Distinct role of AtCuAOß- and RBOHD-driven H2O2 production in wound-induced local and systemic leaf-to-leaf and root-to-leaf stomatal closure.

Polyamines (PAs) are ubiquitous low-molecular-weight aliphatic compounds present in all living organisms and essential for cell growth and differentiation. The developmentally regulated and stress-induced copper amine oxidases (CuAOs) oxidize PAs to aminoaldehydes producing hydrogen peroxide (H2O2) and ammonia. The Arabidopsis thaliana CuAOß (AtCuAOß) was previously reported to be involved in stomatal closure and early root protoxylem differentiation induced by the wound-signal MeJA via apoplastic H2O2 production, suggesting a role of this enzyme in water balance, by modulating xylem-dependent water supply and stomata-dependent water loss under stress conditions. Furthermore, AtCuAOß has been shown to mediate early differentiation of root protoxylem induced by leaf wounding, which suggests a whole-plant systemic coordination of water supply and loss through stress-induced stomatal responses and root protoxylem phenotypic plasticity. Among apoplastic ROS generators, the D isoform of the respiratory burst oxidase homolog (RBOH) has been shown to be involved in stress-mediated modulation of stomatal closure as well. In the present study, the specific role of AtCuAOß and RBOHD in local and systemic perception of leaf and root wounding that triggers stomatal closure was investigated at both injury and distal sites exploiting Atcuaoß and rbohd insertional mutants. Data evidenced that AtCuAOß-driven H2O2 production mediates both local and systemic leaf-to-leaf and root-to-leaf responses in relation to stomatal movement, Atcuaoß mutants being completely unresponsive to leaf or root wounding. Instead, RBOHD-driven ROS production contributes only to systemic leaf-to-leaf and root-to-leaf stomatal closure, with rbohd mutants showing partial unresponsiveness in distal, but not local, responses. Overall, data herein reported allow us to hypothesize that RBOHD may act downstream of and cooperate with AtCuAOß in inducing the oxidative burst that leads to systemic wound-triggered stomatal closure.

Arabidopsis N-acetyltransferase activity 2 preferentially acetylates 1,3-diaminopropane and thialysine.

Polyamine acetylation has an important regulatory role in polyamine metabolism. It is catalysed by GCN5-related N-acetyltransferases, which transfer acetyl groups from acetyl-coenzyme A to the primary amino groups of spermidine, spermine (Spm), or other polyamines and diamines, as was shown for the human Spermidine/Spermine N1-acetyltransferase 1 (HsSSAT1). SSAT homologues specific for thialysine, a cysteine-derived lysine analogue, were also identified (e.g., HsSSAT2). Two HsSSAT1 homologues are present in Arabidopsis, namely N-acetyltransferase activity (AtNATA) 1 and 2. AtNATA1 was previously shown to be specific for 1,3-diaminopropane, ornithine, putrescine and thialysine, rather than Spm and spermidine. In the present study, in an attempt to find a plant Spm-specific SSAT, AtNATA2 was expressed in a heterologous bacterial system and catalytic properties of the recombinant protein were determined. Data indicate that recombinant AtNATA2 preferentially acetylates 1,3-diaminopropane and thialysine, throwing further light on AtNATA1 substrate specificity. Structural analyses evidenced that the preference of AtNATA1, AtNATA2 and HsSSAT2 for short amine substrates can be ascribed to different main-chain conformation or substitution of HsSSAT1 residues interacting with Spm distal regions. Moreover, gene expression studies evidenced that AtNATA1 gene, but not AtNATA2, is up-regulated by cytokinins, thermospermine and Spm, suggesting the existence of a link between AtNATAs and N1-acetyl-Spm metabolism. This study provides insights into polyamine metabolism and structural determinants of substrate specificity of non Spm-specific SSAT homologues.

A New Player in Jasmonate-Mediated Stomatal Closure: The Arabidopsis thaliana Copper Amine Oxidase ß.

