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.


Introduction
Copper amine oxidases (CuAOs) are dimeric proteins of 140-180 kDa, containing a copper ion and a redox-active organic cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) for each monomer. These enzymes catalyze the intracellular and extracellular terminal catabolism of amines, including monoamines, diamines, and polyamines (PAs), by oxidizing the carbon next to the primary amino group, with the subsequent reduction of molecular oxygen to hydrogen peroxide (H 2 O 2 ) and the production of the corresponding aldehydes and ammonia [1,2]. CuAOs have been found at high expression levels in several species of Fabaceae, especially in the cell wall of pea (Pisum sativum), chickpea (Cicer arietinum), lentil (Lens culinaris), and soybean (Glycine max) seedlings, from which these enzymes have been purified and characterized [3]. CuAOs from these species preferentially oxidize the diamine putrescine (Put) and cadaverine [4]. A number of peroxisomal and apoplastic CuAOs have been described in Arabidopsis (Arabidopsis thaliana) [5][6][7][8], tobacco (Nicotiana tabacum) [8,9], and apple (Malus domestica) [10], and a latex CuAO has been characterized from Mediterranean spurge (Euphorbia characias) [11], with diverse substrate affinities and specificities [12]. . The expression of AtCuAOδ gene was analyzed in 12-day-old wild-type (WT) seedlings untreated or treated with 1, 10, and 100 µM ABA for 0, 1, 3, 6, and 24 h. Five independent experiments as biological replicates (mean values ± SD; n = 5) were performed. AtCuAOδ mRNA level after ABA treatment is relative to that of the corresponding untreated plant for each time point. The significance levels between the relative mRNA level at each time and the mRNA level of control untreated plant at time 0, which is assumed to be one, is reported. P values have been calculated with one-way analysis of variance (ANOVA); *, **, ***, and **** p values equal or are less than 0.05, 0.01, 0.001, and 0.0001, respectively.

AtCuAOδ Loss-of-Function Mutants are Unresponsive to ABA-Induced Stomatal Closure
In order to investigate the contribution of AtCuAOδ in ABA-mediated responses, two T-DNA insertional mutant lines for this gene [SALK 072954. 55.00.x line, TAIR (The Arabidopsis Information Resource) accession number 4122972 and GK-011C04-013046 line, TAIR accession number 4242275] were identified from the TAIR database (http://www.arabidopsis.org/; [33]), and obtained, hereafter referred to as Atcuaoδ. 1 and Atcuaoδ.2 ( Figure S1). From evidence available in TAIR, the T-DNA insertion sites are located in the first exon in both mutants ( Figure S1), upstream of the encoded catalytic site active residues ( Figure S2), thereby creating loss-of-function mutants. Mutant plants homozygous for the T-DNA insertions were identified by PCR analysis of genomic DNA ( Figure S1). RT-PCR analysis of the selected plants confirmed the absence of the full-length gene transcripts in Atcuaoδ. 1 and Atcuaoδ.2 ( Figure S1). Analysis of Atcuaoδ mutants under physiological growth conditions did not highlight any apparent different phenotypes, i.e., germination events, stem or root length, and leaf morphology (data not shown).
As ABA has well characterized effects in the regulation of the stomatal aperture, we investigated whether AtCuAOδ-driven PA oxidation in guard cells could be involved in the ABA-mediated stomatal closure. Wild-type (WT), Atcuaoδ.1, and Atcuaoδ.2 plants were treated with ABA (1, 10, and 100 µM) for 2 h and the stomatal aperture was analyzed by measuring the width and the length of the stomatal pore (width/length ratio). In Figure 2A and Table S1, we show that while between control untreated WT (Control WT) and control untreated insertional mutants (Control Atcuaoδ.1 or Control Atcuaoδ.2) no width/length ratio differences were detected, a significant reduction of the ABAmediated stomatal closure was observed in the Atcuaoδ mutants as compared to WT plants. Indeed, stomatal closure was induced in WT by ABA treatments of about 51% at 1 µM and 77% at 100 µM as compared to Control WT, while the ABA-mediated stomatal closure in Atcuaoδ.1 and in Atcuaoδ.2 Figure 1. Analysis of AtCuAOδ gene expression upon abscisic acid (ABA) treatment by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of AtCuAOδ gene was analyzed in 12-day-old wild-type (WT) seedlings untreated or treated with 1, 10, and 100 µM ABA for 0, 1, 3, 6, and 24 h. Five independent experiments as biological replicates (mean values ± SD; n = 5) were performed. AtCuAOδ mRNA level after ABA treatment is relative to that of the corresponding untreated plant for each time point. The significance levels between the relative mRNA level at each time and the mRNA level of control untreated plant at time 0, which is assumed to be one, is reported. P values have been calculated with one-way analysis of variance (ANOVA); *, **, ***, and **** p values equal or are less than 0.05, 0.01, 0.001, and 0.0001, respectively.

