PERTURBATION OF POLYAMINE CATABOLISM CAN STRONGLY AFFECT ROOT DEVELOPMENT AND XYLEM DIFFERENTIATION

Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements and vascular parenchyma and resulted in a higher number of cells resting in G 1 and G 2 phases in the maize primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early-metaxylem (EMX) and late-metaxylem (LMX) precursors. Treatment with either N -prenylagmatine (G3), a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N , N 1 -dimethylthiourea (DMTU), a hydrogen peroxide (H 2 O 2 ) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that G3 inhibited the differentiation of the secondary wall of EMX, LMX elements and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO (A-Zm PAO ) tobacco plants were unaltered, over-expression of Zea mays PAO (S-Zm PAO ) as well as down-regulation of S-adenosyl-L-methionine decarboxylase (RNAi- SAMDC ) in tobacco plants promoted vascular cell differentiation and induced programmed cell death (PCD) in root cap cells. Furthermore, following Spd treatment in maize and Zm PAO over-expression in tobacco, the in vivo H 2 O 2 production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO over-expression, H 2 O 2 derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental PCD. DAB, 3,3’-diaminobenzidine; DAP, 1,3-diaminopropane; DAPI, 4',6-diamidino-2-phenylindole; DMTU, N , N 1 -dimethylthiourea; EMX, early metaxylem; FSC, forward scatter light signal; G3, N -prenylagmatine; HR, hypersensitive response; LMX, late metaxylem; LSCM, laser scanning confocal microscopy; PAs, polyamines; SAMDC, S-adenosyl-L-methionine decarboxylase; PAO, polyamine oxidase; PCD, programmed cell death; PI, propidium iodide; Put, putrescine; Spd, spermidine; Spm, spermine; TEM, transmission electron TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ZmPAO, mays polyamine oxidase. order to achieve full maturity. We also examined the effect of H 2 O 2 derived from PAO-mediated catabolism of Spd, on root growth and cell elongation, cell cycle phase distributions, nuclear condensation and DNA fragmentation. Moreover we investigated the consequences of PAO-mediated H 2 O 2 overproduction on early differentiation of metaxylem and protoxylem precursors, to address the issue of the possible involvement of PAO activity in the PCD of xylem tissues. Finally the effect of the perturbation of PA metabolism on root xylem differentiation was investigated by genetic means, with the aim of discerning the specific contribution of altered PAs on H 2 O 2 levels. allows (w/v) CoCl staining blue Soban, 1982). prior observed under light Control incubated the Spd were pure ZmPAO (Cona al., 2005), used as primary antibody control, and colloidal gold conjugated goat anti-rabbit IgG, used as secondary antibody control, did not show any immunoreactivity. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. The TUNEL assay was carried out on paraffin-embedded cross-sections according to the manufacturer’s (Roche Applied Science, Italy) instructions. Nuclei were stained by incubating in PI (2 μ g/mL) for 2 min. In order to avoid interference due to the binding of PI to ribonucleic acids, samples were incubated in 0.5 mg/mL RNase1-DNase-free for 10 min at 37°C before starting with the TUNEL procedure. As a negative control terminal deoxynucleotidyl transferase enzyme was omitted and as a positive control, DNase1 was added, according to manufacturer’s instructions ( Roche Applied Science, Italy). The percentage of TUNEL-positive nuclei (excitation: 450-500 nm; emission: 515-565 nm) was calculated with respect to the number of nuclei stained with PI in the same sections (excitation: 510-530 nm; emission: 632-675 nm). Nuclear diameter and integrated fluorescence intensity for the estimation of chromatin condensation (Vergani et al., 1998) were measured using Image J Software on digitally acquired images of nuclei stained with PI. apices of wild-type and transgenic S-Zm PAO tobacco plants ( Nicotiana tabacum cv Petit Havana SR1) were also stained with fluoresceine diacetate-SYTOX Orange double fluorescence staining (5 µ M SYTOX Orange in distilled water for 15 min, two washes in distilled water, 10 µ M FDA in distilled water) to discriminate between alive and dead cells. Cell death was visualized in wild-type, transgenic A-Zm PAO and transgenic RNAi- SAMDC tobacco plants ( Nicotiana tabacum cv Xanthi) by SYTOX Orange staining. Root apices stained with PI were observed under LSCM using He-Ne laser emitting at wavelength of 543 nm. The selected emission band ranged from 580 to 680 nm. Root apices stained with fluoresceine diacetate-SYTOX Orange double fluorescence staining were observed under LSCM (argon laser emitting at wavelength of 488 nm and He-Ne laser emitting at wavelength of 543 nm). The selected emission bands ranged from 500 to 530 nm for fluorescein diacetate and from 565 to 580 nm for SYTOX Orange. Z-stacks of 20 µ m were performed with Z-step size of 1 µ m. Shown micrographs are relative to the central Z-stack.


