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Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)beta-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-otaining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific tor the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the, effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.

Tomato plants overexpressing a prosystemin gene that encodes the precursor of a mobile wound signal called systemin have been shown previously to constitutively synthesize extraordinarily high levels of two defensive proteinase inhibitor proteins in leaves in the absence of wounding. We herein report that leaves of these transgenic plants possess enhanced levels of another defensive protein, polyphenol oxidase (PPO) at levels that are up to 70-fold higher than levels found in leaves of wild-type plants. Supplying young wild-type tomato plants with systemin through cut stems induced PPO activity in leaves, and wounding lower leaves of young tomato plants induced PPO activity in both wounded and unwounded leaves to levels equal to those induced by systemin. Exposing young tomato plants to methyl jasmonate vapor caused an increase in PPO activity equivalent to levels found in plants overexpressing the prosystemin gene. The data indicate that PPO and proteinase inhibitor genes are coactivated systemically by wounding via the octadecanoid signal transduction pathway and that systemin has a much broader role in signaling plant defensive genes than was previously known.

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 micrometer antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.

A vacuum infiltration technique was developed that enabled the extraction of apoplastic solution with very little cytoplasmic contamination as evident from a malate dehydrogenase activity of less than 1% in the apoplastic solution relative to that in bulk leaf extracts. The volume of apoplastic water, a prerequisite for determination of the concentration of apoplastic solutes, was determined by vacuum infiltration of indigo carmine with subsequent analysis of the dilution of the dye in apoplastic extracts. Indigo carmine was neither transported across the cell membrane nor significantly adsorbed to the cell walls, ensuring reproducible (SE < 2%) and precise determination of apoplastic water. Analysis of leaves from four different positions on senescing Brassica napus plants showed a similar apoplastic pH of 5.8, while apoplastic NH4+ increased from 1.1 mM in lower leaves to 1.3 mM in upper leaves. Inhibition of glutamine synthetase in young B. napus plants resulted in increasing apoplastic pH from 6.0 to 6.8 and increasing apoplastic NH4+ concentration from 1.0 to 25.6 mM, followed by a marked increase in NH3 emission. Calculating NH3 compensation points for B. napus plants on the basis of measured apoplastic H+ and NH4+ concentrations gave values ranging from 4.3 to 5.9 nmol NH3 mol-1 air, consistent with an estimate of 5.3 +/- 3.6 nmol NH3 mol-1 air obtained by NH3 exchange experiments in growth chambers. A strong linear relationship was found between calculated NH3 compensation points and measured NH3 emission rates in glutamine synthetase-inhibited plants.

HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 micromolar. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression.

Organophosphorus and carbamate insecticides are toxic to insects and mammals by virtue of their ability to inactivate the enzyme acetylcholinesterase. This review addresses the mechanism of inhibition of acetylcholinesterase by organophosphorus and carbamate esters, focusing on structural requirements necessary for anticholinesterase activity. The inhibition of acetylcholinesterase by these compounds is discussed in terms of reactivity and steric effects. The role of metabolic activation or degradation in the overall intoxication process is also discussed.

Tomato plants (Lycopersicon esculentum, var. Better Boy) were stably transformed with a gene consisting of the open reading frame of a prosystemin cDNA under the regulation of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants constitutively produced proteinase inhibitor I and II proteins, which accumulated over time to levels exceeding 1 mg/g of dry leaf weight. This phenotype contrasts with that of untransformed plants, which produce proteinase inhibitor proteins in leaves only in response to wounding or chemical inducers. The transgenic plants were also stunted, although they appeared normal in all other respects. Grafting the upper half (scion) of an untransformed tomato plant onto the lower half (root stock) of a tomato plant expressing the prosystemin transgene resulted in the constitutive expression of proteinase inhibitor proteins in the leaves of both the transformed root stock and the untransformed scion, demonstrating that expression of the prosystemin transgene generates a mobile wound signal. These results show that systemic signal propagation in the transgenic plants does not require wounding, and they support the proposed role of systemin as the mobile wound signal.

Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300-600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercuribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants.