Structure, expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leaves.

The primary leaves of young barley seedlings contain two major, extracellular, acid-soluble proteins of ca. 22 and 23 kDa apparent molecular mass. These proteins disappeared from the intercellular washing fluid upon stress treatments that enhanced H2O2 levels and that induced resistance to subsequent challenge by the powdery mildew fungus Erysiphe graminis f. sp. hordei. A partial peptide sequence of the 22 kDa protein was determined, and a cDNA clone was isolated. The 22 kDa protein belongs the the group of germin-like proteins (GLPs) and was designated HvGLP1. Despite its similarity to germin, i.e. oxalate oxidase, no oxalate oxidase activity of HvGLP1 could be detected. The RNA and soluble protein of HvGLP1 was highly abundant in young leaves, less abundant in older leaves and absent in roots. HvGLP1 RNA oscillated with a circadian rhythm, the minimum and maximum of RNA abundance being at the end of the dark and light periods, respectively. Heat and H2O2 treatment as well as pathogen infection caused disappearance of HvGLP1 protein from the fraction of soluble proteins of the intercellular space. HvGLP1 protein could be re-solubilized from cell walls of heat- or H2O2-treated leaves by boiling in SDS suggesting non-covalent cross linking. Although a physiological role of HvGLP1 insolubilization is still open, the protein may serve as marker for oxidative stress in cereals.

NMR solution structure of the pathogenesis-related protein P14a.

The nuclear magnetic resonance (NMR) structure of the 15 kDa pathogenesis-related protein P14a, which displays antifungicidal activity and is induced in tomato leaves as a response to pathogen infection, was determined using 15N/13C doubly labeled and unlabeled protein samples. In all, 2030 conformational constraints were collected as input for the distance geometry program DIANA. After energy-minimization with the program OPAL the 20 best conformers had an average root-mean-square deviation value relative to the mean coordinates of 0.88 A for the backbone atoms N, C(alpha) and C', and 1.30 A for all heavy atoms. P14a contains four alpha-helices (I to IV) comprising residues 4 to 17, 27 to 40, 64 to 72 and 93 to 98, a short 3(10)-helix of residues 73 to 75 directly following helix III, and a mixed, four-stranded beta-sheet with topology +3x, -2x, +1, containing the residues 24-25, 53 to 58, 104 to 111 and 117 to 124. These regular secondary structure elements form a novel, complex alpha + beta topology in which the alpha-helices I, III and IV and the 3(10)-helix are located above the plane defined by the beta-sheet, and the alpha-helix II lies below this plane. The alpha-helices and beta-strands are thus arranged in three stacked layers, which are stabilized by two distinct hydrophobic cores associated with the two layer interfaces, giving rise to an "alpha-beta-alpha sandwich". The three-dimensional structure of P14a provides initial leads for identification of the so far unknown active sites and the mode of action of the protein, which is of direct interest for the generation of transgenic plants with improved host defense properties.

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

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. infestans 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-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

Initiator-dependent transcription in vitro by a wheat germ chromatin extract.

The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.

[beta]-Aminobutyric Acid Induces the Accumulation of Pathogenesis-Related Proteins in Tomato (Lycopersicon esculentum L.) Plants and Resistance to Late Blight Infection Caused by Phytophthora infestans.

Tomato (Lycopersicon esculentum L.) plants were sprayed with aqueous solutions of isomers of aminobutyric acid and were either analyzed for the accumulation of pathogenesis-related (PR) proteins or challenged with the late blight fungal agent Phytophthora infestans. The [beta] isomer of aminobutyric acid induced the accumulation of high levels of three proteins: P14a, [beta]-1,3 glucanase, and chitinase. These proteins either did not accumulate or accumulated to a much lower level in [alpha]- or [gamma]-aminobutyric acid-treated plants. Plants pretreated with [alpha]-, [beta]-, and [gamma]-aminobutyric acid were protected up to 11 d to an extent of 35, 92, and 6%, respectively, against a challenge infection with P. infestans. Protection by [beta]-aminobutyric acid was afforded against the blight even when the chemical was applied 1 d postinoculation. Examination of ethylene evolution showed that [alpha]-aminobutyric acid induced the production of 3-fold higher levels of ethylene compared with [beta]-aminobutyric acid, whereas [gamma]-aminobutyric acid induced no ethylene production. In addition, silver thiosulfate, a potent inhibitor of ethylene action, did not abolish the resistance induced by [beta]-aminobutyric acid. The results are consistent with the possibility that [beta]-aminobutyric acid protects tomato foliage against the late blight disease by a mechanism that is not mediated by ethylene and that PR proteins can be involved in induced resistance.

Effect of Jasmonic Acid on the Interaction of Barley (Hordeum vulgare L.) with the Powdery Mildew Erysiphe graminis f.sp. hordei.

