatic routes.

A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in arabidopsis.

This work identifies the bHLH transcription factor, JA-ASSOCIATED MYC2-LIKE1 (JAM1), which negatively regulates jasmonate responses independently from known regulators, namely, JASMONATE-ZIM DOMAIN proteins. JAM1 is involved in JA-mediated male fertility and the defense response to insect attack and represses the transcription of the target genes of MYC2 by competing for the binding sequences. Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix–type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.