AtCuAOδ Loss-of-Function Mutants Are Unresponsive to ABA-Induced Stomatal Closure
In order to investigate the contribution of AtCuAOδ in ABA-mediated responses, two T-DNA insertional mutant lines for this gene [SALK 072954.55.00.x line, TAIR (The Arabidopsis Information Resource) accession number 4122972 and GK-011C04-013046 line, TAIR accession number 4242275] were identified from the TAIR database (http://www.arabidopsis.org/; [33]), and obtained, hereafter referred to as Atcuaoδ.1 and Atcuaoδ.2 ( Figure S1). From evidence available in TAIR, the T-DNA insertion sites are located in the first exon in both mutants ( Figure S1), upstream of the encoded catalytic site active residues ( Figure S2), thereby creating loss-of-function mutants. Mutant plants homozygous for the T-DNA insertions were identified by PCR analysis of genomic DNA ( Figure S1). RT-PCR analysis of the selected plants confirmed the absence of the full-length gene transcripts in Atcuaoδ.1 and Atcuaoδ.2 ( Figure S1). Analysis of Atcuaoδ mutants under physiological growth conditions did not highlight any apparent different phenotypes, i.e., germination events, stem or root length, and leaf morphology (data not shown).
As ABA has well characterized effects in the regulation of the stomatal aperture, we investigated whether AtCuAOδ-driven PA oxidation in guard cells could be involved in the ABA-mediated stomatal closure. Wild-type (WT), Atcuaoδ.1, and Atcuaoδ.2 plants were treated with ABA (1, 10, and 100 µM) for 2 h and the stomatal aperture was analyzed by measuring the width and the length of the stomatal pore (width/length ratio). In Figure 2A and Table S1, we show that while between control untreated WT (Control WT) and control untreated insertional mutants (Control Atcuaoδ.1 or Control Atcuaoδ.2) no width/length ratio differences were detected, a significant reduction of the ABA-mediated stomatal closure was observed in the Atcuaoδ mutants as compared to WT plants. Indeed, stomatal closure was induced in WT by ABA treatments of about 51% at 1 µM and 77% at 100 µM as compared to Control WT, while the ABA-mediated stomatal closure in Atcuaoδ.1 and in Atcuaoδ.2 ranged from~9-12% to a maximum of~15-17%, respectively, as compared to the corresponding Control mutant plants ( Figure 2A). Consistent with these data, treatment with the CuAO-specific inhibitors, 2-bromoethylamine (2-BrEtA) and aminoguanidine (AG), inhibited the ABA-mediated stomatal closure in WT plants ( Figure 2B and Table S2). The action of the CuAO-specific inhibitors, 2-BrEtA and AG, on stomatal apertures of Atcuaoδ mutants was also studied. At the two different inhibitor concentrations used, in the presence of 100 µM ABA (the highest hormone concentration used), we observed diverse antagonistic effects. At the 2-BrEtA highest concentration, a considerable reduction of stomatal closure was observed in WT (from 77% to 10%), while the mutant genotypes were further unresponsive to ABA (5% from 15% in Atcuaoδ.1 and 9% from 17% in Atcuaoδ.2). The other CuAO inhibitor, AG, partially prevented the stomatal closure effects induced by ABA in WT (35% and 24% at 0.1 mM and 1 mM, respectively). However, by itself this inhibitor presented a similar effect on stomatal apertures in all the studied genotypes (10% closure in respect to the Control WT and mutant).  Consistent with these data, treatment with the CuAO-specific inhibitors, 2-bromoethylamine (2-BrEtA) and aminoguanidine (AG), inhibited the ABA-mediated stomatal closure in WT plants ( Figure 2B and Table S2). The action of the CuAO-specific inhibitors, 2-BrEtA and AG, on stomatal apertures of Atcuaoδ mutants was also studied. At the two different inhibitor concentrations used, in the presence of 100 µM ABA (the highest hormone concentration used), we observed diverse antagonistic effects. At the 2-BrEtA highest concentration, a considerable reduction of stomatal closure was observed in WT (from 77% to 10%), while the mutant genotypes were further unresponsive to ABA (5% from 15% in Atcuaoδ.1 and 9% from 17% in Atcuaoδ.2). The other CuAO inhibitor, AG, partially prevented the stomatal closure effects induced by ABA in WT (35% and 24% at 0.1 mM and 1 mM, respectively). However, by itself this inhibitor presented a similar effect on stomatal apertures in all the studied genotypes (10% closure in respect to the Control WT and mutant).

