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First published online November 9, 2007; 10.1104/pp.107.108902 Plant Physiology 146:162-177 (2008) © 2008 American Society of Plant Biologists
Involvement of Polyamine Oxidase in Wound Healing1,2,[W]Dipartimento di Biologia, Università Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy (R.A., A.T., M.M.C., R.F., A.C.); Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Monterotondo, 00016 Rome, Italy (G.R.); and Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy (M.B.)
Hydrogen peroxide (H2O2) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H2O2 accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant polyamine oxidases (PAOs) are H2O2 producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H2O2 production in peroxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H2O2 production from that generated by peroxidase, oxalate oxidase, or by NADPH oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H2O2 production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and peroxidase-mediated events during wound healing.
Reactive oxygen species (ROS) are common components of plant developmental processes and defense responses. Their roles in developmental programmed cell death (PCD; for review, see Rogers, 2005  ), stomatal closure (Pei et al., 2000; Kwak et al., 2003), root gravitropism (Joo et al., 2001), and hair cell expansion (Foreman et al., 2003) are at present widely accepted. Rapid generation of ROS is also a basic component of a plant's response to pathogen challenges (for review, see Apel and Hirt, 2004) and wound stress (Orozco-Cárdenas et al., 2001), such as those which occur during physical injury and herbivore or insect attacks. ROS contribute to plant disease resistance either exerting a direct antimicrobical activity and/or by driving peroxidase-mediated oxidative cross-linking of cell wall components. Moreover, ROS induce hypersensitive response (HR) cell death in infected areas (Levine et al., 1994; Solomon et al., 1999; Delledonne et al., 2001) and behave as second messengers in signal transduction pathways leading to increased expression of defense genes (Orozco-Cárdenas et al., 2001; Jih et al., 2003; Takahashi et al., 2004; for review, see Apel and Hirt, 2004). The production of ROS in the cell wall is achieved by different enzymatic sources, some of which are membrane-bound NADPH oxidases (for review, see Torres and Dangl, 2005) and apoplastic oxalate oxidases (for review, see Lane, 2002), peroxidases (for review, see Bolwell et al., 2002), and amine oxidases (for review, see Cona et al., 2006a). All of these ROS delivering systems have been involved in plant defense responses, depending on tissue sources and disease identities. Up to date, NADPH oxidase, the plant homolog of the gp91phox subunit of the mammalian phagocyte NADPH oxidase, is believed to be the principal source of extracellular ROS, in both development and defense. Indeed, NADPH oxidase is involved in pathogen resistance in Arabidopsis (Arabidopsis thaliana; Torres et al., 2002; Love et al., 2005), tobacco (Nicotiana tabacum; Simon-Plas et al., 2002; Yoshioka et al., 2003), and potato (Solanum tuberosum; Kobayashi et al., 2006), wound healing in tomato (Solanum lycopersicum; Sagi et al., 2004), and ABA-induced antioxidant defense in maize (Zea mays) leaves (Jiang and Zhang, 2003). Moreover, this enzyme has recently been demonstrated to also have a role in suppressing the spread of cell death to the uninfected cells surrounding HR sites (Torres et al., 2005). Nevertheless, the specific contribution of each of the other ROS enzymatic sources to the apoplastic ROS production deserves attention and demands further analysis. In this connection an increasing amount of evidence indicates amine oxidases as players in extracellular hydrogen peroxide (H2O2) production during plant development and defense (Allan and Fluhr, 1997; Cona et al., 2006a). Amine oxidases are represented by an heterogeneous group of enzymes, including copper-containing amine oxidases (CuAO) and flavin-containing polyamine oxidases (PAO). Plant CuAO and PAO oxidize polyamines at the primary and secondary amino group, respectively, producing an aminoaldehyde, H2O2, as well as NH3 (CuAO) or 1,3-diaminopropane (Dap; PAO).
