Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn²⁺ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C₅-precursors into phytoalexin biosynthesis. Received: 23 August 1996 / Accepted: 20 March 1997     

Discrimination between cultivars of Vitis vinifera based on molecular variability concerning 5″ untranslated regions of the StSy-CHS genes

 The degree of polymorphism present in 5′ untranslated regions of stilbene synthase (StSy)-like loci was assessed. A ligation-mediated polymerase chain reaction (LM-PCR) cloning strategy was adopted to isolate sequences located immediately upstream of StSy coding regions. Among several clones, 13 randomly chosen fragments were analyzed at the sequence level. Four of the analyzed fragments appeared of particular interest. Two carried sequences reminiscent of micro-satellites, while the remaining fragments contained direct repeats. Oligonucleotides constructed against the specific DNA sequence of these clones disclosed a complex banding pattern when used in polymerase chain reaction (PCR)-analysis of 22 ancient varieties of grapevine. A total of 40 polymorphic bands could be identified and used to calculate coefficients of genetic similarity (GS) between varieties. GS values were used in cluster analysis to differentiate the 22 varieties. The data obtained are in good agreement with available information concerning the relationships between the varieties considered. This suggests the use of the method we have developed in fingerprinting studies of Vitis vinifera germ plasma. Received: 11 April 1996 / Accepted: 14 March 1997     

The cryptochrome family of photoreceptors

CRY1, the primary photoreceptor responsible for blue light-mediated inhibition of hypocotyl elongation in Arabidopsis thaliana, has been characterized. The properties of CRY1, and those of the related protein CRY2, and their relationship to the photolyase family of flavoproteins are discussed.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Influence of Hyperthyroidism on the Activity of Liver Nitric Oxide Synthase in the Rat

Hyperthyroidism enhances the prooxidant activity of the liver by elevating superoxide radical and/or hydrogen peroxide generation in microsomal, mitochondrial, and peroxisomal fractions, with an increased respiratory burst of Kupffer cells. In this study, the influence of daily doses of 0.1 mg 3,3′,5-triiodothyronine (T₃)/kg for three consecutive days on liver nitric oxide (NO) synthase (NOS) was assessed, as a possible contributory mechanism to T₃-induced liver prooxidant activity. Thyroid calorigenesis was paralleled by a progressive increment in the rate of NO generation, with significant increases after 2 (47%) and 3 days (70%) of T₃treatment, and a net 45% (P< 0.05) enhancement in theN^G-methyl-l-arginine-sensitive NO production, compared to control values. These enhancement effects were reversed to control levels after 3 days of hormone withdrawal, concomitantly with the normalization of hepatic respiration. Enhancement of liver NOS activity in hyperthyroid animals was diminished by 27% (P< 0.05) by the selectivein vivoinactivation of Kupffer cells by gadolinium chloride (GdCl₃), without direct actions of GdCl₃on the enzyme. These data demonstrate that hyperthyroidism leads to a significant and reversible enhancement in rat liver NOS activity, an effect that is exerted at hepatocyte and Kupffer cell levels, thus representing an additional source of prooxidants to those of reactive oxygen species.