Induced oleoresin biosynthesis in grand fir as a defense against bark beetles.

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

Molecular mechanisms of defense by rhizobacteria against root disease.

Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases. "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host. Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var. tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat. The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria. Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline. There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes. Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol. The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.