John Browse

JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling

Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCFCOI1 ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl–isoleucine (JA–Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCFCOI1 ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCFCOI1–JAZ1 protein complex as a site of perception of the plant hormone JA–Ile.

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.

The polyunsaturated fatty acids linoleate and alpha-linolenate are important membrane components and are the essential fatty acids of human nutrition. The major enzyme responsible for the synthesis of these compounds is the plant oleate desaturase of the endoplasmic reticulum, and its activity is controlled in Arabidopsis by the fatty acid desaturation 2 (fad2) locus. A fad2 allele was identified in a population of Arabidopsis in which mutations had been created by T-DNA insertions. Genomic DNA flanking the T-DNA was cloned by plasmid rescue and used to isolate cDNA and genomic clones of FAD2. A cDNA containing the entire FAD2 coding sequence was expressed in fad2 mutant plants and shown to complement the mutant fatty acid phenotype. The deduced amino acid sequence from the cDNA showed homology to other plant desaturases, and this confirmed that FAD2 is the structural gene for the desaturase. Gel blot analyses of FAD2 mRNA levels showed that the gene is expressed throughout the plant and suggest that transcript levels are in excess of the amount needed to account for oleate desaturation. Sequence analysis identified histidine-rich motifs that could contribute to an iron binding site in the cytoplasmic domain of the protein. Such a position would facilitate interaction between the desaturase and cytochrome b5, which is the direct source of electrons for the desaturation reaction, but would limit interaction of the active site with the fatty acyl substrate.

Jasmonate Passes Muster: A Receptor and Targets for the Defense Hormone

The oxylipin jasmonate (JA) regulates many aspects of growth, development, and environmental responses in plants, particularly defense responses against herbivores and necrotrophic pathogens. Mutants of Arabidopsis helped researchers define the biochemical pathway for synthesis of jasmonoyl-isoleucine (JA-Ile), the active form of JA hormone, and demonstrated that JA is required for plant survival of insect and pathogen attacks and for plant fertility. Transcriptional profiling led to the discovery of the JASMONATE ZIM-DOMAIN (JAZ) proteins, which are repressors of JA signaling. JA-Ile relieves repression by promoting binding of the JAZ proteins to the F-box protein CORONATINE INSENSITIVE1 (COI1) and their subsequent degradation by the ubiquitination/26S-proteasome pathway. Although we now have a much better understanding of the molecular mechanism of JA action, many questions remain. Experimental answers to these questions will expand our knowledge of oxylipin signaling in plants and animals and will also provide new tools for efforts to improve crop protection and reproductive performance.

Plant defense in the absence of jasmonic acid: The role of cyclopentenones

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

The Critical Requirement for Linolenic Acid Is Pollen Development, Not Photosynthesis, in an Arabidopsis Mutant.

The very high proportions of trienoic fatty acids found in chloroplast membranes of all higher plants suggest that these lipid structures might be essential for photosynthesis. We report here on the production of Arabidopsis triple mutants that contain negligible levels of trienoic fatty acids. Photosynthesis at 22[deg]C was barely affected, and vegetative growth of the mutants was identical with that of the wild type, demonstrating that any requirement for trienoic acyl groups in membrane structure and function is relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditions. Comparisons of pollen development in wild-type and triple mutant flowers established that pollen grains in the mutant developed to the tricellular stage. Exogenous applications of [alpha]-llnolenate or jasmonate restored fertility. Taken together, the results demonstrate that the critical role of trienoic acids in the life cycle of plants is as the precursor of oxylipin, a signaling compound that regulates final maturation processes and the release of pollen.

An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack.

The activation of defense genes in tomato plants has been shown to be mediated by an octadecanoic acid-based signaling pathway in response to herbivore attack or other mechanical wounding. We report here that a tomato mutant (JL5) deficient in the activation of would-inducible defense genes is also compromised in resistance toward the lepidopteran predator Manduca sexta (tobacco hornworm). Thus we propose the name defenseless1 (def1) for the mutation in the JL5 line that mediates this altered defense response. In experiments designed to define the normal function of DEF1, we found that def1 plants are defective in defense gene signaling initiated by prosystemin overexpression in transgenic plants as well as by oligosaccharide (chitosan and polygalacturonide) and polypeptide (systemin) elicitors. Supplementation of plants through their cut stems with intermediates of the octadecanoid pathway indicates that def1 plants are affected in octadecanoid metabolism between the synthesis of hydroperoxylinolenic acid and 12-oxo-phytodienoic acid. Consistent with this defect, def1 plants are also compromised in their ability to accumulate jasmonic acid, the end product of the pathway, in response to wounding and the aforementioned elicitors. Taken together, these results show that octadecanoid metabolism plays an essential role in the transduction of upstream would signals to the activation of antiherbivore plant defenses.

Plant Lipids: Metabolism, Mutants, and Membranes

The mechanisms that regulate plant lipid metabolism determine the dietary and industrial value of storage oils found in economically important species and may control the ability of many plants to survive exposure to temperature extremes. Many of the problems researchers have in defining the pathways, enzymes, and genes involved in plant lipid metabolism appear to be amenable to analysis by genetic approaches. Mutants with alterations in membrane lipid composition have also been used to study the structural and adaptive roles of lipids. The application of genetic engineering methods affords opportunities for researchers to apply knowledge gained about plant lipid metabolism toward enhanced use of plant oils as abundant and renewable sources of reduced carbon.

Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue.

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.

Polyunsaturated fatty acid synthesis: what will they think of next?

Polyunsaturated fatty acids have crucial roles in membrane biology and signaling processes in most living organisms. However, it is only recently that molecular genetic approaches have allowed detailed studies of the enzymes involved in their synthesis. New evidence has revealed a range of pathways in different organisms. These include a complex sequence for synthesis of docosahexaenoic acid (22:6) in mammals and a polyketide synthase pathway in marine microbes.