Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxophytodienoate (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.OPDA reductase ͉ opr3 ͉ Arabidopsis ͉ insect A major objective in plant biology is to develop an integrated understanding of how plants survive in their environment and reproduce. Although it has become clear in the last decade that jasmonic acid (JA) is a key regulator in the development, physiology, and defense of plants, the complexity of the signaling network in which JA evolves is just emerging (1). JA is involved in carbon partitioning (2), in mechanotransduction (3), and the ability of plants to synthesize and perceive JA is absolutely essential for the correct development and release of pollen in Arabidopsis (4-7). Highlighting the regulatory importance of JA, a JA-responsive transcription factor, ORCA3, first found in Catharanthus, provides an important link between primary and secondary metabolism (8). There is also strong evidence supporting a central role of JA in plant defense. Exogenous JA powerfully regulates the expression of many defense genes in plants, and its in vivo production and perception seem to be of vital importance in mounting successful defense against insect attackers (9-11). Together with ethylene, JA also plays a crucial role in defense against necrotrophic fungi (12-14) and in induced systemic resistance in response to nonpathogenic rhizobacteria (15). Broader roles of JA in plant stress responses are likely; it is known that the JA biosynthesis pathway is important in gene activation subsequent to UV damage in plants (16), and JA has been implicated in some responses to water stress (17).The biosynthesis of JA occurs through the octadecanoid pathway (18,19) and is initiated by the addition of molecular oxygen to linolenic acid (18:3) to form 13-hydroperoxylinolenic acid (13-HPOTrE). This fatty acid hydroperoxide is then dehydrated by allene oxide synthase (AOS) and cyclized by ...

The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include: 1) structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen; 2) enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; 3) pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein during the defense response. The PRs have typical physicochemical properties that enable them to resist to acidic pH and proteolytic cleavage and thus survive in the harsh environments where they occur: vacuolar compartment or cell wall or intercellular spaces. Since the discovery of the first PRs in tobacco many other similar proteins have been isolated from tobacco but also from other plant species, including dicots and monocots, the widest range being characterized from hypersensitively reacting tobacco. Based first on serological properties and later on sequence data, the tobacco PRs have been classified in five major groups. Group PR-1 contains the first discovered PRs of 15-17 kDa molecular mass, whose biological activity is still unknown, but some members have been shown recently to have antifungal activity. Group PR-2 contains three structurally distinct classes of 1,3-beta-glucanases, with acidic and basic counterparts, with dramatically different specific activity towards linear 1,3-beta-glucans and with different substrate specificity. Group PR-3 consists of various chitinases-lysozymes that belong to three distinct classes, are vacuolar or extracellular, and exhibit differential chitinase and lysozyme activities. Some of them, either alone or in combination with 1,3-beta-glucanases, have been shown to be antifungal in vitro and in vivo (transgenic plants), probably by hydrolysing their substrates as structural components in the fungal cell wall. Group PR-4 is the less studied, and in tobacco contains four members of 13-14.5 kDa of unknown activity and function. Group PR-5 contains acidic-neutral and very basic members with extracellular and vacuolar localization, respectively, and all members show sequence similarity to the sweet-tasting protein thaumatin. Several members of the PR-5 group from tobacco and other plant species were shown to display significant in vitro activity of inhibiting hyphal growth or spore germination of various fungi probably by a membrane permeabilizing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

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Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3). Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role independent of JA-Ile and its receptor COI1; however, this hypothesis has been challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-didehydro-JA in wounded wild-type and opr3-3 plants. OPR2 was found to reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of OPR3, OPDA enters the β-oxidation pathway to produce 4,5-ddh-JA as a direct precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA biosynthesis.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestam both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-la, PR-lb, and PR-lc. l h e various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestam in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1 g.

The gene family of subtilisin-like serine proteases (subtilases) in Arabidopsis thaliana comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative bioinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co-regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades. Supplemental material is available in the Plant Subtilase Database (PSDB) (http://csbdb.mpimp-golm.mpg.de/psdb.html) , as well as from the CSB.DB (http://csbdb.mpimp-golm.mpg.de).

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