H2O2 deriving from polyamine oxidative catabolism has been correlated to cell wall stiffening events responsible for developmental cell wall maturation in chickpea (Cicer arietinum; Cona et al., 2006a
CuAO have also been involved in extracellular H2O2 production during defense responses to pathogen attacks and wound healing (Cona et al., 2006a
Analogously to what has been demonstrated in human cancer cells, which can undergo PCD through catabolism of intracellular polyamines (Ha et al., 1997
The aim of this study was to analyze the specific contribution of ZmPAO to the apoplastic ROS production during the wound healing response in the maize mesocotyl, exploiting the in vivo use of N-prenylagmatine (G3; Ki = 1.5 x 10–8 M), a powerful inhibitor of ZmPAO activity (Federico et al., 2001
Wounding Induces an Increase in ZmPAO Expression Level
To investigate the involvement of ZmPAO in the extracellular production of H2O2 during wound healing, we performed a time-course analysis of ZmPAO activity levels as well as ZmPAO protein and mRNA accumulation in response to wounding in the outer tissues (i.e. cortical and epidermal tissues) of the nongrowing zone of the maize mesocotyl. These tissues, in which ZmPAO gene expression was previously demonstrated to be up-regulated by light and down-regulated by auxin (Cona et al., 2003 As shown in Figure 1A , the level of extractable ZmPAO activity expressed on a fresh weight basis increased in the mesocotyl outer tissues of wounded plants as compared to the respective unwounded (control) plants until the last time checked (72 h), when its level was 2-fold that of control plants. The greatest difference was detectable 24 h after wounding (Fig. 1A). Moreover, a systemic induction of extractable ZmPAO activity was observed in coleoptiles either at 24 or at 48 h after mesocotyl wounding (Fig. 2 ). Very similar results were obtained when extractable ZmPAO activity was expressed on total protein basis (data not shown). Experiments reported in Figures 1 and 2 were performed using 5-d-old seedlings grown on paper under loam (average stem length = 6.5 cm); however, similar results were obtained when 3-d-old seedlings germinated on paper were kept into a hydroponic culture containing a nutrient solution for 3 d before being wounded (data not shown; average stem length = 6.5 cm).
ZmPAO protein accumulation patterns in the outer tissues taken from wounded and control plants (Fig. 1B, top insert) resembled the relative ZmPAO activity values at each specific time (Fig. 1A). Indeed a ZmPAO protein band of higher intensity was already evident at 12 h after injury (Fig. 1B, top insert). Moreover higher ZmPAO protein levels were observed in the wounded with respect to control seedlings at every time checked (Fig. 1B, top insert) until 72 h from wounding (data not shown).
Northern-blot analysis of the total RNA purified from mesocotyl outer tissues at different time was performed using ZmPAO cDNA (EMBL DataBase accession number AJ002204) as a probe (Fig. 1B, middle insert; Tavladoraki et al., 1998
No difference occurred after wounding in wall-bound to extractable ZmPAO activity ratio, since wall-bound ZmPAO activity increased to the same extent as the extractable enzyme activity at every time checked (data not shown). This result is consistent with previous studies reporting that no significant differences were observed in wall-bound to extractable ZmPAO activity ratio after light or auxin treatments (Cona et al., 2003
To investigate the tissue localization of the wound-induced ZmPAO activity increase, ZmPAO activity was histochemically detected by 3,3'-diaminobenzidine (DAB) staining in transverse sections of mesocotyl from wounded maize plants at 0, 24, 48, and 72 h after wounding (Fig. 3
). The specificity of staining for PAO activity was confirmed by the evidence that sections from wounded plants, incubated in the staining solution lacking spermidine (Spd), were unstained (data not shown). Histochemical ZmPAO activity gradually intensified in the epidermis and in the wound periderm, displaying a strong staining 72 h after injury (Fig. 3D). Cortical parenchyma also exhibited a DAB staining increase from 0 to 72 h after wounding, although to a lower extent. As expected (Cona et al., 2005
ZmPAO Expression Is Induced by JA Treatment
It is well known that JA accumulates locally and systemically in response to tissue damage such as that caused by feeding insect (for review, see León et al., 2001 The dose-response curve (Fig. 4 ) relative to the effects of JA supply on extractable ZmPAO activity levels revealed that optimal JA concentrations averaged around 50 µM, for both mesocotyl outer tissues and coleoptiles. Indeed, despite the fact that levels of ZmPAO activity expressed on a fresh weight basis gradually increased in all treated plants as compared to JA-untreated (control) plants, including the 135 µM JA-treated plants (Fig. 4A), the level of ZmPAO activity expressed on total protein basis decreased in the latter plants as compared to 50 µM JA-treated plants both in mesocotyls (Fig. 4B) and in coleoptiles (data not shown), probably due to a nonspecific effect on total protein content.