Plant defence responses to adverse environmental conditions include different stress signalling, allowing plant acclimation and survival. Among these responses one of the most common, immediate, and effective is the modulation of the stomatal aperture, which integrates different transduction pathways involving hydrogen peroxide (H2O2), calcium (Ca2+), nitric oxide (NO), phytohormones and other signalling components. The Arabidopsis thaliana copper amine oxidases ß (AtCuAOß) encodes an apoplastic CuAO expressed in guard cells and root protoxylem tissues which oxidizes polyamines to aminoaldehydes with the production of H2O2 and ammonia. Here, its role in stomatal closure, signalled by the wound-associated phytohormone methyl-jasmonate (MeJA) was explored by pharmacological and genetic approaches. Obtained data show that AtCuAOß tissue-specific expression is induced by MeJA, especially in stomata guard cells. Interestingly, two Atcuaoß T-DNA insertional mutants are unresponsive to this hormone, showing a compromised MeJA-mediated stomatal closure compared to the wild-type (WT) plants. Coherently, Atcuaoß mutants also show compromised H2O2-production in guard cells upon MeJA treatment. Furthermore, the H2O2 scavenger N,N1-dimethylthiourea (DMTU) and the CuAO-specific inhibitor 2-bromoethylamine (2-BrEtA) both reversed the MeJA-induced stomatal closure and the H2O2 production in WT plants. Our data suggest that AtCuAOß is involved in the H2O2 production implicated in MeJA-induced stomatal closure.

Plant Copper Amine Oxidases: Key Players in Hormone Signaling Leading to Stress-Induced Phenotypic Plasticity.

Polyamines are ubiquitous, low-molecular-weight aliphatic compounds, present in living organisms and essential for cell growth and differentiation. Copper amine oxidases (CuAOs) oxidize polyamines to aminoaldehydes releasing ammonium and hydrogen peroxide, which participates in the complex network of reactive oxygen species acting as signaling molecules involved in responses to biotic and abiotic stresses. CuAOs have been identified and characterized in different plant species, but the most extensive study on a CuAO gene family has been carried out in Arabidopsis thaliana. Growing attention has been devoted in the last years to the investigation of the CuAO expression pattern during development and in response to an array of stress and stress-related hormones, events in which recent studies have highlighted CuAOs to play a key role by modulation of a multilevel phenotypic plasticity expression. In this review, the attention will be focused on the involvement of different AtCuAOs in the IAA/JA/ABA signal transduction pathways which mediate stress-induced phenotypic plasticity events.

Microbiological Quality of Ready-to-Eat Leafy Green Salads during Shelf-Life and Home-Refrigeration.

The market of ready-to-eat leafy green salads is experiencing a noticeable growth in Europe. Since they are intended to be consumed without additional treatments, these ready-to-eat products are associated with a high microbiological risk. The aim of this work was to evaluate the microbiological quality and safety of ready-to-eat leafy green salads sold in widespread supermarket chains in Lazio, Italy, on the packaging date during shelf-life and during home-refrigeration. The study also aimed to determine the differences between low-, medium-, and high-cost products. Salmonella spp. and L. monocytogenes were chosen as safety indicators as specified by European regulations while total aerobic mesophilic bacteria and Escherichia coli were chosen as quality indicators as suggested by national guidelines. Analyses were performed following the ISO standards and in parallel for the evaluation of total aerobic mesophilic bacteria, with an alternative colorimetric system, the Micro Biological Survey method, in order to propose a simple, affordable and accurate alternative for testing the microbiological quality of products, especially suitable for small and medium enterprises and on-site analyses. The study revealed high, unsatisfactory, total bacterial loads in all analyzed samples on the packaging date and expiry date and a very high prevalence of Salmonella spp. (67%) regardless of the selected varieties and cost categories; L. monocytogenes was not recovered aligning with the results obtained in other studies.

