Greenhouse Studies Reveal Increased Aggressiveness of Emergent Canadian Fusarium graminearum Chemotypes in Wheat.

The role of Fusarium graminearum trichothecene-chemotypes in disease outcomes was evaluated by point inoculation in a series of wheat lines with different levels of resistance to Fusarium head blight (FHB). Four inocula, each consisting of a composite of four strains with either 15-acetyldeoxynivalenol (ADON) chemotypes from "traditional" or emergent populations, a 3-ADON chemotype, or a nivalenol (NIV) chemotype, were compared. The evaluated wheat included Canadian lines with different levels of FHB resistance/susceptibility and double haploid lines developed from crosses of these lines. Highly resistant lines were resistant to infection by all of the F. graminearum chemotypes evaluated. In the moderately susceptible/resistant wheat lines, the 3-ADON producers and the emergent 15-ADON population were, in some instances, more aggressive and resulted in higher Fusarium damaged kernel scores and levels of trichothecene accumulation. The data presented in this study demonstrate the importance of growing highly resistant wheat cultivars in the current climate of an evolving F. graminearum population, and suggest that moderate levels of FHB resistance may not be sufficient to minimize trichothecene contamination of grain from F. graminearum-infected wheat.

Cellular machinery of wood production: differentiation of secondary xylem in Pinus contorta var. latifolia.

The objectives of this study were to define cell structure during pine secondary xylem development and to integrate this information with current knowledge of the biochemistry and physiology of secondary cell wall biosynthesis in gymnosperms. Lodgepole pine (Pinus contorta var. latifolia Englem.) cambium and secondary xylem were cryofixed using high pressure freezing and freeze-substitution which allowed excellent preservation of the cell structure of developing secondary xylem and enabled high-resolution transmission electron microscopic viewing of these cells for the first time. In contrast to their precursors in the adjacent cambial zone, developing tracheids were active in secondary wall deposition, with abundant cortical microtubules and developing bordered pits. These cells were also characterized by unusual Golgi structures: the trans-Golgi network was highly developed and the associated vesicles were large and darkly stained. These unusual Golgi structures persisted throughout the period of xylem maturation until programmed cell death occurred. Immuno-cytochemistry and enzyme-gold probes were used to investigate the distribution of key secretory products (mannans) and a lignification-associated enzyme (coniferin beta-glucosidase) during xylogenesis. Mannans were localized to the secondary cell wall, the trans-Golgi cisternae and trans-Golgi network vesicles of developing xylem. Coniferin beta-glucosidase was found only in the secondary cell wall. The cell wall localization of coniferin beta-glucosidase, the enzyme responsible for cleaving glucose from coniferin to generate free coniferyl alcohol, provides a mechanism to de-glucosylate monolignols in muro. A two-step model of lignification of conifer tracheids is proposed. First, Golgi-mediated secretion deposits monolignols into the cell wall, where they polymerize in cell corners and middle lamella. Secondly, cell lysis releases stored, vacuolar monolignol glucosides into the wall where they are deglucosylated and their polymerization is influenced by the wall environment including the lignin deposited earlier.

Induction of phenylalanine ammonia-lyase activity by tryptophan in Ustilago maydis.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.

The phenylalanine ammonia-lyase gene family in raspberry. Structure, expression, and evolution.

In raspberry (Rubus idaeus), development of fruit color and flavor are critically dependent on products of the phenylpropanoid pathway. To determine how these metabolic functions are integrated with the fruit ripening program, we are examining the properties and expression of key genes in the pathway. Here, we report that L- phenylalanine ammonia-lyase (PAL) is encoded in raspberry by a family of two genes (RiPAL1 and RiPAL2). RiPAL1 shares 88% amino acid sequence similarity to RiPAL2, but phylogenetic analysis places RiPAL1 and RiPAL2 in different clusters within the plant PAL gene family. The spatial and temporal expression patterns of the two genes were investigated in various vegetative and floral tissues using the reverse transcriptase competitor polymerase chain reaction assay. Although expression of both genes was detected in all tissues examined, RiPAL1 was associated with early fruit ripening events, whereas expression of RiPAL2 correlated more with later stages of flower and fruit development. Determination of the absolute levels of the two transcripts in various tissues showed that RiPAL1 transcripts were 3- to 10-fold more abundant than those of RiPAL2 in leaves, shoots, roots, young fruits, and ripe fruits. The two RiPAL genes therefore appear to be controlled by different regulatory mechanisms.