AtCuAOδ-Driven Production of H 2 O 2 Is Involved in the ABA-Induced Stomatal Closure
In order to get insights into the possible role played by the AtCuAOδ-driven production of H 2 O 2 in the stomatal closure induced by ABA, WT and Atcuaoδ seedlings were treated with the H 2 O 2 scavenger N,N 1 -dimethylthiourea (DMTU) at the working concentration of 100 µM [26], either alone or in combination with 1, 10, and 100 µM ABA. DMTU reversed the ABA-induced stomatal closure in WT plants (91, 80, and 78%, respectively), whereas it did not significantly affect stomatal aperture under physiological conditions in WT plants, or in 1 and 10 µM ABA-treated and untreated mutants ( Figure 2A and Table S1). At the highest ABA concentration used, DMTU reversion was not complete in the mutant genotypes, where a significant closure effect of 8% and 11% was observed in Atcuaoδ.1 and Atcuaoδ.2, respectively. To further investigate the contribution of AtCuAOδ in the ABA-induced H 2 O 2 production, reactive oxygen species (ROS) levels in guard cells were analyzed using a chloromethyl derivative of 2 ,7 -dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA).

AtCuAOδ-Driven Production of H2O2 is Involved in the ABA-Induced Stomatal Closure
In order to get insights into the possible role played by the AtCuAOδ-driven production of H2O2 in the stomatal closure induced by ABA, WT and Atcuaoδ seedlings were treated with the H2O2 scavenger N,N 1 -dimethylthiourea (DMTU) at the working concentration of 100 µM [26], either alone or in combination with 1, 10, and 100 µM ABA. DMTU reversed the ABA-induced stomatal closure in WT plants (91, 80, and 78%, respectively), whereas it did not significantly affect stomatal aperture under physiological conditions in WT plants, or in 1 and 10 µM ABA-treated and untreated mutants ( Figure 2A and Table S1). At the highest ABA concentration used, DMTU reversion was not complete in the mutant genotypes, where a significant closure effect of 8% and 11% was observed in Atcuaoδ. 1 and Atcuaoδ.2, respectively. To further investigate the contribution of AtCuAOδ in the ABA-induced H2O2 production, reactive oxygen species (ROS) levels in guard cells were analyzed using a chloromethyl derivative of 2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).

AtCuAOδ Over-Expressing Plants Show Enhanced Stomatal Closure and H2O2 Production
The role played by AtCuAOδ-driven production of H2O2 in the stomatal closure has been further investigated through the analysis of transgenic Arabidopsis plants over-expressing AtCuAOδ

AtCuAOδ Over-Expressing Plants Show Enhanced Stomatal Closure and H 2 O 2 Production
The role played by AtCuAOδ-driven production of H 2 O 2 in the stomatal closure has been further investigated through the analysis of transgenic Arabidopsis plants over-expressing AtCuAOδ (overAtCuAOδ). In Figure S3 we show by RT-qPCR and western blot analysis the different levels of the transgene mRNA and protein of the two lines, overAtCuAOδ line P9 and overAtCuAOδ line P17, used in this work. The latter showed higher expression of the transgenic mRNA and protein than overAtCuAOδ line P9 ( Figure S3). As shown in Figure 4, both overAtCuAOδ lines showed constitutively enhanced stomatal closure ( Figure 4A) and similar ROS production in guard cells ( Figure 4B) as compared to WT plants. (overAtCuAOδ). In Figure S3 we show by RT-qPCR and western blot analysis the different levels of the transgene mRNA and protein of the two lines, overAtCuAOδ line P9 and overAtCuAOδ line P17, used in this work. The latter showed higher expression of the transgenic mRNA and protein than overAtCuAOδ line P9 ( Figure S3). As shown in Figure 4, both overAtCuAOδ lines showed constitutively enhanced stomatal closure ( Figure 4A) and similar ROS production in guard cells ( Figure 4B) as compared to WT plants.

