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.

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 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.

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.

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.

Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions.

Polyamines (PAs) are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs), including copper amine oxidases (CuAOs) and flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). The biologically-active hydrogen peroxide (H2O2) is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H2O2 biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H2O2 derived from PA oxidation in xylem tissue maturation under stress-simulated conditions.