TRANSGLUTAMINASES IN SENESCENCE AND PROGRAMMED CELL DEATH OF LEAVES AND PETALS: THEIR SIMILARITIES TO ANIMAL TRANSGLUTAMINASES

Free polyamines (PAs) have a contrasting role in the delay of senescence in animals, whereas in plants its protective effect is better established, eventhough the molecular mechanism is not clarified. We hypothesised that PAs can be conjugated to specific proteins by catalysis of Ca++-dependent transglutaminases (TGases). TGases are involved in PCD in animals, whereas few data are available in plants. Plants utilized were: Nicotiana tabacum corolla, models of short-lived flowers and Lactuca sativa leaves, commercially utilised for food purposes. Many morphological and physiological parameters were examined. The supply of spermine (SM) to excised flowers or entire plant efficiently delayed senescence and PCD in petals or leaves respectively. SM caused an increase also of putrescine (PU) and spermidine (SD) levels. Only in senescent plants a new TGase band was immunodetected and, in concomitance, high molecular mass and other protein bands were PA-modified. During senescence mainly mono-, bis-PU and bis-SD catalysis increased. This data suggests that the protective effect of PAs could be mediated by TGase. SM effect on the retention of leaf proteins and chlorophylls was analysed in Valeriana young and senescent plants. Most of chlorophyll is bound to the antennae of light-harvesting complexes (LHC), known preferential thylakoid TGase substrates under light2. In Valeriana LHC enriched fractions, different TGase isoforms, immunologically related to TGases of animal origin, were co-purified. PA conjugation to both photosystems was light-stimulated. The capacity of plants to express animal TGases and the similarities in the TGase sequences and activity in animals and plants have also been explored. Rat TGase (DP1 ORF) introduced into mature rice embryos by bombardment was expressed by the plant. It is Ca2+-dependent and active in in vitro assay, showing that plants could produce functional mammalian TGases3. Thylakoid TGase recognises animal substrates as well as animal TGases plant substrates, but only the plant TGase/plant substrate catalysis is light regulated2. As in both the investigated senescence models any TGase has been fully-purified until now, another approach was undertaken. On the basis of the identification of a TGase-like domain in an A. thaliana gene, AtPng1p, we have cloned its codifying sequence on E. coli and purified the over-expressed recombinant protein. All biochemical assays showed a clear Ca++, pH, and DTT dependent TGase activity. This TGase was immunodetected by three antibodies raised against animal TGases. Data and bio-informatic analysis also suggest a possible protease activity for this first plant sequenced TGase4. 1 D Serafini-Fracassini, S Del Duca, F Monti, F Poli, G Sacchetti, AM Bregoli, S Biondi and M Della Mea (2002) -Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotian a tabacum) flowers. Cell Death and Diff. 9 (3) 309-321. 2 M. Della Mea, A. Di Sandro, L. Dondini, S. Del Duca, F.Vantini, C.Bergamini, R. Bassi and D. Serafini-Fracassini - A Zea mays 39 kDa thylakoidal transglutaminase catalyses the modification by polyamines of light harvesting complex II in a light-dependent way. Planta, in press. DOI 10.1007/s00425-004-1278-6.