Concerning the time-course analysis (Fig. 5 ), experiments were focused on the effect of JA supply on ZmPAO gene expression in mesocotyl outer tissues, since it represents the model system as regards the analysis of ZmPAO expression regulation (Cona et al., 2003 ). Analogously to what was observed after wounding, extractable ZmPAO activity expressed on a fresh weight basis increased in both 50 and 100 µM JA-treated plants compared to control plants until the last time checked (72 h; Fig. 5A, top insert). However, ZmPAO activity expressed on total protein basis exhibited a lower increase in 100 µM JA-treated plants with respect to the corresponding 50 µM JA-treated plants at either 48 or 72 h from treatment onset (Fig. 5A, bottom insert), further confirming the occurrence of a nonspecific JA effect on total protein content for prolonged JA treatments at concentration higher than 50 µM. Noteworthily, spraying plants with aqueous solution containing JA or methyl-JA resulted in a lower increase of ZmPAO expression likely due to a bad penetration of the chemicals.
Western-blot and northern-blot analysis of JA-treated and control samples showed a ZmPAO protein and mRNA accumulation pattern similar to that exhibited in the corresponding ZmPAO activity values (Fig. 5B, top insert). Indeed, western-blot analysis of 50 µM JA-treated seedlings revealed that the intensity of ZmPAO protein bands were higher at each time checked (Fig. 5B, top insert). Moreover, northern-blot analysis showed more intense hybridization bands in 50 µM JA-treated samples at every time checked, with the highest response at 72 h after treatment onset (Fig. 5B, middle insert).
It has been suggested that salicylic acid (SA)- and JA-signaling pathways are antagonistic to each other and SA has been reported to suppress expression of wound- and JA-induced genes (for review, see Pieterse and Van Loon, 2004 ZmPAO activity was not altered in SA-treated versus SA-untreated plants up to 1 mM SA (data not shown) that represents the highest nontoxic concentration in our experimental system. Otherwise, SA and JA, both 50 and 100 µM, displayed a synergistic effect on ZmPAO activity. In fact, ZmPAO activity in 50 µM JA/1 mM SA- or in 100 µM JA/1 mM SA-treated plants exhibited higher levels after 48 h from the onset of the treatments as compared, respectively, to 50 µM JA- or 100 µM JA-treated plants. Indeed, differences were statistically significant when ZmPAO was expressed either on fresh weight basis or total protein content (Fig. 6, A and B ).
Wound-Induced H2O2 Accumulation Is Inhibited by G3, a Specific ZmPAO Inhibitor
G3 is a specific, powerful inhibitor of ZmPAO activity with a Ki of 1.5 x 10–8 M (Federico et al., 2001
G3 Strongly Reduces Lignin and Suberin Polyphenolic Domain Levels Along the Wound Periderm
Plants respond to wounding by differentiating a lignosuberized layer that constitutes the wound periderm. Besides fortifying cell wall structure, lignin and suberin form important defense barriers against dehydration and pathogens. Lignin is a polyphenolic polymer whose composition is highly variable, differing even among different cell types of a single tissue (Joseleau and Ruel, 1997 To explore the role played by ZmPAO in lignin and suberin deposition along wound lesion, we analyzed the effect of G3 supply on the spatiotemporal accumulation of ester-linked phenolics, lignin and suberin, during wound healing, by the means of a combined approach of UV-light-induced autofluorescence and specific staining. To this scope, hand-cut cross sections from wounded mesocotyls of G3-treated and G3-untreated maize plants were examined for either autofluorescence coupled with ammonium hydroxide treatment or Sudan IV staining observed under light microscopy at 0, 24, 48, and 72 h after wounding.