Cloning and disruption of a phenylalanine ammonia-lyase gene from Ustilago maydis.

The enzyme L-phenylalanine ammonia-lyase (PAL) catalyzes the non-oxidative deamination of L-phenylalanine to form trans-cinnamic acid and ammonia. This enzyme is universally present in higher plants and it catalyzes the starting reaction for a central pathway that generates hundreds of different phenylpropanoid metabolites. Genes encoding PAL have been identified in fungi, but the role of the enzyme has not been determined. We cloned and characterized a gene that encodes PAL from the phytopathogenic fungus Ustilago maydis and we constructed fungal strains carrying a null mutation in the gene. These mutants behaved like wild-type strains in terms of growth, mating, and pathogenicity. These results indicate that PAL does not play a major role in the life cycle of U. maydis under laboratory conditions.

Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase.

Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa x P. deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4H expression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta.

Ozone treatment rapidly activates MAP kinase signalling in plants.

Brief exposure to ozone, a potent cross-inducer of plant stress responses, leads within minutes to activation of an ERK-type MAP kinase (approximately 46 kDa) in tobacco. This activation process is calcium-dependent and can be blocked both by free radical quenchers and by a specific inhibitor of MEK-1 (MAPKK). Hydrogen peroxide and superoxide anion radicals can substitute for ozone as the activation stimulus, which does not appear to require salicylate as an intermediary. The properties of the ozone-induced MAPK suggest that it may be SIPK (salicylate-induced protein kinase), a tobacco MAPK that is activated by a variety of stress treatments. The ability of ozone to activate SIPK indicates that this protein kinase acts as a very early transducer of redox stress signals in plant cells.

U.S. Air force recruit injury and health study.

OBJECTIVES: To assess the types, rates, and risks of injury for male and female USAF recruits. DESIGN: Outpatient visits for female (5250) and male recruits (8656) were collected and analyzed for rate of injury, types of injuries, and risk of injury throughout a 6-week training period. RESULTS: One third of female recruits and one sixth of male recruits were injured at least once during recruit training. The overall rate for injuries in women and men was 63.0 and 27.8 per 1000 person-weeks, respectively. The adjusted relative risk for women versus men for all injuries was 2.22, and was consistent (1.67 to 3.27) across injury sites. Despite declining absolute rates of injury by week (106.1-13.4 for women and 53.7-13.2 for men), relative risk of injury for women versus men remained fairly constant throughout each training week. The relative risk for injury serious enough to result in medical hold was 1.69 for women vis-a-vis men. Approximately half of all medical discharges for women and men were for injuries. CONCLUSIONS: Female recruits were injured twice as often as male recruits, and were 1.5 times more likely to be removed from their training cohort for injury. Relative risk for injuries to specific body areas remained fairly consistent, indicating that no gender-specific injuries were occurring. Further efforts to determine the cause of injuries should be undertaken, and interventions aimed at reducing the disparate risk of injuries in women should be developed and evaluated.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection.

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the "oxidative burst," they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.

Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue.

The site of phosphorylation of phenylalanine ammonia-lyase (PAL) has been identified as a threonine residue. A Ca(2+)-stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease in Vmax in agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545 in the sequence VAKRTLTT (539-546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants.

cDNA cloning and heterologous expression of coniferin beta-glucosidase.

Coniferin beta-glucosidase (CBG) catalyzes the hydrolysis of monolignol glucosides to release the cinnamyl alcohols for oxidative polymerization to lignin. Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus contorta xylem-specific library. The isolated 1909 nucleotide cDNA was confirmed to be that of CBG on the basis of its high homology to family 1 glycosyl hydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate specificity profile displayed by the recombinant protein when expressed in Escherichia coli. The presence of a 23 amino acid N-terminal signal peptide in the deduced 513 amino acid enzyme suggests that CBG is a secretory protein targeted to the ER. The isolation of CBG cDNA will facilitate the evaluation of the importance of this enzyme in the ultimate stages of lignin biosynthesis and could be a valuable tool in manipulating lignin levels in xylem cell walls.

A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell cultures.

