Functional diversity inside the Arabidopsis polyamine oxidase gene family

""Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and\\\/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and\\\/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.. . "

Nitrogen flow into regulatory molecules: The case of Polyamines and Polyamine oxidases.

""\\"Glutamate plays a central role in the flow of nitrogen to various regulatory molecules, such as Polyamines (PAs). PAs are low MW nitrogenous molecules. The most common PAs are the diamine Putrescine (Put), the triamine Spermidine (Spd) and the tetramine Spermine (Spm). In plants, Put and Spd are the obligatory precursors of Spd and Spm, respectively. PAs have been implicated in plant growth and development, abiotic and biotic stress responses (1,2,3,4,5,6). PA oxidation is catalyzed by Di- and PA oxidases (DAO and PAOs, respectively) in the apoplast [terminal catabolism; (3)],or in the cytoplasm and the peroxisomes [PA back-conversion; (4)]. In both cases, H2O2 is generated. Recently, PA catabolism-derived H2O2 was shown to significantly participate in the physiological functions of PAs. Stress induces PA exodus into the apoplast, where it is oxidized by apoplastic PAO leading to increase of H2O2 load; this in turn participates in signalling events, inducing differential gene expression, which results to either PA homeostatic responses or the execution of programmed cell death [PCD; (4,6)]. Thus, the level of apoplastic H2O2 determines whether stress-tolerant mechanisms or the PCD syndrome will be induced. Engineering the PA catabolic pathway led to increased tolerance to biotic but sensitivity to abiotic stress (5,6). The pathway is partially controlled by the phytohormone abscissic acid (ABA), which is involved in stress-dependent signalling cascades, including those controlling stomatal closure (7). ABA induces expression of AtPAO3, a peroxisomal Arabidopsis PAO and GUS reporter analysis, revealed that cis-regulatory elements in the promoter region control this induction (8,9). Interestingly, GUS activity post-treatment with ABA was specifically localized to guard cells, implying a direct role of PAO-derived H2O2 in stomatal closure. Moreover, the complete identification and analysis of AtPAOs in Arabidopsis revealed that all four PAOs (AtPAO1, AtPAO2, AtPAO3 and AtPAO4) are back-converting Spm to Spd and additionally AtPAO2 and AtPAO3 back-convert Spd to Put (8,9). Thus, Arabidopsis seems to lack PAOs involved in terminal catabolism of PAs in contrast to maize, in which the until now characterized PAOs produce 1,3-diaminopropane and 4-aminobutanal or N-(3-aminopropyl)-4-aminobutanal from Spd or Spm oxidation, respectively. H1-NMR studies revealed that AtPAOs, in addition to H2O2 and Spd or Put, produce 3-aminopropanal, which can be further converted to the osmoprotectant molecule β-alanine and pantothenate in a pairwise reaction. The AtPAOs are differentially expressed, as revealed by GUS reporter assays, implying functional diversity inside the AtPAOs family (8,9). Furthermore, Spd Oxidase-derived H2O2 regulates pollen plasma membrane hyperpolarization-activated Ca2+-permeable channels, pollen tube growth and number of seeds (10). Also, PAO-derived H2O2 acts as a signal for deposition of secondary wall and as a mediator of developmental PCD in plant tissues resulting to suppressed primary maize root elongation by inhibiting cell growth and altering cell cycle progression (11). To sum up, all these results along with the recent finding of the involvement of AtPAOs in catabolism of thermospermine, a Spm isomer involved in vascular differentiation and stress adaptation, reveal novel aspects of the PA catabolic pathway and its genetic engineering may result to significant phenotypical and stress-tolerant genotypes. . . REFERENCES. 1.Paschalidis KA & KA Roubelakis-Angelakis (2005) Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division\\\\\\\/expansion, cell cycle progression, and vascular development. Plant Physiol 138(4):2174-84. 2.Paschalidis KA, PN Moschou, I Toumi & KA Roubelakis-Angelakis (2009) Polyamine anabolic\\\\\\\/catabolic regulation along the woody grapevine plant axis is linked to vascular differentiation and to stomatal closure. J Plant Physiol 166(14):1508-19. 3.Moschou, PN, ID Delis, KA Paschalidis & KA Roubelakis-Angelakis (2008) Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors. Physiol Plant 133(2):140-56. 4.Moschou PN, M. Sanmartin, A. H. Andriopoulou, E. Rojo, J. J. Sanchez-Serrano & KA Roubelakis-Angelakis (2008) Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis. Plant Physiol 147(4):1845-57. 5.Moschou, PN, PF Sarris, N Scandalis, AH Andriopoulou, KA Paschalidis, NJ Panopoulos & KA Roubelakis-Angelakis (2009) Engineered polyamine catabolism preinduces tolerance of tobacco to bacteria and oomycetes. Plant Physiol 149(4):1970-81. 6.Moschou, PN, KA Paschalidis, ID Delis, AH Andriopoulou, GD Lagiotis & KA Roubelakis-Angelakis (2008) Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco. Plant Cell 20(6): 1708-24.. 7.Toumi I, PN Moschou, KA Paschalidis, S Daldoul, B Bouamama, S Chenennaoui, A Ghorbel, A Mliki & KA Roubelakis-Angelakis 2010. Abscisic acid signals reorientation of polyamine metabolism to orchestrate stress responses via the polyamine exodus pathway in grapevine. J Plant Physiol. doi:10.1016\\\\\\\/j.jplph. 2009.10.022.. 8. Fincato P, PN Moschou, V Spedatelli, R Tavazza, R Angelini, R Federico, KA Roubelakis-Angelakis & P Tavladoraki (2011) Functional diversity inside the Arabidopsis polyamine oxidase gene family. J Exp Bot 62:1155-1168 doi: 10.1093\\\\\\\/jxb\\\\\\\/erq341. 9.Fincato P, PN Moschou, A Ahou, KA Roubelakis-Angelakis, R Federico & P Tavladoraki (2011) The members of Arabidopsis thaliana PAO gene family exhibit tissue-organ specific expression pattern during seedling growth and flower development. Amino Acids DOI 10.1007\\\\\\\/s00726-011-0999-7.. 10.Wu J, H Qu, Z Shang, X Jiang, PN Moschou, KA Roubelakis-Angelakis & S Zhang (2010) Spermidine oxidase-derived H2O2 activates downstream Ca2+ channels which signal pollen tube growth in Pyrus pyrifolia. Plant J 63:1042-1053, DOI: 10.1111\\\\\\\/j.1365-313X.2010.04301.x. 11.Tisi A, R Federico, S Moreno, S Lucretti, PN Moschou, KA Roubelakis-Angelakis, R Angelini & A Cona (2011) Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation. Plant Physiol. DOI:10.1104\\\\\\\/pp.111.173153.. . . \\""