Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity^,,

An Arabidopsis mutant with defective polyamine oxidase5 exhibits delayed transition from vegetative to reproductive growth caused by lack of thermospermine oxidation.

Abstract

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.

Polyamines (PAs) are low-molecular mass aliphatic amines that are present in almost all living organisms. Cellular PA concentrations are governed primarily by the balance between biosynthesis and catabolism. In plants, the major PAs are the diamine putrescine (Put), the triamine spermidine (Spd), and the tetraamines spermine (Spm) and thermospermine (T-Spm; Kusano et al., 2008; Alcázar et al., 2010; Mattoo et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014). Put is synthesized from Orn by Orn decarboxylase and/or from Arg by three sequential reactions catalyzed by Arg decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Arabidopsis (Arabidopsis thaliana) does not contain an ORNITHINE DECARBOXYLASE gene (Hanfrey et al., 2001) and synthesizes Put from Arg via the ADC pathway. Put is further converted to Spd via an aminopropyltransferase reaction catalyzed by spermidine synthase (SPDS). In this reaction, an aminopropyl residue is transferred to Put from decarboxylated S-adenosyl-Met, which is synthesized by S-adenosyl-Met decarboxylase (SAMDC; Kusano et al., 2008). Spd is then converted to Spm or T-Spm, reactions catalyzed in Arabidopsis by spermine synthase (SPMS; encoded by SPMS) or thermospermine synthase (encoded by Acaulis5 [ACL5]), respectively (Hanzawa et al., 2000; Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010). A recent review reports that T-Spm is ubiquitously present in the plant kingdom (Takano et al., 2012).

The PA catabolic pathway has been extensively studied in mammals. Spm and Spd acetylation by Spd/Spm-N¹-acetyltransferase (Enzyme Commission no. 2.3.1.57) precedes the catabolism of PAs and is a rate-limiting step in the catabolic pathway (Wallace et al., 2003). A mammalian polyamine oxidase (PAO), which requires FAD as a cofactor, oxidizes N¹-acetyl Spm and N¹-acetyl Spd at the carbon on the exo-side of the N⁴-nitrogen to produce Spd and Put, respectively (Wang et al., 2001; Vujcic et al., 2003; Wu et al., 2003; Cona et al., 2006). Mammalian spermine oxidases (SMOs) perform oxidation of the carbon on the exo-side of the N⁴-nitrogen to produce Spd, 3-aminopropanal, and hydrogen peroxide (Vujcic et al., 2002; Cervelli et al., 2003; Wang et al., 2003). Thus, mammalian PAOs and SMOs are classified as back-conversion (BC)-type PAOs.

In plants, Spm, T-Spm, and Spd are catabolized by PAO. Plant PAOs derived from maize (Zea mays) and barley (Hordeum vulgare) catalyze terminal catabolism (TC)-type reactions (Tavladoraki et al., 1998). TC-type PAOs oxidize the carbon at the endo-side of the N⁴-nitrogen of Spm and Spd to produce N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, plus 1,3-diaminopropane and hydrogen peroxide (Cona et al., 2006; Angelini et al., 2008, 2010). The Arabidopsis genome contains five PAO genes, designated as AtPAO1 to AtPAO5. Four recombinant AtPAOs, AtPAO1 to AtPAO4, have been homogenously purified and characterized (Tavladoraki et al., 2006; Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012). AtPAO1 to AtPAO4 possess activities that convert Spm (or T-Spm) to Spd, called partial BC, or they convert Spm (or T-Spm) first to Spd and subsequently to Put, called full BC. Ahou et al. (2014) report that recombinant AtPAO5 also catalyzes a BC-type reaction. Therefore, all Arabidopsis PAOs are BC-type enzymes (Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012; Ahou et al., 2014). Four of the seven PAOs in rice (Oryza sativa; OsPAO1, OsPAO3, OsPAO4, and OsPAO5) catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a), whereas OsPAO7 catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO2 and OsPAO6 remain to be characterized, but may catalyze TC-type reactions based on their structural similarity with OsPAO7. Therefore, plants possess both TC-type and BC-type PAOs.