Cross sections of wounded mesocotyls from G3-untreated seedlings showed UV-induced blue autofluorescence in the phloem as well as in the cortical and pith parenchyma cells (Fig. 8, A, C, and E
) that turned to blue green upon treatment of the sections with ammonium hydroxide (Fig. 9, A, C, and E
). This is indicative of the presence of ester-linked phenolics within cell walls (Harris and Hartley, 1976
As shown in Figure 8, G3 treatment strongly inhibited deposition of poliphenolic associated to lignin and suberin polyphenolic domain occurring at the wound site of G3-untreated plants from 0 h up to 72 h after injury. Indeed the increase in yellow autofluorescence observable along the lesions of G3-untreated plants (Fig. 8, A, C, and E) was greatly impaired by the G3-mediated ZmPAO inhibition (Fig. 8, B, D, and F). The autofluorescence level, after ammonium hydroxide treatment of cross section, was not affected by G3 treatment in phloem as well as in cortical and pith parenchyma, indicating that the level of ester-linked phenolics was unchanged. Nevertheless, G3 treatment inhibited lignin deposition in the innermost cell layers underneath wound periderm (Fig. 9). In fact, the intensity and the tissue extension of UV-induced blue autofluorescence, after ammonium hydroxide treatment, evident along the wound of G3-untreated plants (Fig. 9, A, C, and E) was strongly reduced after G3 treatment (Fig. 9, B, D, and F). Conversely, G3 supply failed to inhibit suberin aliphatic-domain deposition (Fig. 10) as demonstrated by the absence of any detectable differences in Sudan IV staining intensity along the periderm of sections from G3-untreated (Fig. 10, A, C, and E) and G3-treated plants (Fig. 10, B, D, and F).
In plants, polyamine levels are altered by biotic and abiotic stresses (for review, see Walters, 2003
To verify the involvement of ZmPAO in the formation of lignosuberized depositions along wound periderm, we exploited the availability of transgenic tobacco plants constitutively expressing ZmPAO in the cell wall (Rea et al., 2004
In detail, the microscopic analysis of cross sections from wild-type tobacco plants indicated that lignosuberization events occurred early on the external border and late on the innermost part of the wound lesion. Accordingly, after 72 h from wounding a remarkable and similarly intense yellow autofluorescence was already detected under blue light-induced fluorescence microscopy on the external margin of the wound of both wild-type (Fig. 11I) and ZmPAO transgenic plants (data not shown), while the middle and inner boundary cells of the wound periderm displayed a very different lignosuberization level in these two sets of plants for each time examined (Fig. 11, A–C and F–H). Indeed, a very pale yellow autofluorescence was detected only on the outer margins of the wound periderm in wild-type plants at 48 h after injury (data not shown), whose intensity and extension toward the boundary cells of the middle part of the wound increased after 72 h (Fig. 11, A and C) until becoming evident in the innermost terminal part of the wound lesion at 96 h (Fig. 11G). Conversely, in ZmPAO transgenic tobacco plants at 72 h from injury a stronger yellow autofluorescence as compared to the corresponding wild-type plants was revealed on the boundary cells of both the middle part and the innermost terminal part of the wound (Fig. 11, B and F). Moreover, in the same plants at 96 h from wounding, a very strong yellow autofluorescence was present in the whole lesion (Fig. 11H), especially in the innermost part of the wound, indicating that an acceleration of wound healing processes occurred as a consequence of ZmPAO ectopic expression. The presence of lignin in the cell walls of wound periderm was also suggested by the emission of blue autofluorescence under UV light upon ammonium hydroxide treatment both in wild-type (data not shown) and transgenic (Fig. 12, D and F) tobacco plants (Harris and Hartley, 1976 ), as compared to respective wild-type (data not shown) and transgenic (Fig. 12, C and E) untreated plants. A more sensitive investigation performed under confocal laser scanning microscopy allowed to detect healing progressing inside the wound cavity at earlier developmental stages as compared to the blue- or UV-induced fluorescence microscopic analysis (Fig. 11, L and M). The three-dimensional images of cross sections from wild-type and transgenic tobacco plants revealed a remarkable blue-induced autofluorescence on the innermost terminal part of the wound of transgenic plants (Fig. 11M) already 48 h after wounding, that was not present in wild-type plants at the same time (Fig. 11L), confirming the role played by ectopically expressed ZmPAO in accelerating wound healing progression. Spd-treated wild-type plants (Fig. 11D) showed a stronger yellow autofluorescence under blue-induced fluorescence microscopy as compared to the Spd-untreated plants 72 h after wounding (Fig. 11A). As shown in Figure 11D, autofluorescence was present also in the inside end of the lesion, suggesting that an endogenous amine oxidase could be involved in wound healing processes in tobacco plants. As expected, Spd supply in ZmPAO transgenic plants (Fig. 11E) resulted in even stronger yellow autofluorescence as compared to Spd-untreated transgenic plants (Fig. 11B).
