There is accumulating evidence suggesting that PAs interfere with various biological processes through the generation of H₂O₂ during their catabolism [4,7]. In agreement, the participation of PAOs has been reported in many developmental processes where ROS are involved.

Overexpression of the maize (Zea mays) ZmPAO1 resulted in early xylem differentiation and strongly affected root development in transgenic tobacco plants in correlation with augmented H₂O₂ production and increased rate of cell death [44]. This PAO was shown to accumulate in the cell wall of xylem precursors parallel to secondary wall deposition [30]. The AtPAO5 enzyme that specifically accumulates in the vascular system has also been reported to participate in xylem differentiation [45,46]. Although most PAOs generate ROS as a by-product of their activity, AtPAO5 preliminary control PA, especially t-Spm, levels [27,45,46]. Since AtPAO5 acts rather as a dehydrogenase than oxidase, it does not produce excess H₂O₂ [45]. In this way, AtPAO5 indirectly controls xylem differentiation via maintaining t-Spm homeostasis required for normal growth [37,45]. AtPAO5, via controlling the t-Spm level, was hypothesized to contribute to the tightly controlled interplay between auxins and cytokinins during the xylem differentiation process [46].

Nevertheless, there is ample evidence suggesting that the production of H₂O₂ by PA catabolism contributes to the cross-linking of cell wall polysaccharides during cell wall maturation [30,44]. For example, the apoplastic maize ZmPAO enzyme was shown to provide ROS for peroxidase-mediated wall stiffening during wound healing [47,48]. Moreover, rice OsPAO7 was hypothesized to control lignin synthesis in anther cell walls [49]. The polar growth of pollen tubes correlates with ROS accumulation at their tip region, controlling hyperpolarization-activated Ca²⁺ channels and cell wall stiffening [50]. Exogenous polyamines modulate pollen tube growth dependent on ROS generation [51]. In agreement, Spd treatment was reported to promote the opening of Ca²⁺ channels in pollen tubes [52]. Mutations in the AtPAO3 gene blocked the effect of Spd on Ca²⁺ channels and pollen tube growth, indicating that the effect was dependent on AtPAO3-mediated Spd degradation. Untreated pollen tubes of the AtPAO3 mutant also exhibited retarded growth, supporting the view that AtPAO3-generated ROS contributes to pollen tube growth [52]. The observation that the Arabidopsis polyamine transporter ABCG28 is required for the apical accumulation of ROS in growing pollen tubes and root hairs [53] further strengthens this hypothesis. PAs and their catabolism play a role in the induction of Ca²⁺ and K+ fluxes also in roots during stress adaptation [54], supporting the general significance of the PA-PAO-ROS-Ca²⁺ signalling connection.

PAs are well known to be required for and promote in vitro plant regeneration, although the mechanism is largely uncovered (reviewed by [55]). It was suggested that metabolic degradation products of PAs, such as t-Spm and/or H₂O₂, can at least be partly responsible for the observed effects [56]. The expression of AtPAO5, unlike other AtPAO-coding genes, increased in parallel with the conversion of lateral root primordia to shoot meristem during direct in vitro organogenesis [56]. Furthermore, the ectopic expression of AtPAO5 but not AtPAO2 promoted the process. It was hypothesized that AtPAO5 exerted its effect via the modulation of the t-Spm homeostasis rather than H₂O₂ production [56] since AtPAO5, unlike the other AtPAO enzymes, is known to have a stronger dehydrogenase than oxidase activity and has a high affinity for t-Spm as substrate [45]. Interestingly, AtPAO5 had been reported to have a negative effect on indirect (via auxin-induced callus formation) [57] and not direct (via cytokinin-induced meristem conversion) [56] shoot regeneration from Arabidopsis roots. This strengthens the view that maintaining t-Spm homeostasis is the primary function of AtPAO5 [27] since t-Spm was shown to suppress auxin signalling [58], which plays a different role in direct and indirect shoot regeneration from Arabidopsis roots [59]. Furthermore, H₂O₂ as the metabolic product of PAs was found to be essential for the maintenance and propagation of embryogenic calli and their conversion into somatic embryos in cotton [60], indicating a more general role of PA catabolism in plant regeneration in vitro.

The link among PAs, ROS, and leaf senescence has been long established (reviewed in [61]). Augmenting transcription and activity of PA catabolic enzymes has been demonstrated during dark-induced senescence of barley leaves [61,62]. Inhibiting the PAO activity delayed the senescence process in parallel with Spm accumulation and reduced ROS production. In agreement, the Arabidopsis atpao4 mutant exhibited delayed senescence in correlation with high Spm levels but reduced ROS accumulation [38]. Altogether, the observations indicate that PAO-generated H₂O₂ is involved in leaf senescence. PA catabolism has been also associated with fruit ripening, a senescence-like developmental process in grapes, tomatoes, and peaches [63,64,65]. Fruit ripening is associated with the increased expression of genes coding for apoplastic PAO enzymes, catalysing the terminal oxidation of PAs. Inhibition of PAO activity reduced ethylene production and flesh softening of peach fruits and the expression of ripening-related genes, while PA contents were dramatically increased. The role of PAO-generated H₂O₂ as a ripening-promoting signal molecule was hypothesised as one of the potential mechanisms [22]. PAO-generated H₂O₂ was shown to contribute to developmental PCD during xylem differentiation [44,66]. Polyamine oxidases were also found to be key elements in the oxidative burst, leading to programmed cell death in cryptogein-treated tobacco-cultured cells [67]. Moreover, tobacco plants overexpressing the transgene coding for the same maize PAO enzyme had high H₂O₂ levels, which in some cases led to programmed cell death (PCD) [68].