INTRODUCTION
Polyamines (PAs) are ubiquitous, low molecular mass aliphatic polycations. These compounds include the diamine putrescine (Put), the tri-amine spermidine (Spd) and the tetra-amine spermine (Spm) as well as the less common 1,3-diaminopropane (DAP), cadaverine, norspermidine, norspermine and thermospermine. It has been suggested that PAs participate in cell growth and differentiation, by regulating cell cycle progression and physicochemical properties of membranes, as well as by influencing the stabilization and protection of nucleic acid and protein structure (Cohen, 1998). Their essential role in cell growth and differentiation (Wallace et al., 2003;Torrigiani et al., 1987;Kusano et al., 2008) has been strongly supported by the observation that the loss-of-function of genes involved in PA biosynthesis results in early embryonic lethality in mouse (Wang et al., 2004), and in lethal defects in embryonic development in Arabidopsis thaliana (Imai et al., 2004;Urano et al., 2005;Ge et al., 2006). Consistent with the role of PAs as positive regulators of cell growth, these compounds protect cells from apoptosis and their overproduction has been linked to cell over-proliferation and malignant development in animal cells (Wallace et al., 2003). On the other hand, either the increase or the decrease of physiological levels of PAs, induce the execution of the programmed cell death (PCD) syndrome (Seiler and Raul, 2005) either by caspase activation (Stefanelli et al., 1998) or cytochrome c release from mitocondria (Stefanelli et al., 2000), revealing PAs as bivalent regulators of cellular functions.
In plants, it has been suggested that PAs play important roles in diverse developmental processes, including morphogenesis, growth, differentiation, and senescence. Moreover, they are implicated in defence responses to various biotic and abiotic stresses (Bouchereau et al., 1999;Walters, 2003;Kusano et al., 2007;Cuevas et al., 2008) and participate in signaling pathways by regulation of cation channel activities (Kusano et al., 2008) such as during stomatal closure (Liu et al., 2000) and salt stress response (Zhao et al., 2007). As concerns root development, it has been reported that exogenously supplied Spd affects cell elongation and shape as well as the mitotic index in maize primary roots (de Agazio et al., 1995). The involvement of PAs in vascular development (Vera-Sirera et al., 2010) has been recently highlighted by investigation of the 5 morphology of the vessel element and total lack of xylem fibres (Clay and Nelson, 2005). It has been hypothesized that under physiological conditions, ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning (Muñiz et al., 2008).