AtCuAOδ Plays a Role in the Control of Stomatal Closure in Response to ABA
Our data (Figure 1) show that AtCuAOδ gene is regulated by ABA, consistent with the ABAregulated recognition sites identified in its promoter region. In detail, a maximum of 2.5-to four-fold induction, depending on the ABA concentration used, was observed after 6 h from the onset of treatment. In this regard, it is known that ABA, the water-stress hormonal signal that is considered a valid indicator of water potential status in plants, is involved in defense responses against abiotic stresses, such as drought or high soil saline levels [34][35][36]. In line with this, several results have suggested that the ABA-responsive AOs [22,25] are likely involved in salt stress responses [37] and in water balance regulation [28].
Furthermore, one of the major roles of this phytohormone is its action on the regulation of stomatal movement in response to variations in water potential [38,39]. Regarding this aspect of plant responses to ABA, our results demonstrate that alterations in the levels of AtCuAOδ expression by reverse genetics and over-expression approaches, or AtCuAO enzyme activities by pharmacological treatments with the two known CuAO activity inhibitors, 2-BrEtA or AG [40][41][42], caused alterations of the hormonal control of guard cells responses. In fact, it was observed a complete to partial unresponsiveness to ABA in stomatal closure with the two homozygous Atcuaoδ mutants, compared

AtCuAOδ Plays a Role in the Control of Stomatal Closure in Response to ABA
Our data (Figure 1) show that AtCuAOδ gene is regulated by ABA, consistent with the ABA-regulated recognition sites identified in its promoter region. In detail, a maximum of 2.5to four-fold induction, depending on the ABA concentration used, was observed after 6 h from the onset of treatment. In this regard, it is known that ABA, the water-stress hormonal signal that is considered a valid indicator of water potential status in plants, is involved in defense responses against abiotic stresses, such as drought or high soil saline levels [34][35][36]. In line with this, several results have suggested that the ABA-responsive AOs [22,25] are likely involved in salt stress responses [37] and in water balance regulation [28].
Furthermore, one of the major roles of this phytohormone is its action on the regulation of stomatal movement in response to variations in water potential [38,39]. Regarding this aspect of plant responses to ABA, our results demonstrate that alterations in the levels of AtCuAOδ expression by reverse genetics and over-expression approaches, or AtCuAO enzyme activities by pharmacological treatments with the two known CuAO activity inhibitors, 2-BrEtA or AG [40][41][42], caused alterations of the hormonal control of guard cells responses. In fact, it was observed a complete to partial unresponsiveness to ABA in stomatal closure with the two homozygous Atcuaoδ mutants, compared to the WT (Figure 2A and Table S1), and the lack of ABA responsiveness in the WT in combination treatments involving the two inhibitors ( Figure 2B and Table S2). It must be pointed out that this effect was clear even in the case of AG, even if this inhibitor caused by itself a similar stomatal closure effect on all the three studied genotypes (approximately 10% of Control WT and Control mutants). This unspecific effect could be attributed to AG-induced alterations of the plasma membrane potential [43], which could influence stomatal movements. The overAtCuAOδ lines instead showed a significantly reduced (50%) stomatal aperture in respect to WT ( Figure 4A). Figures 3 and 4 show that when compared to the WT, the loss-of-function or the over-expression of the gene encoding the vacuole-resident AtCuAOδ [29] reduced ROS levels of guard cells in ABA-treated mutant genotypes or increased ROS in overAtCuAOδ lines. ROS, such as H 2 O 2 , are ubiquitous metabolites in all aerobic organisms and have been shown to be important signals in many aspects of plant development, including the regulation of stomatal movement [44][45][46]. Thus, the relation between AtCuAOδ-mediated H 2 O 2 production and its involvement in stomatal closure in the ABA transduction pathway is consistent with the role of H 2 O 2 in ABA-signaled phenomena.