Jasmonic acid or its methyl ester induce de novo synthesis of a number of proteins of mostly unknown function in barley (Hordeum vulgare L.). In a topical spray application, 30 [mu]g of jasmonic acid per plant effectively protected barley against subsequent infection by Erysiphe graminis f.sp. hordei. Examination of jasmonic acid-induced barley proteins revealed the presence of several acid-soluble (pH 2.8) proteins. Two prominent groups of 25 kD and 10 to 12 kD apparent molecular mass were present in the intercellular washing fluid. The set of extracellular, induced proteins showed no similarity to barley pathogenesis-related proteins. An in vivo test against E. graminis revealed no antifungal activity of the extracellular jasmonic acid-induced proteins. Experiments with the transcription inhibitor cordycepin showed no correlation between accumulation of jasmonic acid-induced proteins and protection. The application of jasmonic acid and E. graminis simultaneously resulted in independent extracellular accumulation of both jasmonic acid-induced proteins and of pathogenesis-related proteins. The data suggest that jasmonic acid directly inhibits appressoria differentiation of the fungus, and that it is not involved in the signal transduction mechanism leading to induction of pathogenesis-related proteins.

cDNA cloning, in vitro transcription and partial sequence analysis of mRNAs from winter wheat (Triticum aestivum L.) with induced resistance to Erysiphe graminis f. sp. tritici.

Under normal growth conditions wheat shows 100% non-host resistance to the barley powdery mildew Erysiphe graminis f. sp. hordei (Egh.). Primary inoculation of 7-day-old wheat seedlings with this fungus induced partial (60-70%) local resistance to challenge inoculation 12 hours later with the compatible pathogen Erysiphe graminis f. sp. tritici (Egt). mRNA was isolated from induced resistant first leaves (13 hours after primary inoculation) and a cDNA library was established in lambda ZAP. Differential screening of the library with sDNA probes (from induced resistant versus non-inoculated plants) resulted in isolation of 6 cDNA clones corresponding to 6 different induced, plant-encoded mRNA species. Hybridization of in vitro transcripts derived from wheat nuclei to cDNA dot blots showed that the transcription of these genes was induced rapidly, 3 hours after inoculation with either Egt or Egh. At this time point neither fungus had formed appressorial germ tubes yet. When induced resistant first leaves were challenged with the compatible pathogen (Egt), transcription of the host genes was enhanced a second time. No difference in kinetics of induction of transcription could be observed between noninduced and induced resistant leaves. One of the cloned induced mRNAs codes for a peroxidase, as shown by cDNA derived partial peptide sequence analysis. Peroxidase activity increased in intercellular washing fluids of first leaves from 6 to 36 hours after inoculation.

Phytochrome regulation of greening in barley : effects on mRNA abundance and on transcriptional activity of isolated nuclei.

Red light treatment of etiolated barley (Hordeum vulgare L.) seedlings causes an increase in the relative abundance of the mRNA for light-harvesting, chlorophyll a/b-binding polypeptides, and a decrease in the relative abundance of the mRNA for the NADPH:protochlorophyllide oxidoreductase. It also increases transcriptional activity of subsequently isolated nuclei for the mRNA for the chlorophyll-binding polypeptides and reduces it for the reductase. These results confirm those published previously. Fluence-response and kinetic studies support the hypothesis that the abundance of mRNA for the chlorophyll-binding polypeptides may be transcriptionally limited. They do not support the same hypothesis for the reductase. Red light treatments in the very low fluence range significantly decrease transcriptional activity in isolated nuclei for RNAs hybridizable by the reductase probe, but have little effect on mRNA abundance. By contrast, red light treatments in the low fluence range bring about a sharp decrease in reductase mRNA abundance with little further effect on transcription, suggesting light regulation at the level of mRNA stability rather than transcription. The fluence-response relationships for increase in abundance of mRNA for the chlorophyll-binding polypeptides is similar to that published elsewhere for elimination of the lag period of chlorophyll accumulation in barley. However, kinetic studies published elsewhere show that for elimination of the lag in chlorophyll accumulation, the dependence on the far red-absorbing form of phytochrome concentration is significantly different from the dependence of the transcriptional changes, suggesting that although transcription might be what limits mRNA abundance for the chlorophyll-binding proteins, the mRNA abundance can not be what limits chlorophyll accumulation per se.

Phytochrome regulation of greening in barley-effects on chlorophyll accumulation.

Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence-response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far red-absorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure.

Comparison of the effects of exogenous native phytochrome and in-vivo irradiation on in-vitro transcription in isolated nuclei from barley (Hordeum vulgare).

