1.2.21
EARLY POLYAMINE ACCUMULATION DURING TMV-INDUCED HYPERSENSITIVE REACTION IN N-TOBACCO PLANTS

F MARINI1, AL RABITI1, L BETTI1, C BORTOLOTTI2, A CANOVA1, N BAGNI2 and P TORRIGIANI2

1UCI-STAA - Instituto di Patologia Vegetale, Universia degli Studi, Bologna, Italy; 2Dipartimento di Biologia e.s., Universita degli Studi, Bologna, Italy

Background and objectives
Aliphatic polyamines (putrescine, spermidine and spermine) are involved in cellular proliferation and growth in living organisms and are intrinsic components of several viruses. Their mechanism of action at the molecular level remains unclear although, due to their positive charges, they interact with macromolecules such as DNA and RNA. Putrescine, the precursor of the higher polyamines spermidine and spermine, is synthesized in plants from the amino acids arginine and ornithine, via arginine (ADC) and ornithine-decarboxylase (ODC) activities. In several higher plants, especially in the Solanaceae, polyamines can form mono- and di-substituted basic amides with hydroxycinnamic acids, which are metabolic intermediates of phenylpropanoid synthesis. In fact, polyamine hydroxycinnamoyl amides (HCAS) have been reported to be involved in defence mechanisms in several plant/pathogen interactions [1]. In a previous paper, Torrigiani et al. [2] reported that during the hypersensitive reaction (HR; 1, 3 and 5 days from virus inoculation) induced by tobacco mosaic virus (TMV) in Nicotiana tabacum cv. Samsun NN (hypersensitive), free and conjugated polyamine accumulation displayed an increasing concentration gradient towards the hypersensitive lesion. This increase was accompanied by enhanced ADC and especially ODC activities. By contrast, in TMV-infected Samsun nn plants (susceptible), polyamines tended to remain in the free state instead of being conjugated as infection progressed. These findings suggest that polyamine conjugates (HCAS) are involved in the establishment of the HR and thus in virus resistance.

In order to gain a deeper insight into the N-mediated resistance to TMV in tobacco, changes in polyamine metabolism (free and conjugated polyamine levels, putrescine biosynthesis, evaluated through ADC and ODC, and oxidation via diamino oxidase activity, DAO) during the first 24 h from virus inoculation, in both hypersensitive and susceptible Samsun plants, were investigated. Results were compared with those obtained in mock-inoculated control plants.

Results and conclusions
In NN TMV-inoculated plants, which undergo the HR, free putrescine and spermidine levels increased after 5 h compared to controls in which no relevant variations occurred; polyamine conjugates also exhibited an increasing trend compared to controls. In both virus and mock-inoculated plants, ADC and ODC activities generally increased while DAO activity, which was present in controls, was detectable only in trace amounts in inoculated tissues. An opposite trend in polyamine accumulation was observed in TMV-inoculated susceptible tobacco plants. In fact, both free and conjugated polyamine titres decreased, starting 5 h after inoculation. No differences were detected in ADC and ODC activities while DAO activity was absent. Susceptible plants seem to be unable to synthesize and/or accumulate polyamines in response to TMV infection, at least within the first 24 h. These results further support the idea that polyamines play a role in TMV-induced resistance. Moreover, they may be regarded as a contribution to the study of programmed cell death (PCD) since HR is now recognized as a form of PCD.

References
1. Walters DR, Mackintosh CA, 1997. Physiologia Plantarum 100, 689-695.
2. Torrigiani P, Rabiti AL, Bortolotti C et al., 1997. New Phytologist 135, 467-473.