Synthesis of six novel N,N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae.
Six novel N,N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N,N-dimethyl-N1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride (27) and N,N-dimethyl-N1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride (28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.
Ectopic Expression of Maize Polyamine Oxidase and Pea Copper Amine Oxidase in the Cell Wall of Tobacco Plants1
To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H2O2-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H2O2 only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H2O2 produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.
Inhibition of polyamine metabolism in Phytophthora species
The effects of inhibitors of polyamine metabolism on the growth, ornithine decarboxylase (ODC) and polyamine concentration of Phytophthora infestans have been investigated, and comparisons made with Phytophthora cactorum and Phytophthora nicotianae . The enzyme-activated inhibitor of ODC, difluoromethylornithine (DFMO) at 0·1 m m inhibited the radial growth of P. infestans after 21 d to ca 30% of the water control. Using a homologous series of diamines, (NH 2 (CH 2 ) x NH 2 . 2HCl), full reversal of growth inhibition due to DFMO was achieved when x = 4, 5 and 10; and no reversal was found when x = 3 or 7. Spermidine, spermine and agmatine also fully reversed the inhibition due to DFMO. The inhibitor of arginine decarboxylase, monofluoromethyldehydroarginine, was almost as effective as DFMO as a growth inhibitor. The growth of P. cactorum and P. nicotianae was relatively insensitive to DFMO. The in vitro activity of the ODC was reduced by 0·1 m m DFMO, monofluoromethyldehydroomithine or methylacetylenic putrescine to less than 3% and 5% of the control in P. infestans and P. cactorum , respectively. In P. infestans (grown with or without 1 m m DFMO), putrescine, spermidine or spermine were all less than 10 nmol g −1 fresh weight. However, in P. cactorum , spermidine was present at 1·4 mol g −1 fresh weight, and was reduced to 38% by 1 m m DFMO. The results suggest that the greater growth inhibition in P. infestans caused by the inhibitors of polyamine metabolism may be related to the inherently smaller polyamine concentration, which probably results from the relatively low ODC activity found in this fungus.