ABSTRACT DOCTORAL THESIS

“Origin and function of spermidine aminopropyltransferases in Arabidopsis thaliana”

      Polyamines are small and positively-charged molecules at physiological pH. The most abundant and widely spread polyamines in all organisms are putrescine and spermidine, which contain two and three amino groups, respectively. Spermidine is formed by the addition of an aminopropyl group to putrescine. Spermine, a tetraamine present only in eukaryotes, is derived from spermidine by the addition of a second aminopropyl group.

      Polyamines have been involved in several fundamental life processes, suchas as cell division, growth, differentiation, and cell death. In all organisms, polyamines are essential compounds. In plants there are multiple correlations between the variation of polyamine concentration and the extent of germination, embryogenesis, root formation, floral transition, and flower and fruit development.

      Coinciding with the beginning of this work, the identification of the first putative spermine synthase (ACL5) in plants was reported, whose loss of function in Arabidopsis causes a defect in stem elongation and aberrant formation of vascular bundles; however, our detailed analysis of the acl5 mutant revealed that it had not lost the ability to synthesize spermine. In this Thesis we report the isolation and characterization of the SPM gene, a second putative spermine synthase, whose expression is induced by abscisic acid. Loss-of-function mutants in SPM display no obvious differences with respect to the wild type. It is very unlikely that here are additional spermine synthase genes because the double acl5 spm mutant does not produce spermine. However, overexpression of SPM did not alleviate the acl5 phenotype, suggesting that the two genes might not have the same function. In fact, this is the case, because a recent report indicates that ACL5 encodes an aminopropyl transferase with thermospermine synthase activity.

      The detailed analysis of the acl5 mutant that we have carried out, indicates that ACL5 is necessary for the correct development of metaxylem and fibers during secondary growth. Our experiments suggest that the role of ACL5 is to prevent premature cell death, allowing for the xylem differentiation program to be completed.

      The analysis of phylogenetic relationships between the different aminopropyl transferases indicates that the sequence similarity that they show is due to their common evolutionary origin. The model proposed suggests that spermine synthase activity appeared three times during evolution, independently in Animals, Fungi and Plants (only in Angiosperms), through a process of neofunctionalization of a duplicated spermidine synthase gene. Interestingly, putrescine methyltransferases were more recently originated in Solanales by an equivalent process of neofunctionalization. However, thermospermine synthase seems to have been originated in certain prokaryotes, and later acquired only by Plants by horizontal gene transfer. Finally, modelling of the 3D structure of the different active centers has also allowed to pinpoint several key residues to discriminate between the different activities.