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Polyamines (PAs) are ubiquitous low-molecular-weight aliphatic compounds present in all living organisms and essential for cell growth and differentiation. The developmentally regulated and stress-induced copper amine oxidases (CuAOs) oxidize PAs to aminoaldehydes producing hydrogen peroxide (H2O2) and ammonia. The Arabidopsis thaliana CuAOβ (AtCuAOβ) was previously reported to be involved in stomatal closure and early root protoxylem differentiation induced by the wound-signal MeJA via apoplastic H2O2 production, suggesting a role of this enzyme in water balance, by modulating xylem-dependent water supply and stomata-dependent water loss under stress conditions. Furthermore, AtCuAOβ has been shown to mediate early differentiation of root protoxylem induced by leaf wounding, which suggests a whole-plant systemic coordination of water supply and loss through stress-induced stomatal responses and root protoxylem phenotypic plasticity. Among apoplastic ROS generators, the D isoform of the respiratory burst oxidase homolog (RBOH) has been shown to be involved in stress-mediated modulation of stomatal closure as well. In the present study, the specific role of AtCuAOβ and RBOHD in local and systemic perception of leaf and root wounding that triggers stomatal closure was investigated at both injury and distal sites exploiting Atcuaoβ and rbohd insertional mutants. Data evidenced that AtCuAOβ-driven H2O2 production mediates both local and systemic leaf-to-leaf and root-to-leaf responses in relation to stomatal movement, Atcuaoβ mutants being completely unresponsive to leaf or root wounding. Instead, RBOHD-driven ROS production contributes only to systemic leaf-to-leaf and root-to-leaf stomatal closure, with rbohd mutants showing partial unresponsiveness in distal, but not local, responses. Overall, data herein reported allow us to hypothesize that RBOHD may act downstream of and cooperate with AtCuAOβ in inducing the oxidative burst that leads to systemic wound-triggered stomatal closure.

Le piante sono organismi affascinanti in grado di percepire prontamente stimoli endogeni ed ambientali, integrarli e reagire con una rete complessa di risposte fisiologiche. Il concepimento di metodiche innovative di laboratorio è fondamentale per migliorare la conoscenza di questi processi e ideare strategie utili in molteplici settori, come la moderna agricoltura sostenibile e la bioenergia. FISIOLOGIA VEGETALE APPLICATA a cura di Vincenzo Lionetti e Daniela Bellincampi, forti della loro lunga esperienza in campo didattico, sono riusciti a scrivere un testo che sarà di grande aiuto agli studenti e, perché no, anche a curiosi e appassionati, per l’attività pratica di ricerca in laboratorio e per approfondire le procedure analitiche alla base degli studi attuali più rilevanti nel campo della Fisiologia vegetale, curando vari aspetti come la biologia cellulare, la biologia molecolare, la biochimica, l’elettrofisiologia, l’ingegneria genetica e varie applicazioni biotecnologiche. I contenuti sono stati adeguati alle esigenze dei recenti ordinamenti didattici dei corsi di laurea in Scienze biologiche, in Biotecnologie e in Agraria.

""\\"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.. . . \\"""

New linear and cyclic guanidines were synthesized and tested in vitro for their antifungal activity toward clinically relevant strains of Candida species, in comparison to fluconazole. Macrocyclic compounds showed a minimum inhibitory concentration in the micromolar range and a biological activity profile in some cases better than that of fluconazole. One macrocyclic derivative was also tested against Aspergillus species and showed high antifungal activity comparable to that of amphotericin B and itraconazole. © 2009 American Chemical Society.