There is overwhelming evidence that increasing the polyamine content contributes to cell protection under environmental stress conditions. PAs take part in osmoprotection, stabilisation of macromolecular complexes, maintenance of the ion homeostasis, scavenging ROS, and stress and hormone signalling [2,69]. Not only biosynthesis, but PA catabolism has also been shown to have a significant role in various abiotic stress responses [25,70]. These roles can at least partly be attributed to the products of PA catabolism catalysed by DAO and PAO enzymes, such as gamma-aminobutyric acid (GABA) and/or H₂O₂ [70]. GABA, which is mainly synthesized in PA-independent pathways but can also be produced from PA-derived 4-aminobutanal, is an important plant metabolite with various protective functions in stress tolerance [71]. Under hypoxic conditions, PA catabolism with CuAO and PAO enzymes contributed to the GABA content by approximately 30% in Vicia faba [72]. Exogenous GABA, however, was shown to inhibit the breakdown of PAs, indicating negative feedback [71]. Besides GABA, the significance of PAO-dependent H₂O₂ generation has also been described in drought adaptation, namely during ABA, as well as ethylene-mediated stomatal closure in Vitis vinifera and Arabidopsis thaliana, respectively [73,74]. Fine-tuning PA catabolism during stress conditions might be required to control H₂O₂ generation. ROS produced by PA decomposing enzymes can serve as important signalling molecules to boost antioxidative defence reactions, but above a certain level, they can augment the stress-associated cellular damage or even lead to PCD [25,75]. For example, tobacco cells were found to secrete Spd into the apoplast where it was oxidized by PAO, thereby generating H₂O₂ at a level that promoted PCD [32,68]. In citrus (Citrus sinensis), the apoplastic CsPAO4 was shown to produce H₂O₂ and cause oxidative damages under salt stress [41]. In tomato, PA catabolism (both DAO and PAO enzymes) responded stronger to sublethal than lethal doses of the stress hormone salicylic acid in salinity tolerance signalling [76]. Downregulated expression of PAO-coding genes increased the thermotolerance of tobacco likely due to reduced heat-induced H₂O₂ generation [77]. PAO activity was shown to contribute to aluminium- or selenium-induced oxidative stress, further strengthening its pro-oxidant role during severe stresses [78,79].

However, the significance of PA-catabolism in antioxidant defence signalling contributing to the salt tolerance of PA-overproducing transgenic tobacco plants was also demonstrated by different groups [68,80]. Furthermore, contrasting salt stress tolerance of maize genotypes was found to be correlated with PA catabolism-dependent H₂O₂ production during salt stress, but it was rather DAO than PAO activity-dependent [81]. In the leaf blade elongation zone of salinized maize plants, the PAO activity was found to be strongly increased (app. 20-fold) [82]. Together with increased apoplastic PA secretion, the PAO activity resulted in increased apoplastic ROS accumulation, contributing to leaf blade elongation under salt stress. Polyamine oxidase 5 loss-of-function atpao5 mutants of Arabidopsis are salt-stress tolerant; however, their salt tolerance did not show correlation with diminished ROS production but rather with the increased level of t-Spm [37]. However, in the salt-tolerant pao1 pao5 double mutant with no cytoplasmic PAOs, reduced ROS production was observed under NaCl stress [36]. Interestingly, simultaneous mutations in the pao2 and pao4 genes, both coding for peroxisomal PAO, were salt-sensitive, while the pao2 pao3 pao4 triple mutant with no peroxisomal PAO enzymes was not viable [36].

The above observations highlight the differential contribution of the various PAOs with different activities, by-products, and intracellular localisations, to stress tolerance.

PAO-generated H₂O₂ may also contribute to pathogen defence. It may directly act as an anti-microbial agent in the apoplast or serve as a signalling molecule inducing the activation of defence genes [83,84]. PA levels and the activity of PA metabolic enzymes were found to be induced by various (biotrophic, as well as necrotrophic) pathogens infecting plant tissues [85,86,87]. For example, in response to the biotrophic pathogen Pseudomonas syringae, PAO activity was found to be increased in tobacco [88]. The infection also induced Spm secretion that, together with the elevated PAO activity, resulted in strong H₂O₂ accumulation in the apoplast. The apoplastic Spm-mediated disease resistance could be compromised by PAO inhibitors [86,88]. Therefore, in biotrophic plant–pathogen interactions, PAO activity-related H₂O₂ generation might contribute to the hypersensitive response (HR), as described for tobacco mosaic virus or Pseudomonas chicorii-infected tobacco [89,90] and powdery mildew (Blumeria graminis)-infected barley [89]. The oomycete Phytophthora cryptogea secretes cryptogein, a 10-kD protein that induces HR in tobacco. Inhibiting the expression of the gene coding for apoplastic tobacco PAO prevented PA degradation, cryptogein-induced apoplastic H₂O₂ generation, and cell death [67]. The observation that, in these plants, cryptogein-induced kinase signalling was also compromised, highlighted that, besides its cytotoxic effect, PAO-generated H₂O₂ also has a signalling role in the HR. The signalling role of Spm degradation-derived H₂O₂ was also hypothesized in the transcriptional responses of Arabidopsis to the HR-inducing cucumber mosaic virus [91]. While PA degradation and H₂O₂ production might beneficially control the HR response in biotrophic host–pathogen interactions, it might be detrimental in the case of infection by necrotrophic pathogens. In agreement, increased polyamine levels were reported to promote leaf necrosis during fungal infection dependent on PAO activity [86].