Vacuolar AtCuAOδ-Dependent H 2 O 2 Production Is a Necessary Condition for ABA Regulation of Stomatal Aperture
Interestingly, vacuoles have an important role in the regulation of stomatal pore apertures associated with different environmental or hormonal factors signaling water stress [47][48][49]. Moreover, ROS can regulate several channel activities located in the tonoplast, which influence ion fluxes, cytosolic pH, and the uptake and release of calcium [30], all of which are involved in modulation of the stomatal aperture. It is, thus, not surprising that an AtCuAO protein identified in the vacuolar proteome [29] can influence stomatal closure as an element in the ABA transduction pathway regulating this phenomenon. No clear contribution of vacuoles in guard cells to the ROS signaling network has been identified [46]. Nevertheless, reports in the literature indicate that vacuoles can be sites of H 2 O 2 production [50][51][52]. Thus, our data might represent a first indication that ROS in the form of H 2 O 2 produced by a vacuolar AtCuAO have a physiological role in ABA regulation of stomatal movement.

Vacuolar AtCuAOδ Cooperates with Different ROS Sources in Regulation of Stomatal Movement
Among CuAO family member AtCuAOδ is not the only CuAO involved in the control of stomatal aperture levels. Indeed, both AtCuAOβ and AtCuAOζ are expressed in guard cells [25,29], and in the case of AtCuAOζ mutant, a reduced ROS level and stomatal closure in response to ABA are observed [25]. This evidence suggests a potential role (AtCuAOβ) or effective involvement (AtCuAOζ) in the control of the stomatal aperture in Arabidopsis [25,29]. Finally, and in agreement with our data, a CuAO was shown to act in Vicia faba during ABA responses involved in the regulation of stomatal apertures through the production of Put-derived H 2 O 2 [22], suggesting the existence of a common hormonal response pathway in evolutionarily distant taxa.
The complexity of the ABA signal transduction in guard cells is also highlighted by evidence showing that multiple pathways involving several components and compartments are required in the control of stomatal movements in Arabidopsis. In this context, plasma membrane-located nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (AtrbohD and F [53]), regulated by the ABA-induced phospholipase D (PLDα1 [25]), and ABA-activated OST1 [54], peroxisomal AtCuAOζ [25], and vacuolar AtCuAOδ (in this work) represent multiple ROS sources. These different ROS sources are active in different cellular compartments, targeted by ABA, and necessary for this response, suggesting that a strongly coordinated network is needed for the hormonal control of stomatal closure. Indeed, coordination of ROS signaling from different organelles has been reported for ABA-induced stomatal closure [55]. Furthermore, some evidence shows that ABA presents an apparently minor ROS-independent effect on stomatal closure, as its effects could not be entirely counter-balanced by either ROS scavengers or ROS-biosynthesis inhibitors (Figure 2, Table S1) [25].
Our data revealed that another player is necessary in ABA-mediated regulation of stomatal closure and points out the necessity of understanding the hierarchy of action or eventual synergy of actors involved. The contribution of both NADPH oxidases and AOs has also been proposed in the two phases of ROS production during the hypersensitive response to pathogens, with the former involved in the initial burst or first phase and the latter in the second phase [18,19]. It is possible that a similar mechanism involving both AtCuAOδ and AtCuAOζ, as well as NADPH oxidases, have a role in the ABA control of water balance homeostasis acting through cells or tissues responsible for the regulation of water loss in Arabidopsis. Of note is that an apoplastic PAO and a NADPH oxidase are involved in a feed-forward ROS amplification loop in tobacco, suggesting that both enzymes cooperate in ROS homeostasis in plants [37], while a peroxisomal PAO cross-talks with NADPH oxidase in Arabidopsis to activate mitochondrial alternative oxidase, underlining the complexity of ROS homeostasis and biosynthesis involving different enzymatic systems and subcellular compartments [56]. This picture is further enriched by the downstream effect of NO on the involvement of both NADPH oxidase and AOs in PA-induced stomatal closure in the guard cells of Arabidopsis [57]. The complexity of the events involved in the process of stomatal closure in cases where the increase of ABA levels are a signal of both immediate or prolonged reduced water potential could be explained on the basis of required differential and accurate responses at the cellular or tissue level to the different conditions to which plants are commonly exposed during their growth in-field. Clarification of these mechanisms would be helpful in elucidating potential applications for crop adaptation to changing climate, wherein water stress conditions are becoming increasingly relevant.
Plants were grown in soil or in vitro in a growth chamber at a temperature of 23 • C under long-day conditions (16/8 h photoperiod; 50 µmol m −2 s −1 and 55% relative humidity). Soil-grown plants were used for identification of the homozygous insertion mutants, for floral dip transformation to prepare over-expression lines, and in all cases for harvesting seeds of the WT and selected lines. For in vitro growth, seeds were surface sterilized as previously described [60]. Seeds were cold stratified at 4 • C and grown in one-half-strength Murashige and Skoog salt mixture supplemented with 0.5% (w/v) sucrose in presence of 0.8% (w/v) agar. In vitro-grown seedlings were used in the analysis of AtCuAOδ gene expression, in measurements of stomatal apertures, and in ROS detection.
The analysis of AtCuAOδ gene expression upon hormone treatment was performed on Arabidopsis seedlings grown for 12 days on agar medium and then transferred to liquid medium [one-half-strength Murashige and Skoog salt mixture supplemented with 0.5% (w/v) sucrose] containing 1, 10, and 100 µM abscisic acid (ABA; Duchefa) for the described time (0, 1, 3, 6, and 24 h).
The detection of reactive oxygen species (ROS) in guard cells was analyzed on 12-day-old Arabidopsis WT plants, Atcuaoδ mutants, and overAtCuAOδ lines grown on agar medium under control conditions. ROS levels in WT and mutant plants were also analyzed after 2 h treatment with 100 µM ABA.