PAs are catabolized by two classes of amine oxidases, the copper-containing amine oxidases (CuAOs) and the FAD-dependent amine oxidases (PAOs). CuAOs oxidize Put at the primary amino group producing 4-aminobutyraldehyde, H 2 O 2 and ammonia (Medda et al., 2009). PAOs catalyze the oxidation of Spm, Spd, and/or their acetylated derivatives at the secondary amino group (Cona et al., 2006;Angelini et al., 2010). The chemical nature of PAO reaction products depends on the enzyme source and reflects the mode of substrate oxidation. In monocotyledons, PAOs oxidize the carbon at the endo-side of the N 4 -nitrogen of Spd and Spm, producing 4-aminobutyraldehyde and N-(3-aminopropyl)-4-aminobutyraldehyde, respectively, in addition to DAP and H 2 O 2 (Cona et al., 2006;Angelini et al., 2010). In Arabidopsis, PAOs, namely AtPAO1 (Tavladoraki et al., 2006), AtPAO2 (Fincato et al., 2011), AtPAO3 (Moschou et al., 2008c, AtPAO4 (Kamada-Nobusada et al., 2008), oxidize the carbon at the exo-side of the N 4 -nitrogen of Spd and Spm, giving rise to an inter-conversion catabolism, with the production of Spd from Spm and Put from Spd, in addition to 3-aminopropionaldehyde and H 2 O 2 . Thus, H 2 O 2 is the only shared compound in all the AOcatalyzed reactions.
Zea mays PAO (ZmPAO), the best plant PAO characterized so far, is a 53 kD monomeric glycoprotein, containing one FAD molecule and localizing at the apoplast in mature tissues (Cona et al., 2005;Cona et al, 2006). Indeed, using ultrastructural and immunocytochemical techniques, it has been previously reported that ZmPAO protein is localized in both the cytoplasm and cell wall of developing tracheary elements and in the endodermis of maize primary roots, with a progressive increase of cell wall immuno-labeling as the tissues mature (Cona et al., 2005). Until now, three ZmPAO encoding genes (ZmPAO1, ZmPAO2, ZmPAO3) have been identified which show a conserved organization resulting in identical amino acid sequences except for the substitution of one amino acid in ZmPAO3 (Cona et al., 2006). CuAOs and PAOs do not only contribute to important physiological processes through the regulation of cellular PA levels, but do so also through their reaction products: aminoaldehydes, DAP and, markedly, H 2 O 2 (Bouchereau et al., 1999;Walters, 2003;Cona et al., 2006;Angelini et al., 2010). In Arabidopsis, intracellular Spd-derived H 2 O 2 triggers the opening of the hyperpolarization-activated Ca 2+ -permeable channels in pollen, thus regulating pollen tube growth (Wu et al., 2010). In tobacco, H 2 O 2 derived from apoplastic PA catabolism is a key molecule in PCD signaling during the cryptogein-induced hypersensitive response (HR) (Yoda et al., 2006) as 6 well as in the induction of HR marker genes by mitochondrial dysfunction and activation of mitogen-activated protein kinases (Takahashi et al., 2003;Kusano et al., 2007;Moschou et al., 2009). Moreover, it has been recently demonstrated that salinity stress induces the exodus of Spd into the apoplast, resulting in the induction of a PAO-mediated H 2 O 2 burst leading to either tolerance responses or PCD, depending on the levels of intracellular PAs (Moschou et al., 2008b).
The abundant expression of plant CuAOs and ZmPAO in tissues undergoing lignification or extensive cell wall-stiffening events, such as vascular tissues, epidermis and wound periderm, suggests that these enzymes influence plant growth and development as well as taking part in defence responses by affecting cell wall strengthening and rigidity, through H 2 O 2 production (Cona et al., 2005;Cona et al, 2006;Paschalidis and Roubelakis-Angelakis, 2005;Angelini et al., 2008).
Importantly, an additional role for CuAOs and PAOs in PCD associated with developmental differentiation, has been proposed (Møller and McPherson, 1998;Cona et al., 2005). Indeed, the presence of PAO (Cona et al., 2005;this work) and CuAO (Møller and McPherson, 1998) proteins in developing root tracheary elements and sloughed root cap cells of maize and Arabidopsis seedlings, respectively, suggests their potential involvement, as H 2 O 2 delivering sources, in PCD of both cell types. Thus it is evident that both PA anabolism and catabolism through either cell autonomous and non-autonomous pathways may mediate physiological events associated with PAinduced regulation of growth and development as well as induction of PCD involved in tissue differentiation or defence responses.