PAs are involved in plant growth and development. Recent molecular genetic analyses in Arabidopsis indicate that metabolic blocks at the ADC, SPDS, or SAMDC steps lead to embryo lethality (Imai et al., 2004; Urano et al., 2005; Ge et al., 2006). Potato (Solanum tuberosum) plants with suppressed SAMDC expression display abnormal phenotypes (Kumar et al., 1996). It was also reported that hydrogen peroxide derived from PA catabolism affects root development and xylem differentiation (Tisi et al., 2011). These studies indicate that flux through metabolic and catabolic PA pathways is required for growth and development. The Arabidopsis acl5 mutant, which lacks T-Spm synthase activity, displays excessive differentiation of xylem tissues and a dwarf phenotype, especially in stems (Hanzawa et al., 2000; Kakehi et al., 2008, 2010). An allelic ACL5 mutant (thickvein [tkv]) exhibits a similar phenotype as that of acl5 (Clay and Nelson, 2005). These results indicate that T-Spm plays an important role in Arabidopsis xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012).

Here, we demonstrate that Arabidopsis pao5 mutants contain 2-fold higher T-Spm levels and exhibit aerial tissue growth retardation approximately 50 d after sowing compared with that of wild-type plants. Growth inhibition of pao5 stems and leaves at an early stage of development is induced by growth on media containing low T-Spm concentrations. Complementation of pao5 with AtPAO5 rescues T-Spm-induced growth inhibition. We confirm that recombinant AtPAO5 catalyzes BC of T-Spm (or Spm) to Spd. Our data strongly suggest that endogenous T-Spm levels in Arabidopsis are fine tuned, and that AtPAO5 regulates T-Spm homeostasis through a T-Spm oxidation pathway.

RESULTS

AtPAO5 Loss-of-Function Mutants Accumulate High T-Spm Levels

To identify the endogenous AtPAO5 substrate, AtPAO5 transfer DNA (T-DNA) insertion mutants were obtained from the Arabidopsis Biological Resource Center (Ohio State University). Two homozygous T-DNA insertion mutants, pao5-1 (SAIL_664_A11) and pao5-2 (SALK_053110), were selected by genomic PCR analysis (Fig. 1A; Supplemental Figs. S1 and S2), and their AtPAO5 transcript levels were validated by quantitative reverse transcription (RT)-PCR analysis. The AtPAO5 transcript levels in both lines were reduced to approximately null levels compared with that in wild-type ecotype Columbia-0 of Arabidopsis (Col-0) plants (Fig. 1B), whereas T-Spm levels were 2-fold higher compared with those in the wild type and other pao mutants (Fig. 1Cd; Supplemental Fig. S3D). The levels of other PAs in the wild type, pao5-1, and pao5-2 were essentially equivalent (Fig. 1, Ca–Cc). The specific accumulation of T-Spm, but not other PAs, in pao5-1 and pao5-2 stems compared with those of the wild type were confirmed for later growth stages (Supplemental Fig. S3). These results strongly suggest that AtPAO5 preferentially catabolizes T-Spm in planta. In support, AtPAO1 transcript levels in the pao1-1 line were drastically enhanced, indicating that this line is an AtPAO1 overexpressor (Supplemental Fig. S1). In pao1-1, all PA contents including T-Spm were reduced (Supplemental Fig. S1). Recombinant AtPAO1 preferred T-Spm as substrate (Takahashi et al., 2010; Fincato et al., 2011). These results indicate that AtPAO1 is involved in T-Spm catabolism.

Figure 1.

Identification of two T-DNA insertion mutants of AtPAO5 and their PA contents. A, Schematic representation of pao5-1 and pao5-2 mutants. Light-gray and black bars indicate untranslated and coding regions, respectively. White triangles indicate the positions of the T-DNA insertion sites. The position and orientation of the primers used for confirming the T-DNA insertions are indicated by arrows. B, Relative AtPAO5 transcript levels in wild-type, pao5-1, and pao5-2 mutants. Quantitative RT-PCR analysis was performed using primers listed in Supplemental Table S1E; their positions are marked with inverted red arrows in (A). C, PA contents of the aerial parts of wild-type, pao5-1, and pao5-2 plants. Ca, Put; Cb, Spd; Cc, Spm; and Cd, T-Spm. Asterisks indicate a significant difference from the wild type (Col-0; Student’s t test, **P < 0.01). WT, Wild type. [See online article for color version of this figure.]