Identification of the T-DNA Insertional Loss-of-Function Atcuaoδ.1 and Atcuaoδ.2 Mutants
Plants homozygous for the T-DNA insertion were identified by Polymerase Chain Reaction (PCR) on genomic DNA extracted from leaves of soil-grown plants by alkali treatment [61], using gene-and T-DNA-specific primers. AtCuAOδ gene-specific primers (RP-AtCuAOδ/LP-AtCuAOδ) were designed outside of the 5 and 3 ends of the T-DNA insertions and the T-DNA specific primers (LBa1 for Atcuaoδ.1, and RB1-pAC161 for Atcuaoδ.2) were designed at its left border ( Figure S1 and Table S3). Due to the proximity of the insertion points in the two mutants, the same AtCuAOδ gene-specific primers were used for both the mutants and referred to as RP-AtCuAOδ/LP-AtCuAOδ ( Figure S1). The genotype of the Atcuaoδ mutants was ascertained by two sets of PCR reactions: one using RP-AtCuAOδ/LBa1 for Atcuaoδ.1 and LP-AtCuAOδ/RB1-pAC161 for Atcuaoδ.2 to determine the presence of the T-DNA insertion and the other using RP-AtCuAOδ/LP-AtCuAOδ for both the mutants to verify the absence of the fragment indicative of a WT allele, as the T-DNA insertion originates a non-amplifiable long transcript ( Figure S1). The absence of the full-length AtCuAOδ gene transcript in Atcuaoδ.1 and Atcuaoδ.2 seedlings was analyzed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) of total RNA, using rtPCR-AtCuAOδ-for1/RP-AtCuAOδ as gene-specific primers ( Figure S1 and Table S3), which would generate in WT an amplicon of 545 bp.

Construction of the Over-Expressing Transgenic Lines
The transgenic Arabidopsis overAtCuAOδ plants were prepared using Gateway technology. The AtCuAOδ gene sequence was amplified by PCR from Arabidopsis genomic DNA extracted by alkali treatment [61] from agar medium-grown seedlings using the gene-specific primers overAtCuAOδ-for and overAtCuAOδ-rev (Table S3). The overAtCuAOδ-rev primer was designed in order to insert the coding sequence for two Ser residues followed by a 6×His tag prior to the stop codon of the corresponding amplicon. The PCR product was purified and cloned initially into the pDONR 221 vector (Invitrogen), sequenced, and cloned into the pK2GW7 vector [62] through the Gateway recombination system (Invitrogen). The pK2GW7 construct (35SCaMV::AtCuAOδ-6His) was checked by sequencing prior to be transferred to Agrobacterium tumefaciens (strain GV 301) and then used to transform soil-grown Arabidopsis Col-0 WT plants by the floral dip transformation method [63]. Putatively transformed plants were controlled on selective medium (agar medium supplemented with kanamycin at the final concentration of 50 µg/mL) and subsequent PCR analysis of genomic DNA using the gene-specific primer overAtCuAOδ-for and a 6×His tag-specific primer (Table S3). Recombinant AtCuAOδ expression in 35SCaMV::AtCuAOδ-6His transgenic plants was determined by RT-qPCR using the gene-specific primers RTqPCR-AtCuAOδ-for and the RTqPCR-AtCuAOδ-rev (Table S3), as well as western-blot analysis using a rabbit anti-6×His tag antibody conjugated to horseradish peroxidase (Abcam). All the analyses were performed on the third generation (T3) of the lines described herein.