Leaf-Wounding Long-Distance Signaling Targets AtCuAOß Leading to Root Phenotypic Plasticity.

The Arabidopsis gene AtCuAOß (At4g14940) encodes an apoplastic copper amine oxidase (CuAO) highly expressed in guard cells of leaves and flowers and in root vascular tissues, especially in protoxylem and metaxylem precursors, where its expression is strongly induced by the wound signal methyl jasmonate (MeJA). The hydrogen peroxide (H2O2) derived by the AtCuAOß-driven oxidation of the substrate putrescine (Put), mediates the MeJA-induced early root protoxylem differentiation. Considering that early root protoxylem maturation was also induced by both exogenous Put and leaf wounding through a signaling pathway involving H2O2, in the present study we investigated the role of AtCuAOß in the leaf wounding-induced early protoxylem differentiation in combination with Put treatment. Quantitative and tissue specific analysis of AtCuAOß gene expression by RT-qPCR and promoter::green fluorescent protein-ß-glucuronidase fusion analysis revealed that wounding of the cotiledonary leaf induced AtCuAOß gene expression which was particularly evident in root vascular tissues. AtCuAOß loss-of-function mutants were unresponsive to the injury, not showing altered phenotype upon wounding in comparison to wild type seedlings. Exogenous Put and wounding did not show synergy in inducing early root protoxylem maturation, suggesting their involvement in a shared signaling pathway.

Developmental, hormone- and stress-modulated expression profiles of four members of the Arabidopsis copper-amine oxidase gene family.

Copper-containing amine oxidases (CuAOs) catalyze polyamines (PAs) terminal oxidation producing ammonium, an aminoaldehyde and hydrogen peroxide (H2O2). Plant CuAOs are induced by stress-related hormones, methyl-jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA). In the Arabidopsis genome, eight genes encoding CuAOs have been identified. Here, a comprehensive investigation of the expression pattern of four genes encoding AtCuAOs from the α and γ phylogenetic subfamilies, the two peroxisomal AtCuAOα2 (At1g31690) and AtCuAOα3 (At1g31710) and the two apoplastic AtCuAOγ1 (At1g62810) and AtCuAOγ2 (At3g43670), has been carried out by RT-qPCR and promoter::green fluorescent protein-ß-glucuronidase fusion (GFP-GUS). Expression in hydathodes of new emerging leaves (AtCuAOγ1 and AtCuAOγ2) and/or cotyledons (AtCuAOα2, AtCuAOγ1 and AtCuAOγ2) as well as in vascular tissues of new emerging leaves and in cortical root cells at the division/elongation transition zone (AtCuAOγ1), columella cells (AtCuAOγ2) or hypocotyl and root (AtCuAOα3) was identified. Quantitative and tissue-specific gene expression analysis performed by RT-qPCR and GUS-staining in 5- and 7-day-old seedlings under stress conditions or after treatments with hormones or PAs, revealed that all four AtCuAOs were induced during dehydration recovery, wounding, treatment with indoleacetic acid (IAA) and putrescine (Put). AtCuAOα2, AtCuAOα3, AtCuAOγ1 and AtCuAOγ2 expression in vascular tissues and hydathodes involved in water supply and/or loss, along with a dehydration-recovery dependent gene expression, would suggest a role in water balance homeostasis. Moreover, occurrence in zones where an auxin maximum has been observed along with an IAA-induced alteration of expression profiles, support a role in tissue maturation and xylem differentiation events.

The Four FAD-Dependent Histone Demethylases of Arabidopsis Are Differently Involved in the Control of Flowering Time.

