Polyamine metabolism in stressed wheat and inhibition of polyamine biosynthesis in fungal pathogens

Polyamine metabolism in wheat responding to biotic and abiotic stress was examined. Putrescine, spermidine and spermine concentrations increased in Puccinia graminis f.sp. tritici-infected first leaves of wheat (Triticum aestivum L. cv. Sappo). However, an examination of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) revealed that, with the exception of day 3 after inoculation, no increase in activity occurred. ADC activity in both healthy and P. graminis-infected plants was very low. On isolation of pustule and inter-pustule regions of infected first leaves, polyamine concentrations were found to be highest in the pustule areas although inter-pustule regions also exhibited increased polyamine concentrations compared with healthy controls. The activity of ODC was higher in both pustule and inter-pustule regions. The effects of infection by P. graminis on polyamine metabolism in second leaves and stems of wheat were variable. Following a brief study of ADC activity in mannitol- treated wheat leaf segments, a detailed examination was made of polyamine metabolism in osmotic and water-stressed wheat plants. In general, the initial studies revealed an increase in ADC activity during osmotic stress. This increase was prevented by the addition of difluoromethylarginine (DFMA) to the incubation mixture. On examination of ADC and ODC activities in whole wheat plants watered with solutions of mannitol, the second leaves exhibited the greatest increase in enzyme activity. Putrescine, spermine and cadaverine concentrations also increased in the second leaves of treated plants. The concentration of polyamines in the first leaves and roots of treated plants varied throughout the experimental period. On examination of the distribution of 14c-mannitol in wheat plants treated with the radioisotope, greatest accumulation was observed in the roots. The first leaves of treated plants, however, contained a higher concentration of 14c-mannitol than the second leaves. Polyamine metabolism in wheat plants water-stressed using polyethylene glycol (PEG) was examined. The activities of ADC and ODC in water-stressed tissue were not significantly different from enzyme activities in control tissues, with the exception of cytosolic ODC activity which decreased in water-stressed roots. The concentrations of putrescine and cadaverine in water-stressed wheat plants were not significantly different when compared with healthy controls. However, reductions in the concentrations of spermidine and spermine in stressed first leaves and spermidine in stressed second leaves were observed, on comparison with control tissue. A detailed examination was made of the growth of the oat-infecting fungus Pyrenophora avenae, grown on solid and liquid media containing the polyamine biosynthesis inhibitors -difluoromethylornithine (DEMO), methyl-glyoxal bis(guanylhydrazone) (MGBG) and ethylmethylglyoxal bis(guanylhydrazone) (EMGBG). All of the compounds inhibited mycelial growth of the fungus. However, MGBG and EMGBG were more effective than DFMO. The addition of exogenous putrescine and spermidine almost completely prevented inhibition of mycelial growth by DFMO, However, no such effect was observed for inhibition by MGBG or EMGBG. In general, the size of the fungal cells was not significantly affected by either the inhibitors or the exogenous polyamines. DFMO and MGBG, alone and in combination, reduced the activity of ODC in P. avenae and, when grown in media containing EMGBG, the fungus showed reduced activity of S-adenosylmethionine decarboxylase (SAMDC). DFMO significantly increased SAMDC activity in P. avenae. The concentrations of putrescine and spermidine decreased when P. avenae was grown in media containing DFMO or DFMO/MGBG combined, while MGBG reduced spermidine and spermine concentrations. All of the compounds reduced the concentration of cadaverine, which is a significant component of P. avenae. The respiration rate of the fungus decreased when grown in media containing MGBG, MGBG/carnitine or DFMO/MGBG combined. The effects of DFMO and MGBG on infection of wheat leaves by P. graminis were also examined. A range of concentrations of DFMO and MGBG, alone and in combination, were applied as pre- and post-inoculation treatments. By the end of the experiment, 17 days after inoculation, pre- and post-inoculation application of the inhibitors had substantially reduced P. graminis infection. The addition of putrescine or spermidine to inhibitor sprays substantially reduced the effects of the inhibitors and in some instances increased the infection above that of the controls. Leaf dry weight was usually reduced in plants exposed to pre- and post-inoculation application of inhibitors, with the greatest reductions normally occurring at the higher inhibitor concentrations. This effect was normally reversed by the addition of polyamines to the inhibitor sprays