PCR, RT-PCR and RT-Quantitative PCR (RT-qPCR) Analysis
PCR reactions was carried out with the DreamTaqTM DNA Polymerase (Fermentas) in a iCyclerTM ThermalCycler (Bio-Rad) with the following parameters: 2 min of denaturation at 95 • C, 35 cycles of 95 • C for 30 s, 58 • C for 1 min, 72 • C for 1.5 min, and 10 min at 72 • C for the final extension. Total RNA was isolated from 12-day-old whole Arabidopsis seedlings using the RNeasy Plant Mini kit (QIAGEN) following the manufacturer's instructions. DNase digestion was performed during RNA purification using the RNase-Free DNase Set (QIAGEN). For RT-PCR, the first cDNA strand was synthesized from total RNA following the protocol of the ImProm-II Reverse Transcription System (Promega). Ubiquitin-conjugating enzyme 21 (UBC21) [64] was used as the internal control to confirm equal amounts of RNA among the various samples, using the primers UBC21-for and UBC21-rev (Table S3).
RT-qPCR analysis was performed on DNase-treated RNA (4 µg) from 12-day-old whole Arabidopsis seedlings. The cDNA synthesis and PCR amplification were carried out using GoTaq®2-Step RT-qPCR System200 (Promega) according to the manufacturer's protocol. The PCRs were run in a Corbett RG6000 (Corbett Life Science, QIAGEN) utilizing the following program: 95 • C for 2 min, then 40 cycles of 95 • C for 7 s and 60 • C for 40 s. The melting program ramps from 60 • C to 95 • C. rising by 1 • C each step. AtCuAOδ specific primers were RTqPCR-AtCuAOδ-for and RTqPCR-AtCuAOδ-rev (Table S3). UBC21 (At5g25760) was used as reference gene and specific primers were prepared [64] (UBC21-For and UBC21-Rev; Table S3). Fold change in the expression of the AtCuAOδ was calculated according to the ∆∆C q method as follows [65], where: C q refers to the quantification cycle, Expression fold-induction = ∆∆C q treated sample / ∆∆C q non-treated sample .
The value of ∆C q at time 0 (control sample) has been assumed to be the reference value for both treated and untreated samples at each experimental time. Accordingly, ∆∆C q has been calculated as indicated above for both treated and untreated samples at each experimental time. The reported values of expression fold-inductions after treatment are relative to the corresponding expression values of non-treated plants for each time point, with the value for time zero assumed to be one. The software used to control the thermocycler and to analyze data was the Corbett Rotor-Gene 6000 Application Software (version 1.7, Build 87; Corbett Life Science, QIAGEN, Milan, Italy).

Measurement of Stomatal Aperture
Measurement of stomatal aperture was performed as described previously [66], with slight modifications. In detail, seedlings from 12-day-old Arabidopsis WT plants, Atcuaoδ mutants, and AtCuAOδ over-expressing lines grown on agar medium were incubated in opening solution (30 mM KCl, 10 mM MES-Tris, pH 6.15) for 3 h under light to allow stomatal opening. Then, seedlings from WT plants and Atcuaoδ mutants were incubated for 2 h under light in liquid medium in the absence or presence of ABA 1, 10, and 100 µM, DMTU 100 µM, and ABA 1, 10, and 100 µM/DMTU 100 µM to analyze stomatal aperture.
Treatments with CuAOs inhibitors 2-BrEtA and AG were performed as follows: after 3 h incubation with opening solution, seedlings from WT plants and Atcuaoδ mutants were incubated in liquid medium supplemented or not with 2-BrEtA (0.5, 5 mM) or AG (0.1, 1 mM) for 30 min under light, after which ABA at the final concentration of 100 µM was added and further incubated for 2 h under light.
Based on preliminary experiments of ABA dose-response curve, independent experiments were carried out grouping treatments in separate blocks by ABA concentration or CuAO inhibitor type.
Following the various treatments, seedlings from WT plants, Atcuaoδ mutants, and AtCuAOδ over-expressing lines were treated with a fixing solution (1% glutaraldehyde, 10 mM NaPi pH 7.0, 5 mM MgCl 2 , and 5 mM EDTA) and incubated for 30 min under light. Images of stomata with the outline of stomatal pores in the focal plane were acquired by a Leica DFC 450C digital camera applied to a Zeiss Axiophot 2 microscope at the magnification of 20×, and stomatal apertures (width/length) were measured using a digital ruler (ImageJ 1.44). Width and length of stomata pores were measured, and stomatal apertures were expressed as the width/length ratio.