In the present work, we focused on the role exerted by PAO in root growth and development, and especially in vascular tissue differentiation, by means of both pharmacological and genetic approaches. In particular, we studied PAO expression, by a combined histochemical and immunocytochemical approach, in differentiating maize root tissues focusing on those tissues undergoing developmental PCD, in order to achieve full maturity. We also examined the effect of H 2 O 2 derived from PAO-mediated catabolism of Spd, on root growth and cell elongation, cell cycle phase distributions, nuclear condensation and DNA fragmentation. Moreover we investigated the consequences of PAO-mediated H 2 O 2 overproduction on early differentiation of metaxylem and protoxylem precursors, to address the issue of the possible involvement of PAO activity in the PCD of xylem tissues. Finally the effect of the perturbation of PA metabolism on root xylem differentiation was investigated by genetic means, with the aim of discerning the specific contribution of altered PAs on H 2 O 2 levels.
Overall, our data suggest that H 2 O 2 derived from PA catabolism strongly affects root development and xylem differentiation by inducing differentiation of secondary wall and precocious cell death in xylem precursors of Spd-treated maize primary root as well as by inducing 8 compound has been shown not to affect other cell wall or plasma membrane enzymatic sources of H 2 O 2 , namely NADPH oxidase, peroxidase or oxalate oxidase, or proteases, such as papaine (Cona et al., 2006). In order to discriminate the role of the different reaction products, plants were also treated with either two H 2 O 2 scavengers, N,N 1 -dimethylthiourea (DMTU) and L-ascorbic acid, or chemically synthesized 4-aminobutyraldehyde (ABAL), the aldehyde released during PAOmediated Spd oxidation. Spd strongly inhibited root growth (Supplemental Table S1) by the simultaneous modulation of both cell elongation (Table I) and cell cycle phase distributions (Fig. 2). Table S1, 1 μ M Spd slightly affected root growth while 10 μ M Spd was the lowest concentration at which almost complete inhibition of root total growth (94 ± 9 %) was observed when compared to untreated controls, and hence this concentration was hereafter used in combined hydroponics culture treatments, such as Spd and G3, Spd and DMTU, as well as Spd and L-ascorbic acid. Put, Spm and ABAL were supplied at a concentration equimolar to 10 μ M Spd, while G3 and DMTU were used at the working concentration of 10 μ M and 5 mM, respectively, as previously reported (Angelini et al., 2008;Zacchini and De Agazio, 2001). Treatment with 10 μ M or 1 mM Spd resulted in a decrease in the average length of cortical parenchymal cells (Table I) without affecting PAO enzymatic activity, protein abundance (Supplemental Fig. S1) or PAO immunocytochemical localization (data not shown). Put (10 μ M) did not affect root cell growth, while 10 μ M Spm exerted a similar effect to 10 μ M Spd (data not shown). Supply of 5 mM DMTU or 10 μ M G3 partially reverted the 10 μ M Spd-induced inhibition of both root growth and cortical parenchyma cell elongation (Table I), whereas 10 μ M L-ascorbic acid unexpectedly inhibited root elongation, even at lower concentrations such as 1 μ M, therefore being ineffective in antagonizing Spd action (Supplemental Table S2).

As shown in Supplemental
Furthermore, flow cytometry analysis of root tip nuclei stained with 4',6-diamidino-2phenylindole (DAPI) revealed that a 10 μ M Spd-treatment altered cell cycle phase distributions, with a reduced percentage of nuclei in S phase and a higher number of cells resting in G 1 and G 2 phases compared to untreated control ( Fig. 2A Figure S2 and

Spermidine oxidation induces precocious cell death of early-metaxylem and latemetaxylem precursors and this hinders full differentiation of the secondary cell wall in maize primary roots
With the aim of further studying the effect of Spd on differentiation and the cell death of xylem tissues, we performed electron microscopy on the apex or mature zone of maize primary roots grown in hydroponics either in the presence of or in the absence of 10 μ M Spd and/or G3. Figure   4C shows that G3 treatment did not affect cytoplasm ultrastructure of cells in the apical zone at 1000 µm from the apical meristem, as compared to untreated controls (Fig. 4A), while Spd induced apparent cytoplasm degradation (Fig. 4B), an effect which was only partly reverted by G3 (Fig.   4D). Owing to the considerable difference in total length of Spd-treated roots as compared to untreated controls, sections of mature zones were prepared from a root portion that already existed at the treatment onset, namely 1.5 cm from the seed. At this distance from the seed, EMX and LMX respectively at 1500 µm and 3000 µm from the apical meristem. In control plants they were detectable much further than 3000 µm (image not shown; Fig. 6G).