Growth Phenotypes of pao5 Mutants

The phenotypes of 8-week-old pao5-2 mutants clearly differed from those of the wild-type Col-0 plants (Fig. 2). Mutant pao5-2 inflorescence stems were shorter (Fig. 2, A and B), older rosette leaves were dark green (Fig. 2, C and D), and the number of rosette leaves was approximately 1.5-fold higher (Fig. 2, E and F). The fresh weights of the 8-week-old wild type, pao5-1, and pao5-2 differed (Fig. 3Aa). The total number of leaves for pao5 clearly increased at 7 weeks compared with that of the wild type (Fig. 3, Ab and B; Supplemental Fig. S4). Shoot lengths of pao5-1 and pao5-2 were shorter compared with that of the wild type (Fig. 3Ac; Supplemental Fig. S4Cc). Leaf lengths of the wild type, pao5-1, and pao5-2 were comparable, whereas the rosette leaf distribution patterns differed (Fig. 3B; Supplemental Fig. S4). Petiole lengths of the wild type and pao5 were comparable up to 6 weeks, whereas their distribution patterns differed in 7- and 8-week-old plants (Supplemental Fig. S5). Leaves of pao5 became darker than those of the wild type, turning from green to dark red (Fig. 2F; Supplemental Fig. S4C). The chlorophyll contents of the wild type, pao5-1, and pao5-2 were comparable in 4- to 8-week-old plants (measured with the SPAD-502 m; Konica Minolta; data not shown). However, the anthocyanin content of 8-week-old pao5-1 and pao5-2 was approximately 3-fold (pao5-2) to 4-fold (pao5-1) higher compared with that of the wild type (data not shown; Teng et al., 2005). These growth parameters indicate that the time of transition from vegetative to reproductive growth is delayed in pao5 mutants.

Figure 2.

Growth phenotypes of the pao5 mutant. Phenotypes of fully grown wild-type (A and C) and pao5-2 (B and D) plants. A and B, Side view. C and D, Top view. E and F, Shapes and numbers of leaves representative of wild-type (E) and pao5-2 (F) plants. Bar = 5 cm. [See online article for color version of this figure.]

Figure 3.

Growth parameters of the wild type, pao5-1, and pao5-2. Aa, Fresh weight changes of above-ground tissues during plant development from 2 to 8 weeks old. Ab, Changes in total leaf numbers in 2- to 8-week-old plants. Asterisks indicate a significant difference from the wild type (Col-0). P < 0.05 (*) and P < 0.01 (**) based on Student's t test. Ac, Shoot length of wild-type, pao5-1, and pao5-2 plants at 7 to 8 weeks old. One dot represents the length (in centimeters) of one plant. Ba, Four-week-old plants; Bb, 6-week-old plants; Bc, 7-week-old plants; Bd, 8-week-old plants. Leaf position is indicated from the lower to upper leaves. In Bc and Bd, a couple of the older leaves were dried out. WT, Wild type. [See online article for color version of this figure.]

Growth of pao5 Seedlings Is Inhibited by T-Spm

We tested the growth responses of wild-type, pao5-1, pao5-2, and other pao mutants in response to low T-Spm concentrations (5 or 10 μM). Sterilized seeds were placed onto one-half-strength Murashige and Skoog (MS) agar media without (control) or with T-Spm at 5 or 10 μM, and were then incubated in a vertical position for 18 d at 22°C under a 14-h-light/10-h-dark photocycle. In control media, the growth of wild-type plants and all pao mutants was essentially equivalent. In media containing 5 μm T-Spm, the growth of aerial parts of pao5-1 and pao5-2 was inhibited compared with those of wild-type plants and other pao mutants such as pao1-2, pao2-4, pao3-1, and pao4-1 (Fig. 4A). In media containing 10 μm T-Spm, the growth of aerial parts of pao5-1 and pao5-2 mutants was severely inhibited compared with that of the wild type (Supplemental Fig. S6A). The growth inhibition affected aerial plant parts but not roots (Fig. 4A; Supplemental Fig. S6). No significant growth differences were observed when plants were grown on media containing 100 μm Spm (Fig. 4A), 1 mm Put, or 1 mm Spd (Supplemental Fig. S6B). These results indicate that T-Spm specifically inhibits pao5 growth.

