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

Abstract

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-β-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.

MeJA-induced protoxylem differentiation in Arabidopsis roots is mediated by AtAO1-produced H₂O₂

Polyamines (PAs) are aliphatic amines involved in multiple roles in plants during both physiological and pathological events,^1,2 which may be secreted in the cell wall at specific developmental stages or under stress conditions.³ In the apoplast, these compounds are terminally oxidized by apoplastic amine oxidases (AOs), which include copper amine oxidases (CuAOs) and flavin containing polyamine oxidases, generating, among other reaction products, the biologically active compound hydrogen peroxide (hereafter referred as PA-derived H₂O₂).^3-6 It has been shown that H₂O₂ produced by apoplastic AOs plays a dual role both in cell wall maturation events by triggering peroxidase-driven wall stiffening events and in defense responses by signaling modulation of defense gene expression.^3-5 Cell-wall resident AOs show high levels of expression in root vascular tissues of different plant species, especially in developing xylem tissues.^7-10 In this regard, it has been suggested that PA-derived H₂O₂ may play a role in maturation of xylem cells by triggering the peroxidase-driven polymerisation of lignin and by signaling developmental programmed cell death (PCD) in differentiating xylem precursors. Growing evidence allows us to hypothesize that apoplastic PA-derived H₂O₂ has a role in inducing xylem differentiation under stress conditions, while its involvement under physiological conditions needs to be further explored.^8,10-12

The Arabidopsis thaliana At4g14940 gene (AtAO1) encodes for an apoplastic MeJA-inducible CuAO that has been revealed to be expressed in root protoxylem and metaxylem precursors. We have recently demonstrated, by pharmacological and forward/reverse genetic approaches, that AtAO1-driven H₂O₂ production has a key role in the early protoxylem differentiation occurring in the primary root apex upon treatment with the wound-signal MeJA.⁸ In this regard, it has been shown that in roots of MeJA-treated seedlings, fully differentiated protoxylem vessels, in addition to display higher AtAO1 expression levels, appear closer to the root apex with respect to roots from control plants. Conversely, Atao1 loss-of-function mutants have been shown to be unresponsive to the MeJA-induced early protoxylem differentiation. The involvement of AtAO1 in this event is further supported by the reduction of the levels of the AtAO1 substrate putrescine in roots of MeJA-treated wild type (WT) plants.⁸ A key role in MeJA-induced early differentiation of root protoxylem has been ascribed to the AtAO1-produced H₂O₂ considering that the effect observed in roots of MeJA-treated WT plants is reversible upon H₂O₂removal and that an accumulation of H₂O₂ at the site of appearance of protoxylem cells with fully developed secondary walls has been revealed in the same roots.⁸ Early protoxylem differentiation was also induced in AtAO1 over-expressing plants and in WT plants upon treatment with putrescine.⁸

AtAO1 is strongly expressed in guard cells of leaves and flowers

The involvement of apoplastic AOs in stomatal movement has been revealed in different plant species. In Vicia faba guard cells an apoplastic CuAO behaves as H₂O₂ source in ABA-induced stomatal closure.¹³ Spd-derived production of H₂O₂ in guard cells was shown to induce stomatal closure in grapevine (Vitis vinifera L.) as a consequence of ABA-induced exodus of PAs in the cell wall.^14,15

Here, AtAO1 expression pattern has been analyzed in leaves and flowers of AtAO1::GFP-GUS Arabidopsis transgenic plants, which have been prepared using a region of 2.6 kb upstream of ATG, as described by Ghuge et al. 2015.⁸ Analysis of both promoter-driven GFP signal and GUS staining revealed a strong AtAO1 gene expression in guard cells of different tissues, such as leaves from 4-day-old seedlings and flowers (Fig. 1).

Figure 1.

AtAO1 tissue expression pattern in stomata of 4-day-old AtAO1::GFP-GUS transgenic plants. (A) Analysis of GFP signal and propidium iodide staining by Laser Scanning Confocal Microscopy in leaf guard cells. (B and C) Light microscopy analysis by GUS staining in leaves (B) and flowers (C) guard cells. The GUS staining reaction was allowed to proceed overnight. Highly reproducible results were obtained from 3 independent transgenic lines. Micrographs are representative of those obtained from 5 independent experiments. Bars = 10 µm (A), 20 µm (B), 200 µm (C).

Conclusions and Future Perspectives

The occurrence of a strong constitutive AtAO1 gene expression in leaves and flowers of guard cells may suggest a possible role of this gene in stomatal closure. Considering our previous observation showing the involvement of AtAO1 in MeJA-mediated early protoxylem differentiation in the root apex, we propose a hypothetic model of the role played by this gene in tissues and cell types involved in water balance maintenance, such as xylem and guard cells. In this regard, the reason for which MeJA induces early protoxylem differentiation in root is unclear and deserves a more comprehensive investigation. A reasonable hypothesis could be that after a MeJA-signaled stress, such as wounding, a more efficient water supply represents an advantage for the plants. Thus, the observed reorganization of the xylem tissues could be functional to achieve an improved water uptake. In stomata cells, AtAO1-dependent PA oxidation could be triggered by a controlled secretion of PAs in the apoplast. A further increase of AtAO1 expression and/or PA secretion in the apoplast of guard cells may occur upon injury, leading to a MeJA-induced stomatal closure.

Overall, the constitutive expression of AtAO1 in the guard cells, together with its MeJA-inducible expression in xylem tissues, suggests a role of this gene in homeostatic mechanisms leading to a balance between xylem-dependent water supply and stomata-dependent water loss under stress conditions. Stomatal closure may represent a fast response to injury followed by a remodelling of xylem tissue organization allowing plant acclimation to adverse conditions. Considering this, the involvement of AtAO1 in stomatal closure and xylem differentiation under different stress conditions deserves to be the object of future investigations.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.