To verify whether PAO-mediated production of H 2 O 2 resulted in detectable H 2 O 2 levels in xylem differentiating tissues, in situ H 2 O 2 localization by in vivo DAB-staining was performed in untreated control and Spd-treated maize roots. To detect the in vivo Spd-induced production of H 2 O 2 , DAB-staining was achieved by simultaneously supplying Spd and DAB in hydroponics. As shown in Figure  13 significantly different in all Z-stacks analysed). Consistent with the results found in Spd-treated maize roots, a significantly higher generation of H 2 O 2 was detected in S-ZmPAO tobacco roots (Fig.   7E, F) as compared to wild-type roots (Fig. 7G, H). In particular, H 2 O 2 production was visualized by Amplex Ultra Red (AUR) staining in xylem tissue at about 370 µm from the root cap in transgenic plants (Fig. 7E), whereas in wild-type tobacco roots the first detectable H 2 O 2 appeared at about 1200 µm from the root cap (Fig. 7G). These results further support the role of H 2 O 2 generated by the PAO-mediated PA oxidation in early xylem tissue differentiation and cell death.

DISCUSSION
In this work we show that ZmPAO is mainly expressed in primary root tissues undergoing developmental PCD, such as EMX, LMX and associated paratracheal parenchyma, as well as in sloughed root cap cells (Fig. 1). In xylem precursors, PAO mainly has a cytoplasmic localization and it was shown that as the cells mature, the enzyme will accumulate in the cell wall parallel to 14 secondary wall deposition and developmental cell death (Cona et al., 2005). Development of vascular tissues is associated with cell cycle/endocycle progression and amine oxidase expression in tobacco (Paschalidis and Roubelakis-Angelakis, 2005). It has been proposed that these events are to be linked with the physiological need for the higher production of H 2 O 2 via amine oxidases in the secretion pathway (Cona et al., 2005) and in the apoplast to drive peroxidase-catalyzed cross-linking of cell wall polysaccharides and proteins to complete cell wall maturation, to stimulate endoreduplication and possibly to act as a signal triggering xylem precursor cell death (Cona et al., 2005;Paschalidis and Roubelakis-Angelakis, 2005). Accordingly, Spd treatment at a concentration similar to that found in maize roots (Cohen, 1998) suppressed root growth by inhibiting cell elongation and altering cell cycle phase distributions in maize primary root apices, thus allowing cells to initiate their differentiation programs (Table I; Fig. 2). Importantly, the PAO inhibitor G3 or the H 2 O 2 scavenger DMTU, reverted Spd-induced inhibition of cell growth and reduction of percentage of nuclei in S phase, while ABAL, G3 or DMTU alone failed to affect these events, thus indicating that the observed effect was exclusively mediated by H 2 O 2 generated by PAO activity.
