A study on the physiological roles of arabidopsis thaliana polyamine oxidasses

Abstract

The polyamines 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, polyamines are also implicated in defence responses to various biotic and abiotic stresses. Polyamine homeostasis is strictly regulated through anabolic and catabolic processes, but also through conjugation, transport and compartimentalization. Polyamine oxidases (PAOs) are FAD-dependent enzymes which oxidatively de-aminate the polyamines Spm and Spd. The chemical identity of PAO reaction products depends on the enzyme source and reflects the mode of substrate oxidation. In particular, PAOs from the monocotyledonous plants so far characterized, such as the apoplastic maize PAO (ZmPAO), oxidise the carbon on the endo-side of the secondary amino group of Spm and Spd to produce 1,3- diaminopropane, H2O2 and an aminoaldehyde, and are thus considered to be involved in a terminal polyamine catabolic pathway. Instead, animal PAOs and spermine oxidases (SMOs) oxidise the carbon on the exo-side of the secondary amino groups of Spd and Spm (and/or their acetylated derivatives) to produce Put and Spd, respectively, in addition to an aminoaldehyde and H2O2, and are considered to be involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAO genes (AtPAO1-5) are present with varying amino acid sequence homology to ZmPAO and subcellular localization. In particular, AtPAO1 and AtPAO5 have a predicted cytosolic localization, while for AtPAO2, AtPAO3 and AtPAO4 a peroxisomal localization was shown. Furthermore, AtPAO2, AtPAO3 and AtPAO4, having a high sequence homology to each other and a very similar intron/exon organization, are considered to form a distinct PAO subfamily (AtPAO2-4 subfamily). Heterologous expression and biochemical characterization of recombinant AtPAO1, AtPAO2, AtPAO3 and AtPAO4 showed that AtPAO1 oxidises only Spm, whereas AtPAO2, AtPAO3 and AtPAO4 oxidise both Spm and Spd. AtPAO5 catalytic properties have not been determined yet. Another important characteristic of the four Arabidopsis PAOs until now characterised is their involvement in a polyamine backconversion pathway. Indeed, they produce Spd from Spm and Put from Spd similarly to the animal PAOs / SMOs and contrary to ZmPAO. Accumulating data indicate that in plants polyamine catabolism is more than a biochemical process aiming to control polyamine homeostasis. Indeed, it has been demonstrated that extracellular PAOs constitute a nodal point during plant growth under physiological as well as abiotic and biotic stress conditions, giving rise to increased apoplastic H2O2, which signals primary and secondary developmental and defense responses. In contrast to the so far characterized apoplastic PAOs, little is known until now on the physiological roles of the intracellular PAOs. In the present study, in an attempt to determine the physiological roles of the various AtPAOs, their tissue- and organ-specific expression pattern was analysed in detail through analysis of promoter activity in transgenic Arabidopsis plants expressing AtPAO::β-glucuronidase. Histochemical analysis of the transgenic plants revealed a distinct expression pattern for each one of the five AtPAOs, such as in the transition region between meristematic and elongation zones of roots and in anther tapetum for AtPAO1, in columella, stipules and pollen for AtPAO2, in hypocotyls, roots, stipules, columella, trichomes, guard cells and pollen for AtPAO3, in the meristematic/elongation transition zone of roots, guard cells, anther tapetum and pollen for AtPAO4, and in the vascular system of roots, hypocotyls and anther tapetum for AtPAO5. These studies also evidenced abscisic acid (ABA)-inducible expression of AtPAO1 in the meristematic/elongation transition zone of the roots and of AtPAO2 in guard cells, inducible expression of AtPAO1 in roots under gravity stimulus, as well as cold- and Spm-inducible expression of AtPAO5 in cotyledons. These data altogether indicate distinct physiological roles for the various AtPAOs during seedling growth and flower development and suggest functional diversity inside the AtPAO gene family. In parallel, studies were performed to determine the physiological roles of the various AtPAOs using loss-offunction T-DNA insertional mutants. In particular, considering the expression of AtPAO1, AtPAO2, AtPAO3 and AtPAO4 in root apex, their involvement in gravitropism was analysed. The obtained data, however, evidenced no significant variation in root bending of single loss-of-function mutants comparing to the wild-type plants. Experiments are in progress with multiple loss-of-function mutants. Since the plant hormone ABA plays a protective role in response to abiotic stresses, acting as a key regulator of stomatal apertures to restrict transpiration and reduce water loss, the ABA-inducible expression of AtPAO2 in guard cells, together with the constitutive expression of AtPAO3 and AtPAO4 in the same cells, led us to hypothesize involvement of these enzymes in the control of stomatal opening and in plant defence responses to abiotic stresses. To test this hypothesis, the transpiration rate and the stomatal opening of atpao loss-of-function mutants were analysed. Our data showed no difference in the transpiration rate between atpao mutants and wild-type plants, but evidenced a reduced ability to close stomata upon ABA supply for atpao2, atpao3, atpao4 single mutants, as well as of atpao2/atpao4 and atpao3/atpao4 double mutants, comparing to the wild-type plants. Studies are in progress to make clear the underlying mechanisms in the contribution of the peroxisomal AtPAOs to the regulation of stomata movement. Studies are also in progress to determine AtPAO involvement in plant development and defence responses to various abiotic stresses. In conclusion, the present study provides evidence for important differences in the spatial and temporal expression pattern of the various Arabidopsis PAOs, which, together with the distinct catalytic properties, suggest distinct physiological roles for each member of the A. thaliana PAO gene family. This study will contribute to a detailed analysis of the physiological roles of the polyamine catabolic pathways in plants.