To counteract chewing insects or larger herbivores as well as invasion by microbial pathogens, plants make use of preexisting physical barriers and inducible defense mechanisms, which largely depend on the transcriptional activation of specific genes, directed to the healing of the damaged tissues and to prevent further damage. In this connection, owing to the ease of microbe penetration allowed for the physical injury consequent to either environmental stresses or animal feeding, plants respond to wounding by enhancing defense capacity against microbial pathogens. Therefore, defense pathways activated in response to physical injury or pathogen attack cross communicate each other, leading to the activation of distinct or partially overlapping sets of genes. A common feature of these defense strategies is a transient production of ROS, whose chemical identity is spatially and temporally regulated. In this regard, it is known that superoxide anion is produced in the damaged tissues of wounded plants only a few minutes after injury, while H2O2 is produced both locally and systemically, reaching a peak after 4 to 6 h (for review, see León et al., 2001 ).
Here, it is suggested that ZmPAO represents the main source of apoplastic H2O2 production during wound healing. Indeed, the increase in ZmPAO enzyme activity, protein, and mRNA levels in mesocotyls from wounded plants (Fig. 1) clearly demonstrates the occurrence of a positive regulation of ZmPAO expression in damaged tissues. Noteworthily, the temporal features of the increase of ZmPAO expression suggest that ZmPAO is involved in the late phase of the oxidative burst. Indeed, the increase in ZmPAO mRNA accumulation that precedes the rise in ZmPAO enzyme activity and protein levels is strongly evident 6 h after wounding and persists for several hours, up to 48 h. This result is in keeping with previous studies, suggesting that H2O2 produced by polyamine degradation contributes to the second phase of ROS production during TMV-induced HR in tobacco plants (Yoda et al., 2003
Noteworthy is the wound-induced increase of ZmPAO activity; besides confirming the ability of epidermal ZmPAO to be rapidly regulated by various external stimuli, it presents interesting analogies with either the light-induced increase of ZmPAO gene expression or the ZmPAO-mediated H2O2 production upon cantharidin treatments, both also specifically occurring in the outer tissues of the maize mesocotyls. Indole-3-acetic acid, whose levels are known to decline in maize mesocotyl epidermis upon light exposure (Barker-Bridgers et al., 1998
With the aim of analyzing the molecular signals responsible for the control of ZmPAO expression upon mechanical injury, we tested the effect of plant treatments with JA and SA, well known mediators of wound and pathogen signaling, respectively. In line with the hypothesis of ZmPAO involvement in wound healing processes, JA treatments resulted in an increase of ZmPAO expression, either in coleoptiles or in mesocotyl outer tissues (Figs. 4 and 5). Conversely, SA treatments had no effect on ZmPAO activity (data not shown), confirming the specificity of the role played by JA in the activation of the wound-responsive ZmPAO gene. Worth noting is that JA-mediated increase of ZmPAO gene expression could be ascribed to the presence of a G-box motif in its promoter region (Cervelli et al., 2000
The availability of a specific and selective inhibitor of ZmPAO activity, such as G3 (Ki = 1.5 x 10–8 M; Federico et al., 2001 The absence of variation in polyamine content after wounding in mesocotyl outer tissues (Supplemental Table S1) could be ascribed to the occurrence of a strictly balanced rate of polyamine biosynthesis and degradation, which results in no detectable polyamine accumulation. On the other hand, this phenomenon could arise also from variations occurring only in tissues directly involved in wound healing processes, such as the epidermis and wound periderm, and therefore not measurable in a whole tissue extract.
A stronger causal relationship between ZmPAO activity and lignosuberized depositions along wound lesions has been established by the analysis of transgenic tobacco plants constitutively expressing ZmPAO in the cell wall. As previously reported (Rea et al., 2004 Overall these results suggest the involvement of ZmPAO in wound healing processes, as a main provider of apoplastic H2O2 needed for cell wall strengthening and lignosuberization events.