In barley seedlings the transcription of genes coding for the light-harvesting chlorophyll a/b protein (LHCP) is stimulated and the transcription of genes coding for the NADPH-protochlorophyllide oxidoreductase (reductase) is repressed by light working via the phytochrome system. This phytochrome-mediated control of gene expression has been studied by monitoring in-vitro transcription in isolated nuclei. Two different experimental approaches have been used to elucidate the function of phytochrome (Pfr) during the transduction of the light signal. Concentrations of phytochrome were varied experimentally either by illuminating intact plants or macerated plant material prior to the isolation of nuclei or by adding purified phytochrome (Pfr) in its native 124-kDa form to the isolated nuclei. Our results indicate that there are at least two different steps involved in the phytochrome control of specific gene expression. (i) There is a rapid and transient change in the transcription rate which is saturated by very low levels of Pfr. (ii) There is a change in the duration and the maximum range of the transient change; this step requires relatively high Pfr concentrations and thus reacts very sensitively and rapidly to changes in Pfr levels as induced by secondary irradiations. This second step, but not the first one, could be triggered by the addition of purified oat phytochrome to a reconstituted nuclear system. This effect of purified phytochrome could only be shown if nuclei isolated from red-light (R)-irradiated seedlings were used. It was thus possible to simulate the effect of an in-vivo-applied second R pulse by the addition of Pfr to nuclei isolated from R-preirradiated plants.

The implication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll a/b protein.

In carotenoid-deficient albina mutants of barley and in barley plants treated with the herbicide Norflurazon the light-dependent accumulation of the mRNA for the light-harvesting chlorophyll a/b protein (LHCP) is blocked. Thus, the elimination of a functional chloroplast, either as a result of mutation or as a result of herbicide treatment, can lead to the specific suppression of the expression of a nuclear gene encoding a plastid-localized protein. These results confirm and extend earlier observations on maize [Mayfield and Taylor (1984) Eur. J. Biochem. 144, 79-84]. The inhibition of mRNA accumulation appears to be specific for the LHCP; the mRNAs encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the NADPH: protochlorophyllide oxidoreductase are relatively unaffected. The failure of the albina mutants and of Norflurazon-treated plants to accumulate the LHCP mRNA is not exclusively caused by an instability of the transcript but rather by the inability of the plants to enhance the rate of transcription of the LHCP genes during illumination. Several chlorophyll-deficient xantha mutants of barley, which are blocked after protoporphyrin IX or Mg-protoporphyrin, and the chlorophyll-b-less mutant chlorina f2 accumulate the LHCP mRNA to almost normal levels during illumination. Thus, if any of the reactions leading to chlorophyll formation is involved in the control of LHCP mRNA accumulation it should be one between the formation of protochlorophyllide and the esterification of chlorophyllide a. While the nature of the regulatory factor(s) has not been identified our results suggest that, in addition to phytochrome (Pfr), plastid-dependent factors are required for a continuous light-dependent transcription of nuclear genes encoding the LHCP.

Phytochrome control of in vitro transcription of specific genes in isolated nuclei from barley (Hordeum vulgare).

The transcriptional rates of four different genes in shoots of barley grown under different light regimes were quantified by monitoring nuclear RNA transcripts using gene-specific hybridization probes. Isolated nuclei were pulse-labelled with [alpha-32P]UTP and the relative rates of light-harvesting chlorophyll a/b protein (LHCP) mRNA, NADPH:protochlorophyllide oxidoreductase mRNA, B1 hordein mRNA, and 26-S rRNA synthesis were measured. Irradiation of dark-grown plants with a red light pulse increased the rate of LHCP mRNA synthesis tenfold within 3 h, and the rate of rRNA synthesis more than twofold within 9 h. The relative rate of synthesis of the oxidoreductase mRNA decreased following a red light pulse reaching a minimum after 3-6 h. As a direct proof of phytochrome involvement in the light-induced stimulation of LHCP and the repression of the oxidoreductase transcripts for both responses, red/far-red reversibility could be demonstrated. We conclude that phytochrome is able both to increase the transcription of certain nuclear genes and decrease the transcription of others.

In-vivo phytochrome control of in vitro transcription rates in isolated nuclei from oat seedlings.

The transcription rate of isolated nuclei from oat seedlings was analysed by measuring the incorporation of radioactive label in trichloroacetic-acid-pelletable material. It was observed that this transcription rate depends on the light pretreatment of the plants: 7.5 h after a 5-min red or far-red light pulse maximum rates were found, but at this time far-red light induced only about half of the stimulation observed after a red light pulse. The far-red reversibility of the effect of the red light pulse indicates that phytochrome controls the capacity for transcription by isolated nuclei. Besides this slow reaction, phytochrome regulated the transcription rates in a very fast response when homogenates were irradiated.

Polysome assembly and RNA synthesis during phytochrome-mediated photomorphogenesis in mustard cotyledons.