In Arabidopsis thaliana, four FAD-dependent lysine-specific histone demethylases (LDL1, LDL2, LDL3, and FLD) are present, bearing both a SWIRM and an amine oxidase domain. In this study, a comparative analysis of gene structure, evolutionary relationships, tissue- and organ-specific expression patterns, physiological roles and target genes for the four Arabidopsis LDL/FLDs is reported. Phylogenetic analysis evidences a different evolutionary history for the four LDL/FLDs, while promoter activity data show that LDL/FLDs are strongly expressed during plant development and embryogenesis, with some gene-specific expression patterns. Furthermore, phenotypical analysis of loss-of-function mutants indicates a role of all four Arabidopsis LDL/FLD genes in the control of flowering time, though for some of them with opposing effects. This study contributes toward a better understanding of the LDL/FLD physiological roles and may provide biotechnological strategies for crop improvement.

The Copper Amine Oxidase AtCuAOδ Participates in Abscisic Acid-Induced Stomatal Closure in Arabidopsis.

Plant copper amine oxidases (CuAOs) are involved in wound healing, defense against pathogens, methyl-jasmonate-induced protoxylem differentiation, and abscisic acid (ABA)-induced stomatal closure. In the present study, we investigated the role of the Arabidopsis thaliana CuAOδ (AtCuAOδ; At4g12290) in the ABA-mediated stomatal closure by genetic and pharmacological approaches. Obtained data show that AtCuAOδ is up-regulated by ABA and that two Atcuaoδ T-DNA insertional mutants are less responsive to this hormone, showing reduced ABA-mediated stomatal closure and H2O2 accumulation in guard cells as compared to the wild-type (WT) plants. Furthermore, CuAO inhibitors, as well as the hydrogen peroxide (H2O2) scavenger N,N1-dimethylthiourea, reversed most of the ABA-induced stomatal closure in WT plants. Consistently, AtCuAOδ over-expressing transgenic plants display a constitutively increased stomatal closure and increased H2O2 production compared to WT plants. Our data suggest that AtCuAOδ is involved in the H2O2 production related to ABA-induced stomatal closure.

Maize polyamine oxidase in the presence of spermine/spermidine induces the apoptosis of LoVo human colon adenocarcinoma cells.

Amine oxidases, which contribute to the regulation of polyamine levels, catalyze the oxidative deamination of polyamines to generate H2O2 and aldehyde(s). In this study, and at least to the best of our knowledge, maize polyamine oxidase (ZmPAO) was used for the first time with the aim of identifying a novel strategy for cancer therapy. The cytotoxicity and the mechanisms of cell death induced by the enzymatic oxidation products of polyamine generated by ZmPAO were investigated. Exogenous spermine and ZmPAO treatment decreased cell viability in a spermine dose­ and time­dependent manner, particularly, the viability of the multidrug­resistant (MDR) colon adenocarcinoma cells, LoVo DX, when compared with drug­sensitive ones (LoVo WT). Further analyses revealed that H2O2 derived from spermine was mainly responsible for the cytotoxicity. Flow cytometric analysis revealed that treatment with ZmPAO and spermine increased the apoptotic population of LoVo WT and LoVo DX cells. In addition, we found that treatment with ZmPAO and spermine markedly reduced mitochondrial membrane potential in the LoVo DX cells, in agreement with the results of cell viability and apoptosis assays. Transmission electron microscopic observations supported the involvement of mitochondrial depolarization in the apoptotic process. Therefore, the dysregulation of polyamine metabolism in tumor cells may be a potential therapeutic target. In addition, the development of MDR tumor cells is recognized as a major obstacle in cancer therapy. Therefore, the design of a novel therapeutic strategy based on the use of this combination may be taken into account, making this approach attractive mainly in treating MDR cancer patients.

Stress-Triggered Long-Distance Communication Leads to Phenotypic Plasticity: The Case of the Early Root Protoxylem Maturation Induced by Leaf Wounding in Arabidopsis.