Figure 4.

Growth arrest of pao5 aerial parts on 5 μm T-Spm-containing media. A, Growth phenotypes of wild-type and pao mutants, including pao5-1 and pao5-2. Top row, Control (normal MS agar medium); middle row, medium containing 5 μm T-Spm; bottom row, medium containing 100 μm Spm. B, PA contents of aerial parts of wild-type (Col-0), pao5-1, and pao5-2 mutants in the absence or presence of 5 μm T-Spm. PAs were extracted, and the respective contents were measured by HPLC. Mean ± sd values are shown. A, Put; B, Spd; C, Spm; D, T-Spm. Gray and black columns indicate control growth conditions and 5 μm T-Spm media, respectively. Asterisks indicate significant differences from the wild type (Col-0; Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001). WT, Wild type.

We analyzed the PA contents in aerial parts of wild-type, pao5-1, and pao5-2 seedlings grown in one-half-strength MS agar media without (control) or with 5 μm T-Spm. In the presence of 5 μm T-Spm, the endogenous T-Spm contents increased in all plants, reaching 4 nmol g⁻¹ fresh weight in the wild type and 7 to 8 nmol g⁻¹ fresh weight in pao5-1 and pao5-2 (Fig. 4Bd). Although there were no major changes in Spm contents (Fig. 4Bc), there were concomitant increases in Put and reductions in Spd contents in mutants grown on 5 μm T-Spm (Fig. 4, Ba and Bb). The previous experiment showed that high Put or Spd concentrations (up to 1 mM) did not adversely affect pao5 growth (Supplemental Fig. S6B). Therefore, we propose that endogenous T-Spm concentrations over a particular threshold adversely affect aerial growth of Arabidopsis plants.

AtPAO5 Complementation of pao5-2 Rescues T-Spm-Induced Growth Inhibition

To confirm whether the T-Spm-induced aerial growth inhibition of pao5 mutants was due to AtPAO5 dysfunction, we performed complementation analysis by transforming the wild-type genomic AtPAO5 into pao5-2 mutants. Parallel experiments replaced the intron-less genomic AtPAO5 open reading frame (ORF) with AtPAO1 to AtPAO4 ORFs, and these hybrid constructs were transformed into pao5-2 mutants. Transgene expression was first confirmed by RT-PCR analysis (Fig. 5A). Subsequently, the selected transgenic lines were subjected to growth assays without (control) and with 5 μm T-Spm. AtPAO5 complemented pao5-2 growth defects, clearly indicating that the observed T-Spm-induced growth inhibition was a direct result of AtPAO5 dysfunction. The AtPAO1 ORF under the control of the AtPAO5 promoter also complemented T-Spm-induced pao5-2 growth inhibition to a similar extent as that of AtPAO5 (Fig. 5B). This result is consistent with the observed AtPAO1 preference for T-Spm as substrate (Takahashi et al., 2010; Fincato et al., 2011).

Figure 5.

AtPAO5 and AtPAO1 complementation of T-Spm-induced growth-arrested pao5-2. A, Expression of complementation transgenes in pao5-2 transformants. The pao5-2 mutants were transformed with the empty vector (pPZP2H-lac; Fuse et al., 2001) or with the respective AtPAOs under the control of the AtPAO5 promoter including its 5′-untranslated region. RT-PCR was performed using the AtPAO5 5′-untranslated region forward primer and the respective PAO gene-specific primers (Supplemental Table S1F). B, Growth phenotypes of the wild type and pao5-2 and pao5-2 transformants. C, Vascular networks of representative rosette leaves of the wild type and pao5-2 at the six-leaf stage; plants were grown on media with or without 5 µM T-Spm. Leaves were fixed in ethanol to acetic acid and then cleared in chloral hydrate to glycerol to water. D, PA contents of the wild type and pao5-2 and pao5-2 transformants in the absence or presence of 5 μm T-Spm. Da, Put; Db, Spd; Dc, Spm; Dd, T-Spm. Also see the legend for Figure 4B. Gray column, Control media; black column, media containing 5 μm T-Spm. Asterisks indicate significant differences from the wild type (Col-0; Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001). EV, Empty vector; WT, wild type. Bar = 1 mm.

