Does polyamine catabolism influence root development and xylem differentiation under stress conditions?

Abstract

Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H₂O₂). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H₂O₂ production. Here, we suggest that H₂O₂ derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H₂O₂ balance.

Amine oxidases are involved in polyamine homeostasis and hydrogen peroxide production

Copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs) catalyze the oxidative deamination of polyamines (PAs) at the primary and secondary amino group, respectively, producing an aldehyde, the removed amine moiety and hydrogen peroxide (H₂O₂) in stoichiometric amounts. Identity of the reaction products varies depending on the oxidized substrate and the mode of oxidation, H₂O₂ being the only shared product in all the amine oxidase (AO)-mediated polyamine oxidations.¹ Notwithstanding heterogeneity in molecular properties, sub-cellular localization, tissue-specific expression and species distribution, CuAOs and PAOs share common physiological roles in plant development and responses to biotic and abiotic stresses.¹ Indeed, AOs behave as crucial check-points in the modulation of PAs and H₂O₂ levels, which both represent basic cellular mediators of programmed cell death (PCD) and cell differentiation.^2-5

PAs, which are essential factors for cell growth and differentiation,^4,6 are well known to induce PCD upon depletion/overproduction with respect to their physiological levels.⁷ On the other hand, the oxidative stress, triggered by an imbalance in the production and metabolism of reactive oxygen species (ROS), is a primary response following pathogen challenge, H2O2 representing a key molecule in the PCD signaling during hypersensitive response (HR).^8,9 Moreover, redox processes have been involved in root growth and differentiation and notably ROS signaling has been proposed to control transition from proliferation to differentiation zone in the Arabidopsis (Arabidopsis thaliana) root.⁵

The duality of PA action in the regulation of cell growth and cell death as well as the involvement of both PAs and H₂O₂ in plant differentiation events and PCD signaling raise the still unresolved question whether AOs play their roles mainly through modulation of PA homeostasis or H₂O₂ production via PA catabolism. Recently, it has been proposed that the PA/H2O2 ratio could be the essential factor involved in salinity induced cell death in tobacco (Nicotiana tabacum) plants by governing cell fate decision leading to either induction of PCD or to tolerance mechanisms.¹⁰ Concerning this, AOs may represent a key switch in the balancing of the relative concentrations of these basic signaling compounds.

Alteration of apoplastic polyamine catabolism by pharmacological and genetic approaches affects root development inducing early differentiation of xylem tissues

Our recent study highlighted that exogenous spermidine (Spd) strongly affected maize (Zea mays) primary root growth and development by inhibiting cell elongation and altering cell cycle phase distributions in root apices, thus allowing cells to initiate their differentiation programs.11 Indeed, Spd treatment promoted deposition of phenolics in cell walls of rhizodermis, xylem elements and vascular parenchyma, likely associated with early maturation of cell wall and inhibition of cell expansion, and resulted in a higher number of cells resting in G1 and G2 phases. Importantly, the PAO inhibitor N-prenylagmatine (G3) or the H₂O₂ scavenger N, N¹-dimethylthiourea (DMTU), reverted the Spd-induced alteration of maize root growth, revealing the PAO-driven H₂O₂ production as the responsible for the observed root growth inhibition.11 Moreover, Spd treatment induced early differentiation and precocious cell death of early-metaxylem (EMX) and late metaxylem precursors (LMX) in the maize root apex associated with enhanced H₂O₂ production in xylem tissues.¹¹ In agreement with the above reported findings, maize PAO overexpression in the cell wall of tobacco plants resulted in early differentiation of root xylem precursors and increased cell death of sloughed root cap cells, as well as enhanced in vivo H₂O₂ production in xylem tissues as compared with wild-type (WT) plants.¹¹ Overall, our data suggest that, after Spd supply in maize or PAO overexpression in tobacco, H₂O₂ derived from PA catabolism strongly affects root development and xylem differentiation, behaving as a signal for deposition of secondary wall and for induction of developmental PCD.¹¹

Unexpectedly, tobacco antisense PAO plants, downregulating endogenous PAO, showed a similar spatio-temporal pattern of vascular terminal differentiation as compared with WT plants.¹¹ Even though we can argue that additional and/or alternative H₂O₂ sources, such as apoplastic CuAOs, could be involved in tobacco vascular tissue differentiation,¹² the lack of a defective phenotype showed by antisense PAO plants strongly contrast with the hypothesis that PA catabolism represents a key element in xylem differentiation during development under physiological conditions. However, this incongruity could be explained bearing in mind that Spd supply and PAO overexpression can be both considered un-physiological conditions in which a high rate of PA oxidation has been artificially forced to occur in the apoplast, thus mirroring a stress-like conditions. Indeed, it has been previously demonstrated that a coordinated enhancement of polyamine metabolism and transport to the apoplast occurs during plant responses to biotic and abiotic stresses, leading to a higher production of H₂O₂ in the cell wall,^10,13 also depending on the spatio-temporal pattern of AO expression levels (Fig. 1). Taking into account the above considerations, we can hypothesize that H₂O₂ derived from PA catabolism has a role in inducing xylem differentiation under stress conditions, while further investigations are needed to establish its involvement under physiological conditions. Notably, AOs are stress-responsive genes, strongly induced by both pathogen infection and abiotic stresses.^2,10,14 Furthermore, it has been previously reported that the expression of atao1, a gene encoding an extracellular CuAO expressed at the early stages of vascular tissue development in Arabidopsis,¹⁵ is induced after root invasion by nematode parasites, suggesting that ATAO1 may be responsible for cell-wall cross-linking during vascular re-differentiation needed to counter the effects of mechanical pressure caused by the pathogen.¹⁶

Figure 1.