In Situ Detection of Reactive Oxygen Species (ROS) in Guard Cells
ROS production in guard cells was analyzed using a chloromethyl derivative of 2 ,7 -dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA; Molecular Probes, Invitrogen) as previously described [25,67], with slight modifications. Arabidopsis leaves from 12-day-old seedlings from WT, as well as AtCuAOδ insertional mutants and over-expressing lines grown on agar medium, were detached and incubated for 3 h in the assay solution containing 5 mM KCl, 50 µM CaCl 2 , and 10 mM MES-Tris (pH 6.15), and then 50 µM CM-H 2 DCFDA was added to the sample. Leaves were incubated for 30 min at room temperature and then the excess dye was washed out with the fresh assay solution. Collected tissues were again incubated in the assay solution containing 100 µM ABA for 20 min in dark conditions. Images were captured by Laser Scanning Confocal Microscopy (LSCM), using a Leica TCS-SP5 equipped with an Argon laser (Excitation/Emission:~492-495/517-527 nm) and the Leica Application Suite Advanced Fluorescence (LAS-AF; Leica Microsystems, Milan, Italy).

Statistics
For RT-qPCR analysis, five independent experiments (in this case representing the biological replicates; n = 5) were performed; in each experiment, the seedlings (~200 mg) from four agar plates for every time point and treatment were used. For each cDNA obtained, qPCR was performed in triplicate (technical replicates) and the triplicate mean values have been used in the statistical analysis for each of the five independent experiments.
For the stomatal aperture measurements, three independent experiments were performed for each treatment on the different genotypes. Independent experiments were carried out grouping treatments in separate blocks by ABA concentration or CuAO inhibitor type. A Control and a 100 µM ABA treatment for each of the genotypes analyzed were always included to verify data reproducibility between blocks. For each time, five similarly-sized leaves were harvested from different seedlings for each genotype and treatment. In this case, each of the five leaves from the three experiments was considered a biological replicate for a total of fifteen biological replicates for each genotype and treatment (n = 15). For each leaf, four random chosen fields (430 µm × 325 µm) were acquired and approximately 60 stomata were measured, and the mean values were used in the statistical analysis. The results presented are supported by preliminary experiments of ABA dose-response curves. Statistical tests were performed using GraphPad Prism (GraphPad Software) with one-way ANOVA analysis, followed by Sidak's multiple comparison tests. Statistical significance of differences was evaluated by p level, with ns showing not significant, and *, **, ***, and **** p values representing equal or less than 0.05, 0.01, 0.001, and 0.0001, respectively. The LSCM analysis of ROS production by CM-H 2 DCFDA-staining was performed on plants from five independent experiments, each of which analyzed leaves from five plants per plant genotype and per treatment, yielding reproducible results. Images from single representative experiments are shown.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/8/6/183/s1. Figure S1: Characterization of Atcuaoδ.1 and δ.2 mutants. Figure S2: Nucleotide and deduced amino acid sequences of AtCuAOδ gene (At4g12290, TAIR accession number 2139069) retrieved from TAIR database. Figure S3: Characterization of the lines over-expressing the AtCuAOδ gene, showing both protein and mRNA levels and the positive correlation between them. Table S1: Data presented in Figure 2A with the effect of ABA and DMTU on stomata pore width/length ratio. Results from three independent experiments are reported (mean values, SD, and SE). Table S2: Data presented in Figure 2B with the effect of ABA and CuAO inhibitors (2-BrEtA and AG) on stomata pore width/length ratio. Results from three independent experiments are reported (mean values, SD, and SE). Table S3