Root architecture and xylem phenotypic plasticity influence crop productivity by affecting water and nutrient uptake, especially under those environmental stress, which limit water supply or imply excessive water losses. Xylem maturation depends on coordinated events of cell wall lignification and developmental programmed cell death (PCD), which could both be triggered by developmental- and/or stress-driven hydrogen peroxide (H2O2) production. Here, the effect of wounding of the cotyledonary leaf on root protoxylem maturation was explored in Arabidopsis thaliana by analysis under Laser Scanning Confocal Microscope (LSCM). Leaf wounding induced early root protoxylem maturation within 3 days from the injury, as after this time protoxylem position was found closer to the tip. The effect of leaf wounding on protoxylem maturation was independent from root growth or meristem size, that did not change after wounding. A strong H2O2 accumulation was detected in root protoxylem 6 h after leaf wounding. Furthermore, the H2O2 trap N,N¹-dimethylthiourea (DMTU) reversed wound-induced early protoxylem maturation, confirming the need for H2O2 production in this signaling pathway.

Determination of Copper Amine Oxidase Activity in Plant Tissues.

Copper amine oxidases (CuAOs) involved in polyamine catabolism are emerging as physiologically relevant enzymes for their involvement in plant growth, differentiation and defence responses to biotic and abiotic stress. In this chapter, we describe two spectrophotometric and one polarographic method for determining CuAO activity in plant tissues. Some aspects related to cell wall association of apoplastic CuAOs and possible interference of plant metabolites with the enzymatic activity assays are also considered.

The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation.

In plants, the polyamines putrescine, spermidine, spermine (Spm), and thermospermine (Therm-Spm) participate in several physiological processes. In particular, Therm-Spm is involved in the control of xylem differentiation, having an auxin antagonizing effect. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis, five PAOs are present, among which AtPAO5 catalyzes the back-conversion of Spm, Therm-Spm, and N1-acetyl-Spm to spermidine. In the present study, it is shown that two loss-of-function atpao5 mutants and a 35S::AtPAO5 Arabidopsis transgenic line present phenotypical differences from the wild-type plants with regard to stem and root elongation, differences that are accompanied by changes in polyamine levels and the number of xylem vessels. It is additionally shown that cytokinin treatment, which up-regulates AtPAO5 expression in roots, differentially affects protoxylem differentiation in 35S::AtPAO5, atpao5, and wild-type roots. Together with these findings, Therm-Spm biosynthetic genes, as well as auxin-, xylem-, and cytokinin-related genes (such as ACL5, SAMDC4, PIN1, PIN6, VND6, VND7, ATHB8, PHB, CNA, PXY, XTH3, XCP1, and AHP6) are shown to be differentially expressed in the various genotypes. These data suggest that AtPAO5, being involved in the control of Therm-Spm homeostasis, participates in the tightly controlled interplay between auxin and cytokinins that is necessary for proper xylem differentiation.

Copper-Containing Amine Oxidases and FAD-Dependent Polyamine Oxidases Are Key Players in Plant Tissue Differentiation and Organ Development.

Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell-wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development.

Molecular Evolution of Alternative Oxidase Proteins: A Phylogenetic and Structure Modeling Approach.

Alternative oxidases (AOXs) are mitochondrial cyanide-resistant membrane-bound metallo-proteins catalyzing the oxidation of ubiquinol and the reduction of oxygen to water bypassing two sites of proton pumping, thus dissipating a major part of redox energy into heat. Here, the structure of Arabidopsis thaliana AOX 1A has been modeled using the crystal structure of Trypanosoma brucei AOX as a template. Analysis of this model and multiple sequence alignment of members of the AOX family from all kingdoms of Life indicate that AOXs display a high degree of conservation of the catalytic core, which is formed by a four-α-helix bundle, hosting the di-iron catalytic site, and is flanked by two additional α-helices anchoring the protein to the membrane. Plant AOXs display a peculiar covalent dimerization mode due to the conservation in the N-terminal region of a Cys residue forming the inter-monomer disulfide bond. The multiple sequence alignment has also been used to infer a phylogenetic tree of AOXs whose analysis shows a polyphyletic origin for the AOXs found in Fungi and a monophyletic origin of the AOXs of Eubacteria, Mycetozoa, Euglenozoa, Metazoa, and Land Plants. This suggests that AOXs evolved from a common ancestral protein in each of these kingdoms. Within the Plant AOX clade, the AOXs of monocotyledon plants form two distinct clades which have unresolved relationships relative to the monophyletic clade of the AOXs of dicotyledonous plants. This reflects the sequence divergence of the N-terminal region, probably due to a low selective pressure for sequence conservation linked to the covalent homo-dimerization mode.