Chemicals
N-prenylagmatine [G3; N-(4-aminobutyl)-N'-(3-methyl-2-butenyl)guanidine] was prepared as previously described (Corelli et al., 2002
Maize (Zea mays ‘Corona’; from Monsanto Agricoltura) seeds were soaked for 12 h in running water and germinated on paper under 2 cm of loam, at 22°C in a growth chamber in the dark. Five-day-old seedlings (average stem length = 6.5 cm; average mesocotyl length = 4 cm) were longitudinally wounded (wound length = 2 cm) on the nongrowing zone of the mesocotyl with a blade, precisely from 1 cm above the seed up to 1 cm below the node. After injury some seedlings were treated with 1 x 10–5 M G3 in aqueous solution, by brushing 50 µL of the inhibitor along the wound at 0, 24, and 48 h after wounding. In detail, the inhibitor was first applied with a pipette over the wound surface and then brushed to avoid dripping. G3-untreated plants were supplied with 50 µL of distilled water. Five-, 6-, 7- and 8-d-old plants were collected at 0, 24, 48, and 72 h after wounding, respectively. Alternatively, after soaking, maize seeds were germinated on paper at 22°C in a growth chamber in the dark for 3 d (average stem length = 1 cm) and then transferred into an aerated hydroponic culture supplied with a nutrient solution. Nutrient solution was prepared as follows: 4 mM KNO3, 1 mM Na2HPO4, 1 mM MgSO4, 0.5 mM CaCl2, 1 mg L–1 Fe-citrate, pH value was adjusted to 6 with NaOH. Hormone treatments were performed by adding JA and SA at the appropriate concentration into the nutrient solution. Nutrient solution alone (control plants) or nutrient solution plus JA or SA or JA/SA were replaced every day. Four-, 5-, and 6-d-old plants were harvested after 24, 48, and 72 h from the onset of hormone treatments, respectively. Coleoptiles from unwounded (control) and wounded maize plants were collected at 0, 24, and 48 h after wounding, as well as coleoptiles from untreated (control) and JA-treated plants were collected at 48 h from the onset of JA treatments and then used for extractable ZmPAO activity analysis. Two-centimeter-long mesocotyl segments were excised after eliminating 1 cm above the seed from control and wounded maize plants at the indicated times after wounding. Mesocotyl segments of variable length depending on plant age were excised after eliminating 0.3 cm above the seed and 0.3 cm below the node from control and hormone-treated maize plants at the indicated times after the onset of the treatments. Cortical plus epidermal tissues (outer tissues) from the mesocotyl were obtained by drawing out the stele and used for determination of enzyme activity, western-blot, northern-blot, and polyamine level analysis. Maize plants were kept in the dark until the end of the treatments. All technical operations were performed under photomorphogenically inactive green light.
Plants of tobacco (Nicotiana tabacum ‘Petit Havana SR1’) constitutively expressing ZmPAO in the cell wall (Rea et al., 2004
Plant material was ground with mortar and pestle at 4°C in 0.2 M sodium phosphate buffer, pH 6.5 (tissue to buffer ratio 1:3, w/v). Homogenates were centrifuged at 12,000g for 10 min at 4°C. Supernatants were used for the determination of protein concentration, extractable ZmPAO activity, and western-blot analysis. For determination of wall-bound ZmPAO activity, pellets obtained after centrifugation of crude homogenates were resuspended in the appropriate volume of 0.2 M sodium phosphate buffer, pH 6.5, containing 0.01% Triton X-100 and then filtered onto Miracloth. This step was repeated three times to remove traces of extractable enzyme. The washed pellets were resuspended in 0.2 M sodium phosphate buffer, pH 6.5 [1 mL (g fresh weight)–1], and the suspension used for the polarographic determination of wall-bound ZmPAO activity as described in the following paragraph.
Extractable ZmPAO enzyme activity was measured spectrophotometrically by following the formation of a pink adduct ( Enzyme activities were expressed in International Units (U; 1 unit is the amount of enzyme that catalyzes the oxidation of 1 µmol substrate per min). The reported data are the average of values obtained in five different experiments, each performed with two technical replicates. All data were obtained at 25°C.
Protein content was evaluated by the method of Bradford (1976)
RNA-blot analysis was performed on maize mesocotyl outer tissues from wounded, JA-treated, and control plants at different times from the onset of the treatment.
Total RNA was isolated using the TRIzol reagent, following the manufacturer's instructions. Blotting and hybridization procedures were performed as reported by Sambrook et al. (1989)
H2O2 was directly visualized in maize mesocotyls by DAB staining. Etiolated maize seedlings (average stem length = 6 cm) were longitudinally wounded on the mesocotyl with a razor blade. Alternatively, an epidermal strip was removed. Afterward wounded seedlings were deprived of the roots and then incubated for 30 min in 10 mM NaH2PO4 containing 60 µg mL–1 peroxidase in the presence or absence of 1 x 10–5 M G3, prior to be supplied with 1 mg mL–1 DAB for 8 h. Reactions were stopped by thoroughly washing in distilled water. Experiments were performed independently three times and a minimum of three plants for each treatment were observed, yielding reproducible results. Single representative experiments are shown in the figures.