Hypothetical signal transduction pathway leading to root xylem differentiation under plant stress conditions. Biotic and/or abiotic stresses increase cell wall amine oxidase levels simultaneously inducing polyamine (PA) secretion in the apoplast. H₂O₂ derived from PA catabolism induces early xylem differentiation by triggering cell wall stiffening and PCD in xylem precursors.

PAs affect plant development and stress responses depending on AO levels

We have recently demonstrated that transgenic tobacco plants overexpressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall showed increased cell death, as detected by SYTOX Orange staining, in root cap cells and in the rhizodermis of the subapical region (up to 300 μm from the root cap) in comparison to the same tissues of WT roots, with a S-ZmPAO/WT ratio of 3/1.¹¹ The observed increase of cell death has been reasonably ascribed to the higher rate of PA oxidation consequent to PAO overexpression and has been associated to PCD events occurring in the sloughed root cap cells of growing roots.¹¹ Unexpectedly, exogenously supplied Spd was able to induce cell death in WT (Fig. 2A,C) but not in S-ZmPAO plants (Fig. 2B,D), that showed a lower degree of SYTOX Orange staining as compared with Spd-treated WT plants. In particular, by the Z-stack analysis under laser scanning confocal microscope (LSCM) of the whole root (Fig. 2C,D) extensive cell death was detectable after Spd treatment in root apices of WT plants (Fig. 2C), while in S-ZmPAO plants (Fig. 2D) cell death occurred to a lesser extent, once more raising the question whether PAs play a role by themselves and/or through the AO-mediated H₂O₂ production. Notably, the Z-stack analysis under LSCM of the central zone of Spd-treated roots (Figs. 2A,B) revealed SYTOX Orange staining in root cap cells and in the rhizodermis of the subapical region of both WT and S-ZmPAO plants with about a 6-fold increase of the number of stained nuclei in Spd-treated WT plants (Fig. 2A) as compared with control Spd-untreated WT plants,¹¹ the extent of nuclei staining being almost unchanged upon Spd supply (Fig. 2B) in transformed plants.¹¹ As shown in Figure 2E, the S-ZmPAO/WT ratio after Spd treatment was 1/3, with a reversal in comparison to the ratio of 3/1 previously observed for the corresponding control plants, namely Spd-untreated WT and S-ZmPAO plants.11 Presumably, the abnormal PA accumulation consequent to Spd supply was the responsible for the widespread cell death observed in Spd-treated WT plants. Conversely, in Spd-treated S-ZmPAO plants, the rapid removal of the exogenously supplied Spd via the PAO-mediated PA oxidation along with the enhanced antioxidant capacity previously described in these transgenic plants¹⁷ protected cells from widespread death events caused by over-accumulation and possible toxicity of PAs.¹⁸ Taking into account the above-mentioned observations, we propose that in S-ZmPAO plants, the alteration of the PAs/H₂O₂ balance protects cells against extensive death associated to PA over-accumulation stress (Fig. 2) and/or drives them toward developmental PCD in competent tissues.¹¹

Figure 2.

LSCM analysis of cell death detected by SYTOX Orange fluorescence staining in root apices up to 300 μm from the root cap of Spd-treated tobacco plants (Nicotiana tabacum cv Petit Havana SR1) both wild-type (WT) (A, C) and overexpressing maize (Zea mays) PAO (S-ZmPAO) (B, D) in the cell wall. Tobacco plants were grown for 3 weeks in sterile ½ MS salt mixture supplemented with 0.5% (w/v) sucrose and 0.8% (w/v) agar and then hydroponically treated for 24 h with 10 μM Spd in aerated distilled water. A and B represent a Z-stack analysis (thickness 8 µm with a Z-step size of 1 µm) of the central longitudinal plane. (C and D) represent a Z-stack analysis (Z-step size of 1 µm) of the whole root. (E) mean values ± SD relative to the number of nuclei stained with SYTOX Orange in the central 8 µm-Z-stack. (A and B) bar = 30 µm; C and D, bar = 50 µm. P values have been calculated with Student’s t test analysis, comparing the number of SYTOX Orange-labeled nuclei in transgenic tobacco plants with respect to WT plants. ns, not significant, P value > 0.05; *, ** and ***, P values ≤ 0.05, 0.01 and 0.001, respectively.

Tisi A, Federico R, Moreno S, Lucretti S, Moschou PN, Roubelakis-Angelakis KA, et al. Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation. Plant Physiol. 2011;157:200–15. doi: 10.1104/pp.111.173153.