Unexpectedly, the H 2 O 2 scavenger L-ascorbic acid inhibited root elongation by itself, being ineffective in antagonizing Spd action (Supplemental Table S2). In fact L-ascorbic acid, although it is an effective antioxidant in vivo (Pignocchi and Foyer, 2003), is rapidly oxidized by the apoplastic ascorbate oxidase to dehydroascorbate, which can be transported into the cytoplasm to negatively affect cell cycle progression (de Pinto et al., 1999), or can be further degraded in the cell wall with concomitant generation of H 2 O 2 (Kärkönen and Fry, 2006). Moreover, Spd treatment induced DNA fragmentation, which was also reverted by G3 or DMTU (Fig. 3). The apparent lack of DNA laddering in Spd-treated roots could be explained by the fact that this event in plants is not universal. In this regard, there is no evidence of DNA laddering in PCD of tracheary elements, in which S1-type nucleases may degrade chromosomal DNA instead of the ladder-forming nucleases seen in animal apoptosis (Ito and Fukuda, 2002). The early differentiation of xylem precursors (Fig.   4, 5 and 6) associated with enhanced H 2 O 2 production (Fig. 6) in Spd-treated maize primary roots, further supports the hypothesis that root differentiation could be mediated by the PAO-driven Spd oxidation. Moreover, the incomplete differentiation of the secondary wall in EMX and LMX elements observed after Spd treatment, could be interpreted as the result of a sudden H 2 O 2 burst leading to premature cell death of the precursor, occurring prior to the onset of secondary wall deposition (Fig. 4). Accordingly, PAO over-expression in the cell wall of tobacco plants resulted in early differentiation of root xylem precursors and cell death of sloughed root cap cells and rhizodermis of the subapical region, as well as enhanced in vivo H 2 O 2 production in xylem tissues (Fig. 7). On the other hand, the incomplete cell wall differentiation observed in these tissues after G3 treatment (Fig. 4) could be ascribed to the inhibition of PAO activity and lack of H 2 O 2 production which represents an essential signal for the biosynthesis of the secondary wall (Potikha et al., 1999). Indeed, combined G3/Spd treatment resulted in an intermediate degree of differentiation of the secondary wall (Fig. 4). In apparent contradiction with our hypothesis about the role played by PA in xylem differentiation and cell death, is the finding that exogenous Spm  The proposed scenario is supported by several studies performed in both plants or animals, where a role of PA oxidation products in mitochondrial damage and cell death has been reported. In particular, in plants, it has been shown that PAs accumulate in the intercellular space during HR in tobacco, and that H 2 O 2 produced by PAO-mediated oxidation initiates a signaling transduction pathway, mediated by mitochondrial dysfunction, leading to activation of mitogen-activated protein kinases SIPK and WIPK, up-regulation of HR associated genes and ultimately to cell death (Takahashi et al., 2003;Takahashi et al., 2004;Yoda et al., 2006). Other data also have emphasised  Further studies addressing the molecular mechanisms underlying these processes will be fundamental to identifying potential targets of intervention aimed at ameliorating defence responses and improving the morpho-functional traits of plants. ABAL was prepared by acid hydrolysis of ABAL diethyl acetal in 0.5 M HCl at room temperature. ABAL production was determined spectrophotometrically by measuring the formation of a yellow adduct (ε 430nm = 1.86 × 10 3 M -1 cm -1 ) produced through the reaction of containing 5 mM 2-aminobenzaldehyde at 37°C. Reaction was blocked after 20 min by adding trichloroacetic acid to a final concentration of 4% (w/v).

Plant materials and treatments
Maize (Zea mays "CORONA"; from Monsanto Agricoltura) seeds were soaked in tap water for 12 h and germinated in the dark, at 21°C over three layers of filter paper moistened with distilled water. A stock of 5-d-old maize seedlings was selected on the basis of root length (3 cm) and transferred into an aerated hydroponics culture with magnetic stirring. Seedlings were grown hydroponically for 24 h in distilled water (control plants) or alternatively supplied with Spd

Enzyme activity, protein assays and DNA ladder analysis
Western blot, PAO enzymatic activity and protein level determination were carried out in maize primary root apex extracts as previously described (Angelini et al., 2008). For DNA ladder detection, genomic DNA was isolated from maize primary root apices and run on an agarose gel according to Gunawardena et al. (2001).