Spatiotemporal expression analysis of AtPAO1 and AtPAO5 revealed that AtPAO1 was expressed at a minimal level during all growth stages examined, and was specifically expressed at high levels in anthers, whereas AtPAO5 was abundantly expressed at all growth stages (Takahashi et al., 2010; Fincato et al., 2012). We quantitatively examined AtPAO1 and AtPAO5 transcript levels using 2- to 8-week-old Arabidopsis plants. AtPAO1 transcripts were approximately 2-fold higher in 6-week-old plants compared with those in 2- and 3-week-old plants, and the higher expression levels were maintained until later growth stages. AtPAO5 transcript levels were approximately 3- and 10-fold higher than those of AtPAO1 in 2- and 3-week-old plants, respectively (Supplemental Fig. S7). In 4- to 8-week-old plants, AtPAO5 transcript levels were approximately 2- to 4-fold higher than those of AtPAO1, except for 6-week-old plants (Supplemental Fig. S7). Ectopic expression of the AtPAO1 ORF under the control of the AtPAO5 promoter could lead to efficient T-Spm catabolism. By contrast, AtPAO2, AtPAO3, and AtPAO4 transgenes did not complement T-Spm-induced pao5-2 growth inhibition (Fig. 5B).

The pao5-2 mutants and transgenic pao5-2 carrying either empty vector or AtPAO2, AtPAO3, or AtPAO4 transgenes exhibited the dark-brown coloration in the presence of 5 μm T-Spm, even after treatment with acetone/methanol to remove chlorophyll pigments (Fig. 5C; Supplemental Fig. S8). They also had thicker veins compared with wild-type and red trichomes (Fig. 5C; Supplemental Fig. S8). By contrast, control (Col-0) wild-type plants and transgenic pao5-2 expressing AtPAO5 or AtPAO1 did not accumulate red or dark-brown pigments, and their vascular networks were normal (Fig. 5C; Supplemental Fig. S8).

We measured PA contents in the aerial parts of plants grown without or with 5 μm T-Spm. When T-Spm-induced inhibition of aerial growth was observed, endogenous T-Spm contents were >2.5 nmol g⁻¹ fresh weight, whereas T-Spm levels in wild-type plants and transgenic pao5-2 expressing AtPAO5 or AtPAO1 were <1.5 nmol g⁻¹ fresh weight (Fig. 5Dd). The growth-inhibited plants also had significantly higher Put contents (Fig. 5Da). However, 1 mm Put did not affect aerial growth of pao5 mutants (Supplemental Fig. S6B).

Recombinant AtPAO5 Prefers Tetraamine Substrates But Not Spd

To determine whether AtPAO5 utilizes T-Spm as substrate, the AtPAO5 coding region was subcloned into the pCold expression vector and introduced into the Escherichia coli Rosetta2 strain. The extracted and highly purified recombinant AtPAO5 protein (Fig. 6A) displayed FAD-specific absorption peaks at approximately 380 and 460 nm (Fig. 6B). The optimal pH and temperature for maximum AtPAO5 activity using T-Spm as substrate were then investigated. AtPAO5 catabolism of T-Spm was detected in the pH 6.0 to 10.0 range. Maximum AtPAO5 activity with T-Spm as substrate occurred at pH 6.5 and 45°C (Fig. 6, C and D). AtPAO5 activity for Spm was detected in the pH 6.5 to 10.0 range, with maximum activity at pH 7.5 (Fig. 6E), and only marginal activity in acidic pH below 6.0 (Fig. 6E). The highest AtPAO5 activity with Spm as substrate occurred at pH 7.5 and a temperature of 37°C to 42°C (Fig. 6F).

Figure 6.