Hand-cut cross sections (approximately 100 µm thick) obtained from the wounded zone of the maize mesocotyl at 0, 24, 48, and 72 h after wounding were utilized for light and fluorescence microscopic investigation of ZmPAO histochemistry as well as cell wall-associated ester-linked phenolics, lignin and suberin aliphatic and polyphenolic domain. Moreover, hand-cut cross sections (approximately 100 µm thick) obtained from the wounded zone of tobacco second internodes at 48, 72, and 96 h after wounding were used for fluorescence and confocal laser scanning microscopic investigation of lignosuberized deposition. To this purpose, some sections were mounted on glass slides and observed in an Axioplan 2 Zeiss microscope equipped with a video camera (Delta Sistemi). Digitized images were acquired by an IAS 2000 software (Delta Sistemi). Other sections were analyzed under Leica TCS-SP5 confocal microscope supplied with the Leica Application Suite Advanced Fluorescence software. Experiments were performed independently five times and a minimum of three plants for each time and each treatment were observed, yielding reproducible results. A minimum of 10 sections for mesocotyl or internode was analyzed. The reported micrographs are representative of single experiments.
Histochemical Visualization of ZmPAO Activity
Autofluorescence Analysis Coupled with Ammonium Hydroxide Treatment and Light Microscopic Sudan IV Staining
The levels of free and soluble-conjugated polyamines were determined in the outer tissues obtained from the wounded zone of the maize mesocotyl and from the corresponding mesocotyl zone of unwounded (control) plants, at 0, 24, 48, and 72 h after wounding. Polyamines were extracted overnight from mesocotyl outer tissues in 5% (w/v) perchloric acid (PCA) containing 0.12 mM 1,6-diaminohexane as an internal standard (tissue to 5% PCA ratio 1:3 [w/v]). The extracts were centrifuged at 20,000g for 15 min and then PCA-soluble free polyamines were analyzed in one-half of the supernatant. The remaining supernatant was acid hydrolyzed in 6 M HCl for 18 h at 110°C to obtain PCA-soluble conjugates of polyamines (Slocum et al., 1989
Polyamines were quantified after derivatization with dansyl chloride according to Smith and Davies (1985) The reported data are the average of values obtained in three different experiments, each performed with two technical replicates.
All statistical tests were performed using ANOVA using GraphPad Prism. Statistical significance of differences was evaluated by P level. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AJ002204 and AF067732.
The following material is available in the online version of this article.
We wish to thank Paraskevi Tavladoraki (Biology Department, University Roma Tre, Italy) for critical reading of the manuscript. Received September 13, 2007; accepted October 29, 2007; published November 9, 2007.
1 This work was supported by the Italian Ministry for University and Research (PRIN 2005, project contract 2005052297_002 to R.A.). 
2 This work is dedicated to the memory of Daniele Liberatori. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alessandra Cona (cona{at}uniroma3.it).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.108902 * Corresponding author; e-mail cona{at}uniroma3.it.
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ABSTRACT
RESULTS
-difluoromethyl-Orn or guazatine, respectively, inhibitors of Orn decarboxylase and PAO, reduced HR cell death caused by TMV infection (Yoda et al., 2003
0.05, 0.01, and 0.001, respectively. B, Western-blot and northern-blot analyses (samples as in A). Western immunoblotting of extractable ZmPAO fractions was performed after SDS-PAGE loaded on the basis of the total protein content (20 µg/well; top insert). Total RNA was fractionated by agarose/formaldehyde gel electrophoresis, blotted onto a nylon membrane, and hybridized with 32P-labeled maize PAO cDNA probe (middle insert). As a loading control, samples were also hybridized with the cDNA of the S13 ribosomal protein (bottom insert). C, Control; W, wounded; subscripted numbers indicate sampling time.
515 nm = 2.6 x 104 M–1 cm–1) as a result of the H2O2-dependent oxidation of AAP catalyzed by horseradish peroxidase and the subsequent condensation of the oxidized AAP with DCHBS (Artiss and Entwistle, 1981