The effect of phytochrome (high irradiance reaction, elicited by continuous far-red light) on cellular polysome levels was investigated using ribosome-isolation procedures which prevent the methodological artifacts inherent in previous studies on polysomes. By including the large pool of ribosomal subunits in the analysis and using the ratio (polysomes: monomers + subunits) as a quantitative estimate of the translational capacity of the ribosomes in mustard (Sinapis alba L.) cotyledons, we found the following results: 1) After a lag-phase of less than 30 min, phytochrome induces a massive increase in the relative amount of cytosolic (free) polysomes at the expense of ribosomal subunits. 2) Cytosolic and membrane-bound polysomes are increased by phytochrome in constant proportions (constant ratio of 65:35 in light and darkness). 3) Simultaneously with the light-mediated increase of the polysome level there is an increased incorporation of newly synthesized (labeled) non-ribosomal RNA, presumably mRNA, into the polysomes which can be kinetically discriminated from the slower incorporation of newly synthesized (labeled) rRNA. 4) Cordycepin strongly inhibits the synthesis of RNA and completely prevents the light-mediated increase of polysomes. 5) The electrophoretic patterns of the in-vitro translation products obtained with polysomal polyadenylated RNA from dark-grown and light-grown cotyledons showed no significant qualitative differences. We conclude from these results that photomorphogenesis of mustard cotyledons is related to a massive increase of newly synthesized mRNA leading to a correspondingly increased recruitment of ribosomal subunits into polysomes. The phytochrome-induced increase of translatable mRNA involves mainly quantitative changes in the production of mRNA species which are also present in the dark-grown cotyledons.

Evidence against the involvement of DNA synthesis in phytochrome-mediated photomorphogenesis.

In order to clarify the relationship between photomorphogenesis and DNA replication we investigated the effect of continuous far-red or white light on the synthesis of DNA in the cotyledons and the hypocotyl of mustard seedlings between 36 and 108 h after sowing. The total DNA content of the cotyledons (about 2.2 pg cell(-1)) did not significantly change during this period although long-term labeling experiments revealed newly synthesized DNA of nuclear, plastid, and mitochondrial origin. Light had no detectable effect on total DNA content and on the labeling of either DNA fraction. Histoautoradiography indicated that nuclear DNA synthesis was exclusively localized in dividing stomatal cells and in sieve tube companion cells undergoing endopolyploidization. The DNA content of the hypocotyl increased continuously but likewise showed no detectable effect of light. It is concluded that cell growth and differentiation during photomorphogenesis is independent of DNA synthesis.

Impairment of renal function in rats with generalized lipidosis as induced by chlorphentermine.

The purpose of this work was to study the impact of experimentally induced lipidosis upon renal function. Rats were orally treated up to 12 weeks with the anorectic drug chlorphentermine at dosages of 20 and 50 mg/kg, respectively. During the course of treatments the following parameters were determined: plasma urea level, creatinine clearance, ability of the kidneys to concentrate the urine after acute deprivation of drinking water, and to dilute the urine after acute water load. Morphological examination confirmed generalized lipidosis affecting kidney and many other organs. Chronic treatments with both drug dosages caused a rise of plasma urea level. Treatment with the high dosage caused a significant reduction of creatinine clearance and significant impairment of both abilities to concentrate and to dilute the urine. The findings demonstrate that serve generalized lipidosis as induced by high doses of chlorphentermine is associated with significant impairment of renal function.

Pharmacokinetic studies of tobramycin and gentamicin.

Broth dilution susceptibility tests of 100 isolates of Pseudomonas aeruginosa and 101 isolates of Staphylococcus aureus against tobramycin (formerly nebramycin factor 6) and gentamicin showed that tobramycin was more effective against P. aeruginosa and less effective against S. aureus. The minimal inhibitory concentration of tobramycin against the Pseudomonas sp. isolates that required 5 mug of gentamicin per ml for inhibition ranged from 0.63 to 0.31 mug/ml. Peak concentrations in the blood of 10 healthy adults after intramuscular injection of 80 and 40 mg of tobramycin averaged 3.7 +/- 0.62 and 2.4 +/- 0.27 mug/ml, and declined to 0.56 +/- 0.05 and 0.26 +/- 0.02 mug/ml, respectively, after 6 h. The urine recovery averaged 60%. The half-life was 1.6 h. During continuous intravenous infusion of tobramycin and gentamicin (infusion rate 6.6 mg per h), blood levels at steady state were 0.94 +/- 0.10 and 1.04 +/- 0.06 mug/ml, respectively. For both antibiotics, the calculated distribution volume ranged from 15 to 17 liters. The renal clearance to tobramycin averaged 76% and that of gentamicin averaged 85% of the total clearance, indicating that the drugs are primarily eliminated by the kidneys. The present results suggest that tobramycin may be more successful in the treatment of Pseudomonas infections than gentamicin at the same dosage (80 mg intramuscularly three to four times daily).