Characterization of recombinant AtPAO5. A, Purification of AtPAO5. Lane 1, Crude extract after sonication; lane 2, supernatant fraction; lane 3, insoluble fraction; lane 4, AtPAO5 purified by nickel-affinity chromatography (25-fold concentrated sample compared with that of lanes 1 to 3). B, Absorbance spectrum of the lane 4 sample in Figure 4A ranging from 300 to 550 nm. C, Optimal pH for AtPAO5 activity with T-Spm as substrate. The buffers used are as follows: pH 4.0 to 5.5, 100 mm MES buffer (triangles); pH 5.5 to 8.5, 100 mm phosphate buffer (squares); and pH 8.5 to 10.0, 100 mm HEPES buffer (circles). D, Optimal temperature for AtPAO5 activity with T-Spm as substrate at pH 7.5. E, Optimal pH for AtPAO5 activity with Spm as substrate. F, Optimal temperature for AtPAO5 activity with Spm as a substrate at pH 6.5. ABS, Absorbance spectrum. [See online article for color version of this figure.]

AtPAO5 activity was highest at pH 6.5 for T-Spm and pH 7.5 for Spm (Fig. 6, C and E). Therefore, we examined recombinant AtPAO5 substrate specificity at pH 6.5 and 7.5 with 500 µM substrate. At pH 6.5, AtPAO5 preference was in the following decreasing order: T-Spm > N¹-acetyl Spm > Norspermine (NorSpm) > Spm > Spd, with Spd catabolized at a very low rate (Fig. 7A). At pH 7.5, AtPAO5 preference was in the following decreasing order: Spm > NorSpm > N¹-acetyl Spm > T-Spm > Spd, with Spd catalyzed at a very low rate (Fig. 7B). Kinetic analyses of AtPAO5 were performed at pH 7.5 and 6.5. At pH 7.5, AtPAO5 activity obeyed typical Michaelis-Menten curves for Spm and NorSpm. After conversion to Lineweaver-Burk plots, K_m values for Spm and NorSpm were calculated as 25.56 and 25.54 μM, respectively, whereas turnover number (k_cat) values for Spm and NorSpm were 0.069 and 0.086 s⁻¹, respectively (Table I). By contrast, AtPAO5 activity behaved unusually for T-Spm and N¹-acetyl Spm. In both cases, within the lower substrate (S) concentration, AtPAO5 catalyzed a one-order reaction, but enzyme activity became less efficient at [S] ≥ 20 μM. At [S] = 125 μM, enzyme activity was only approximately one-third of the highest measured activity, which was obtained at [S] = 7.8 μM. Converting the one-order reaction to Lineweaver-Burk plots, K_m values for T-Spm and N¹-acetyl Spm were calculated as 5.09 and 1.92 μM, respectively. The k_cat values for T-Spm and N¹-acetyl Spm were 0.115 s⁻¹ and 0.084 s⁻¹, respectively (Table I). K_m and k_cat values for Spd were 68.3 μm and 0.009 s⁻¹, respectively (Table I). At pH 6.5, k_cat values of AtPAO5 for Spm and NorSpm were 0.009 s⁻¹ and 0.012 s⁻¹, respectively, and K_m values were 78.67 μm and 15.82 μM, respectively (Table I). These results indicate that AtPAO5 activity toward Spm and NorSpm dramatically decreases at pH 6.5. Conversely, at pH 6.5, AtPAO5 converted T-Spm in a typical Michaelis-Menten fashion. K_m and k_cat values were 13.65 μm and 0.009 s⁻¹, respectively (Table I). At higher N¹-acetyl Spm concentrations, AtPAO5 activity was slightly reduced, but this was not significant compared with that observed at pH 7.5. The K_m and k_cat values for N¹-acetyl Spm calculated from the one-order reaction were 2.18 μm and 0.025 s⁻¹, respectively (Table I). At this pH, kinetic parameters for Spd were not determined because enzyme activity was too low (Table I). Spm and NorSpm are symmetric molecules containing a 3-4-3 and 3-3-3 carbon backbone, respectively, whereas T-Spm and N¹-acetyl Spm are asymmetric molecules containing a 3-3-4 and 3*-4-3 (asterisk indicates acetyl residue) backbone, respectively. T-Spm and N¹-acetyl Spm may function as negative effectors for AtPAO5 at higher concentrations, especially in alkaline pH (pH 7.5).

Figure 7.