Using degenerate PCR primers that target evolutionarily conserved sequences in pal genes, we show that in the gymnosperm, Pinus banksiana, phenylalanine ammonia-lyase (PAL) is encoded by a multigene family of at least eight to ten loci. Five classes of pal sequence were easily distinguished among 28 clones sequenced from the products of PCR amplification of haploid genomic DNA. The dominant sequence from each class was named, yielding pal1 to pal5 loci. These genes shared 68.8% to 94.0% nucleotide identity over the 366 bp region compared. All of pal1 to pal5 were expressed in cell suspension cultures treated with a fungal elicitor and all but pal3 were expressed in differentiating xylem tissue of a mature tree. Only pall was expressed in unelicited cell cultures. While these P. banksiana genes are quite divergent, they are still more similar to each other than to any angiosperm pal gene cloned to date. For its roles in development and defense, PAL production in P. banksiana is coordinated from a large, diverse multigene family. We discuss evidence suggesting that other pines have similar pal gene family structures.

4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes.

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa x Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

Pre-activating wounding response in tobacco prior to high-level ozone exposure prevents necrotic injury.

In tobacco, both wounding and treatment with jasmonates prior to exposure of the tissue to high concentrations of ozone (250 to 500 p.p.b.) produce a dramatic decrease in ozone injury. A systemic pattern of increased ozone tolerance developed within 3-6 h after wounding and also after local application of jasmonates. Ozone treatment of transgenic (NahG) tobacco plants showed that the inability of these plants to accumulate salicylic acid is also accompanied by increased ozone tolerance. Expression of mRNA encoding the anti-oxidant enzyme ascorbate peroxidase is upregulated by ozone challenge, wounding and by methyl jasmonate exposure within 3-4 h, while levels of carbonic anhydrase mRNA are simultaneously depressed following ozone exposure and methyl jasmonate treatment. The pattern of these results shows that the response to ozone challenge in tobacco involves signalling mechanisms similar to those induced in plants by other environmental stresses that generate reactive oxygen species.

Expression of poplar phenylalanine ammonia-lyase in insect cell cultures.

A cDNA encoding one of the phenylalanine ammonia-lyase genes from Populus trichocarpa x deltoides was inserted into a baculovirus expression vector and the PAL protein was successfully expressed in insect cell cultures. High levels of active holoenzyme were obtained that could be purified in a single chromatographic step. Site-directed mutagenesis and expression of the mutant enzyme confirmed that conversion of the putative active site serine202 residue to alanine is sufficient to destroy the catalytic activity of PAL.

A beta-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin.

Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.

Risk of developing life-threatening ventricular arrhythmia associated with tefenadine in comparison with over-the-counter antihistamines, ibuprofen and clemastine.

An observational, historical cohort evaluation was performed to examine the hypothesis that terfenadine (Seldane) exposure increases the risk of developing life-threatening ventricular arrhythmias. The study population consisted of Medicaid recipients from 4 states that were included in the Computerized On-Line Medical Pharmaceutical Analysis and Surveillance System (COMPASS). The drug exposure period was defined prospectively as 30 days in all treatment cohorts. The primary end point was the development of life-threatening ventricular arrhythmias (ventricular tachycardia, fibrillation and flutter, and cardiac arrest and sudden death). The comparison cohorts included terfenadine (n = 181,672), over-the-counter antihistamines (n = 150,689), ibuprofen (n = 181,672) and clemastine (Tavist; n = 83,156). Over the exposure period, a total of 317 life-threatening ventricular arrhythmic events occurred, 244 of which were cardiac arrests. The incidence of total life-threatening ventricular arrhythmic events and cardiac arrests were more frequent in patients receiving over-the-counter antihistamines (relative risk 0.36) than in those receiving terfenadine, a finding that was consistent across all subgroups. There was no increased risk of life-threatening ventricular arrhythmias in the terfenadine cohort as compared with the ibuprofen cohort (relative risk 0.62), and in some analyses, the ibuprofen cohort had a significantly higher arrhythmic event rate. In all comparisons with the clemastine cohort, the terfenadine cohort had a statistically indistinguishable relative risk (1.08). Age, race, sex and cardiovascular risk were all considered in the adjusted relative-risk analyses. No baseline historical characteristic or imbalance of baseline medications explained the differences between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures.

A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both (14)C-phenylalanine and (14)C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.