1. The mechanism of toxicity of Norspermidine in Arabidopsis Sayas E 1*, Alabadí D 1, del Pozo JC 2, Ferrando A 1, Serrano R 1 1 Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C 2 Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid *ensamon@upvnet.upv.es Polyamines are small polycationic molecules found in all organisms where they play essential functions not well understood. On the other hand, high concentrations of polyamines are toxic by mechanisms also unknown. The uncommon polyamine norspermidine (NE) has been used as toxic cation to select resistant mutants affected in cation homeostasis (Alejandro et al, 2007, EMBO J 26: 3203) and therefore it is important to know the mechanisms of NE toxicity. This is also important to ascertain the use of NE as anti-tumoral drug (Prakash et al, 1988, Anticancer Res 8: 563). NE treatment triggers a transcriptional response similar to that observed when cytosolic misfolded proteins accumulate. Accordingly, seedlings treated with NE have increased levels of insoluble proteins. This toxic effect can be explained if NE were denaturing proteins or disturbing the Ubiquitin Proteasome System (UPS). In vivo NE treatment reduced the levels of polyubiquitinated proteins and in vitro NE inhibited the ubiquitination reaction, probably interfering with E3 enzymes. Further evidence was obtained with a DELLA protein fused with Green Fluorescence Protein (GFP-RGA), that is degraded in the presence of gibberellins (GA) via the UPS (Silverstone et al, 2001, Plant Cell 13: 1555). By using confocal microscopy and western blot we showed that after a previous NE treatment, RGA is stabilized in the presence of GAs. These results suggest that one mechanism of NE toxicity is blocking the degradation of misfolded proteins by inhibition of their ubiquitination. The structural similarity between NE and lysine residues of substrate proteins of the UPS may explain this effect. NE inhibits poliubiquitination. Western blot of total poliubiquitinated protein A) seedlings subjected to NE (24h, 3mM) or mock treated with or without the proteasome inhibitor MG132 (50uM, 6h) B) time course of seedlings treated with NE (3mM). NE stabilizes DELLA protein GFP-RGA in the presence of Giberellins. Confocal images of root tips of seedlings treated with A) mock B) 3mM, 24h NE C) 1uM, 3h GA 3 D) 1uM, 3h GA 3, 3mM, 24h NE. E. Western blot showing GFP-RGA levels. 1) mock treated seedlings 2) NE (3mM, 24h) 3) GA 3 (1uM, 3h) and NE (3mM, 24h) 4) GA 3 (1uM, 3h) A B NE inhibits ubiquitination of E3 enzyme in vitro. Immunoblot analysis of the in vitro ubiquitination of a recombinant E3 enzyme. kDa 72 55 43 34 26 95 130 kDa 1 2 34 5 6 NE treatment increases the insoluble protein fraction. Total protein of seedlings treated with 3mM, 24h NE (1) or mock (2). Insoluble protein fraction of seedlings treated with NE (3) or mock (4). Soluble protein fraction of seedlings treated with NE (5) or mock (6) E Suggested model of NE toxicity in Arabidopsis. Under normal conditions, proteins targeted for degradation are polyubiquitinated by the E2-E3 enzyme complex. NE, due to its similarity to a free lysine residue, could interact with the E2-E3 enzyme complex and thus preventing the correct degradation of misfolded or denaturated proteins by these enzymes. These misfolded proteins could then form insoluble toxic aggregates, which in turn would trigger a Heat Shock response.