The involvement of Arabidopsis thaliana polyamine oxidases in plant development and defence responses

Abstract

The polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm) are small aliphatic polycations found in all living cells. They are involved in several cellular processes and play important roles in morphogenesis, growth, differentiation and senescence. In plants, they are also implicated in defence responses to various biotic and abiotic stresses. PA homeostasis is strictly regulated through anabolic and catabolic processes, but also through conjugation, transport and compartimentalization. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in PA catabolism. PAOs from monocotyledonous plants, such as the apoplastic maize PAO (ZmPAO), oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H2O2 and an aminoaldehyde and are considered involved in a terminal catabolic pathway of PAs. Conversely, animal PAOs and spermine oxidases (SMOs) oxidize Spd, Spm and/or their acetyl-derivatives to produce Put and Spd, respectively, in addition to H2O2 and 3-aminopropanal and are thus considered involved in a PA back-conversion pathway. In Arabidopsis thaliana, five PAO genes (AtPAO1-5) are present with a varying amino acid sequence homology to ZmPAO and subcellular localization (putative cytosolic for AtPAO1 and AtPAO5 and peroxisomal for AtPAO2, AtPAO3 and AtPAO4). Furthermore, following heterologous expression in bacteria it was shown that AtPAO1 oxidizes Spm but not Spd, whereas AtPAO2, AtPAO3 and AtPAO4 oxidize both Spm and Spd. Conversely, AtPAO5 substrate specificity has not been determined so far since production of the recombinant protein in various heterologous systems has not been successful. The four characterized AtPAOs are also active towards the uncommon PAs thermospermine (Ther-Spm) and norspermine (Nor-Spm). In particular, AtPAO1 shows a higher catalytic activity towards Ther-Spm and NorSpm than towards Spm, which suggests that these two uncommon PAs may be the physiological substrates of this enzyme. This is of particular interest because it has been recently shown the existence in Arabidopsis of an enzyme able to synthesize Ther-Spm and a loss-of-function mutant for this gene shows a severely dwarfed phenotype. Another important characteristic of the four Arabidopsis PAOs is their involvement in a PA back-conversion pathway, producing Spd from Spm and Put from Spd, similarly to the animal PAOs / SMOs and contrary to ZmPAO. Studies on the tissue- and organ-specific expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 using AtPAO::GFP-GUS transgenic Arabidopsis plants showed some distinct expression patterns for each one of the four AtPAOs, such as in the transition region between cell division and elongation zones of roots and anther tapetum for AtPAO1, in columella, stipules and pollen for AtPAO2, in hypocotyls and roots, stipules, columella, trichomes, guard cells and pollen for AtPAO3, and in the vascular system of roots and hypocotyls for AtPAO5. These studies also evidenced increased expression of AtPAO1 in roots and of AtPAO2 in guard cells following treatment with the stress-related plant hormone abscisic acid (ABA). In the present work, the study on the tissue- and organ-specific expression pattern of the five AtPAOs was completed analysing AtPAO4 promoter activity. In particular, histochemical GUS staining of AtPAO4::GFP-GUS transgenic Arabidopsis plants evidenced that AtPAO4 is expressed in the roots (from the meristematic/elongation transition region up to the hypocotyl–root junction site), in the guard cells, in the base of very young and completely closed flower buds, in anther tapetum and in mature pollen grains. These data together with data from promoter analysis of the other four AtPAOs indicate distinct physiological roles for the various AtPAOs during seedling growth and flower development and suggest functional diversity inside the AtPAO gene family. To determine the physiological roles of the various AtPAOs, loss-of-function T-DNA insertional mutants (atpao1, atpao2, atpao3, atpao4 and atpao5) have been previously obtained from the NASC collection of Arabidopsis seeds and homozygous mutant lines have been selected. In the present study, double (atpao2/atpao4 and atpao3/atpao4, atpao3/atpao2) and triple (atpao2/atpao4/atpao3) mutants for the peroxisomal AtPAOs as well as the double atpao1/atpao5 mutant for the two AtPAOs with predicted cytosolic localization were also obtained through sexual crossings. The atpao1 single mutant was analyzed for the levels of the common PAs Put, Spd and Spm as well as of the uncommon PA Ther-Spm, evidencing no statistically significant variation comparing to the wild-type plants. This may be due either to gene redundancy or to the activation of homeostatic mechanisms and may exclude the possibility that Ther-Spm is the physiological substrate of AtPAO1. The atpao1 single mutant was also analyzed for germination and growth rate under physiological and stress conditions, but also in these cases no variation was observed as compared to the wild-type plants. Similar studies on the atpao1/atpao5 double mutant are in progress. Analysis of PA levels in the single and triple mutants for the three peroxisomal AtPAOs showed some alterations in the atpao2/atpao4/atpao3 triple mutant. Furthermore, since all three AtPAO2, AtPAO3 and AtPAO4 are highly expressed in the guard cells, specialized cells surrounding stomata pores, stomata movements were evaluated evidencing reduced ABA- and PA-mediated stomata closure in the corresponding mutant plants as compared to wild-type plants. Furthermore, the atpao2/atpao4/atpao3 triple mutant appeared more tolerant to dehydration and ABA treatment. Altogether, these data suggest the involvement of the three peroxisomal AtPAOs in the ABA-mediated signaling network. On the other hand, germination and growth rate of the atpao2/atpao4/atpao3 triple mutant in the absence of sucrose was shown to be delayed comparing to the wild-type plants. The underlying mechanisms in these phenotypical alterations in the atpao2/atpao4/atpao3 triple mutant are currently under investigation. In the present study, it was also possible to express AtPAO5 in 35S::AtPAO5-6His transgenic Arabidopsis plants, to partially purify the corresponding recombinant protein and to determine substrate specificity and reaction products. In particular, it was shown that AtPAO5 has indeed PAO activity, catalyzing the oxidation of Spm, N1-acetyl-Spm, Ther-Spm and Nor-Spm through a PA back-conversion pathway. Furthermore, confocal analysis of 35S::GFP-AtPAO5 and 35S::AtPAO5-GFP transgenic Arabidopsis plants indicated that AtPAO5 is a cytoplasmic protein undergoing proteasomal control. It was also shown cytokinin-inducible expression of AtPAO5 as well as AtPAO5 involvement in the control of xylogenesis by cytokinins. Experiments are in progress to determine the physiological significance of AtPAO5 regulation by the proteasomal pathway as well as to unravel the mechanisms by which AtPAO5 is involved in the cytokinin-mediated pathways. This study represents the starting point to understand the distinct physiological roles of the different PA catabolic pathways in plants during development and defense responses which may permit the application of biotechnological strategies to transfer increased yield and stress-tolerance traits to crops of agronomical relevance.