Histochemical visualization under light microscopy of ZmPAO enzyme activity
Apical segments (5-mm-long) from maize primary root were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde and 0.05% sucrose in 0.1 M sodium phosphate buffer (pH 6.5), for 1 h at room temperature under vacuum. After fixation, specimens were embedded in 0.4% agar. ZmPAO activity was histochemically detected in longitudinal sections using a peroxidase-coupled assay with the chromogenic peroxidase substrate DAB that is oxidised to a brown compound upon PAO-mediated
ZmPAO immunohistochemistry. Paraffin-embedded cross-sections were de-waxed and hydrated as described above and then processed for immunohistochemistry using a rabbit polyclonal antiserum against ZmPAO, fractionated by affinity chromatography through a CNBr-activated Sepharose 4B column coupled to bromelain, according to the manufacturer's instructions, to eliminate anti-glycan antibodies (Cona et al., 2005). The resulting anti-serum against ZmPAO polypeptide recognised a single protein band corresponding to ZmPAO when probed against maize crude extract. As the three maize genes encoding ZmPAO express the same protein product, the antiserum recognised all the ZmPAO gene products. After washing first in distilled water (30 min) and in 0.1M sodium phosphate buffer (pH 7; buffer A) for 10 min, sections were incubated in blocking solution (5% non-fat dry milk in buffer A) for 3 h. Afterwards, sections were incubated in the primary antibody diluted 1:2000 in buffer A containing 2.5% non-fat dry milk for 24 h at 4°C.
Control sections were incubated in the absence of primary antibody. Subsequently, sections were washed in buffer A (for 3 times 10 min each) and then incubated in goat anti-rabbit IgG conjugated to 5-nm colloidal gold particles diluted 1:200 (w/v) in medium A containing 1% normal goat serum for 2 h at room temperature. The reaction was finally visualized by using a silver enhancement kit.
Anti-ZmPAO antiserum pre-adsorbed onto a column of CNBr-activated Sepharose 4B conjugated to pure ZmPAO (Cona et al., 2005), used as primary antibody control, and colloidal gold conjugated goat anti-rabbit IgG, used as secondary antibody control, did not show any immunoreactivity.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. The
TUNEL assay was carried out on paraffin-embedded cross-sections according to the manufacturer's (Roche Applied Science, Italy) instructions. Nuclei were stained by incubating in PI (2 μ g/mL) for 2 min. In order to avoid interference due to the binding of PI to ribonucleic acids, samples were incubated in 0.5 mg/mL RNase1-DNase-free for 10 min at 37°C before starting with the TUNEL procedure. As a negative control terminal deoxynucleotidyl transferase enzyme was omitted and as Microsystems, Italy) were contrasted by uranyl acetate and lead citrate and collected on collodiumcoated nickel grids. Sections were observed in a CM120 electron microscope (Philips, Italy) and images were electronically captured.

Propidium iodide staining of cell wall and cell death/vitality analysis
Longitudinal sections (150-μm-thick) were cut with a vibratome from agar-embedded maize primary root apices (5-mm-long) from untreated (control) and Spd-treated plants, stained with PI and observed under LSCM at 543 nm (He-Ne laser) to reveal the outlines of cells in the root apex.
The selected emission band ranged from 580 to 680 nm.

In situ detection of H 2 O 2 in maize and tobacco root
In situ detection of H 2 O 2 in the maize primary root was achieved by in vivo DAB-staining (Graham and Karnowsky, 1966). Maize seedlings were grown hydroponically for 24 h in distilled water (control plants)

Supplemental Data
The following material is available in the online version of this article. Supplemental Figure S3. UV light-induced autofluorescence of maize primary root after H 2 O 2 or ABAL treatment.
Supplemental Figure S4. Effect of exogenously supplied Spd on in vivo H 2 O 2 production in maize primary root.
Supplemental Table S1. Effect of Spd treatment on maize primary root growth, dose-response relationship.
Supplemental Table S2. Effect of L-ascorbic acid treatment on maize primary root growth.