Different disulfide bridge connectivity drives alternative folds in highly homologous Brassicaceae trypsin inhibitors.

Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C1-C3, C2-C4, C5-C6, and C7-C8) is different from that of ATTI Cys (C1-C8, C2-C5, C3-C6, and C4-C7). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different three-dimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments.

The MeJA-inducible copper amine oxidase AtAO1 is expressed in xylem tissue and guard cells.

Copper amine oxidases oxidize the polyamine putrescine to 4-aminobutanal with the production of the plant signal molecule hydrogen peroxide (H2O2) and ammonia. The Arabidopsis (Arabidopsis thaliana) gene At4g14940 (AtAO1, previously referred to as ATAO1) encodes an apoplastic copper amine oxidase expressed in lateral root cap cells and developing xylem, especially in root protoxylem and metaxylem precursors. In our recent study, we demonstrated that AtAO1 expression is strongly induced in the root vascular tissues by the wound-signal hormone methyl jasmonate (MeJA). Furthermore, we also demonstrated that the H2O2 derived by the AtAO1-driven oxidation of putrescine, mediates the MeJA-induced early protoxylem differentiation in Arabidopsis roots. H2O2 may contribute to protoxylem differentiation by signaling developmental cell death and by acting as co-substrate in peroxidase-mediated cell wall stiffening and lignin polymerization. Here, by the means of AtAO1 promoter::green fluorescent protein-ß-glucuronidase (AtAO1::GFP-GUS) fusion analysis, we show that a strong AtAO1 gene expression occurs also in guard cells of leaves and flowers. The high expression levels of AtAO1 in tissues or cell types regulating water supply and water loss may suggest a role of the encoded protein in water balance homeostasis, by modulating coordinated adjustments in anatomical and functional features of xylem tissue and guard cells during acclimation to adverse environmental conditions.

POLYAMINE OXIDASE2 of Arabidopsis contributes to ABA mediated plant developmental processes.

Polyamines (PA) are catabolised by two groups of amine oxidases, the copper-binding amine oxidases (CuAOs) and the FAD-binding polyamine oxidases (PAOs). Previously, we have shown that CuAO1 is involved in ABA associated growth responses and ABA- and PA-mediated rapid nitric oxide (NO) production. Here we report the differential regulation of expression of POLYAMINE OXIDASE2 of Arabidopsis (AtPAO2) in interaction with ABA, nitrate and ammonium. Without ABA treatment germination, cotyledon growth and fresh weight of pao2 knockdown mutants as well as PAO2OX over-expressor plants were comparable to those of the wild type (WT) plants irrespective of the N source. In the presence of ABA, in pao2 mutants cotyledon growth and fresh weights were more sensitive to inhibition by ABA while PAO2OX over-expressor plants showed a rather similar response to WT. When NO3(-) was the only N source primary root lengths and lateral root numbers were lower in pao2 mutants both without and with exogenous ABA. PAO2OX showed enhanced primary and lateral root growth in media with NO3(-) or NH4(+). Vigorous root growth of PAO2OX and the hypersensitivity of pao2 mutants to ABA suggest a positive function of AtPAO2 in root growth. ABA-induced NO production in pao2 mutants was lower indicating a potential contributory function of AtPAO2 in NO-mediated effects on root growth.

The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots.

Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-ß-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N(1)-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N(1)-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root.