Substrate specificity of recombinant AtPAO5 and AtPAO5-catalyzed reaction product(s) of T-Spm and Spm. The enzymatic activity of recombinant AtPAO5 was determined in 100 mm phosphate buffer either at pH 6.5 (A) or pH 7.5 (B). A and B, Substrate (500 μm each) was incubated at 25°C for 10 min, and the resulting production of hydrogen peroxide was measured. Enzyme activity using Spm as substrate was set as 1, and the relative activities toward the other substrates are shown. Experiments were repeated at least three times, and the mean ± sd values are displayed. C, HPLC analysis after conversion of T-Spm at pH 6.5. D, HPLC analysis after conversion of Spm at pH 7.5. C and D, Top rows show the PA (Put, Spd, T-Spm, and Spm) standard; the second, third, and bottom rows show product analysis after incubation with AtPAO5 for 0, 15, and 30 min, respectively.

Table I. Kinetic parameters of recombinant AtPAO5.

k_cat and K_m values were obtained from the Lineweaver-Burk plot analyses. Experiments were repeated at least three times and the mean ± sd values are displayed. ND, not determined.

Substrate	pH 7.5			pH 6.5
	kcat	Km	kcat/Km	kcat	Km	kcat/Km
	S−1	μM	M−1 s−1	S−1	μM	M−1 s−1
Spm	0.069 ± 0.003	25.56 ± 2.07	2,700 ± 142	0.009 ± 0.001	78.67 ± 7.13	115 ± 18
T-Spm	0.115 ± 0.007a	5.09 ± 1.06a	23,256 ± 3,299a	0.035 ± 0.003	13.65 ± 1.50	2,596 ± 68
NorSpm	0.086 ± 0.006	25.54 ± 1.81	3,386 ± 111	0.012 ± 0.001	15.81 ± 0.64	747 ± 17
N1-AcSpm	0.084 ± 0.004a	1.92 ± 0.16a	44,020 ± 1,943a	0.025 ± 0.003a	2.18 ± 0.57a	12,323 ± 2,409a
Spd	0.009 ± 0.002	68.30 ± 35.61	185 ± 98	ND	ND	ND

^aThe reaction occurred in a noncanonical Michaelis-Menten fashion. In <10 μm substrate concentration, the reaction occurred linearly. In cases with >20 μm substrate concentration, each reaction was markedly repressed. In those cases, the values were calculated from the data with one-order reaction.

AtPAO5 Is a BC-Type Enzyme

To determine the AtPAO5 reaction type, we used HPLC to analyze the reaction product(s) catalyzed by recombinant AtPAO5 with T-Spm and Spm substrates. At pH 6.5, all T-Spm substrate was converted to Spd within 30 min; a very low Put level was detected at 30 min (Fig. 7C). The conversion of T-Spm to Spd was fairly reduced at pH 6.0 (Supplemental Fig. S9B), and only tiny amounts of Put were observed (Supplemental Fig. S9, A and B). Similar HPLC analyses performed for Spm showed that AtPAO5 catalyzed the conversion of Spm to Spd at pH 7.5, whereas the conversion to Put was less efficient (Fig. 7D). Below pH 6.5, AtPAO5 activity toward Spm diminished very rapidly (Supplemental Fig. S9, C and D). These results indicate that AtPAO5 catabolizes the T-Spm and Spm tetraamines to Spd and marginally to Put, indicating that AtPAO5 is a partial BC-type PAO. This is consistent with a recent report showing that AtPAO5 is a BC enzyme (Ahou et al., 2014).

Spatial Expression of PAO5, ACL5, and SPMS in Arabidopsis

To examine how AtPAO5 specifically recognizes T-Spm in planta, we investigated the spatial expression of AtPAO5, ACL5, and SPMS in Arabidopsis using GUS fusion constructs. Transgene expression was driven by the AtPAO5 promoter (AtPAO5-GUS), the ACL5 promoter (ACL5-GUS), and the SPMS promoter (SPMS-GUS; Clay and Nelson, 2005; Sagor et al., 2011). We also generated an AtPAO5 promoter-GFP::AtPAO5-ORF construct, in which the AtPAO5 coding region was fused to the back of GFP, and the whole fused fragment was under the control of the AtPAO5 promoter. The fusion constructs were transformed into wild-type plants. Inflorescence stems and mature rosette leaf petioles of the transgenic plants were examined for GUS (or GFP). AtPAO5 expression was detected in the cortex and vascular bundles, especially procambium (Fig. 8, Aa, Ad, and B; Benítez and Hejátko, 2013). ACL5 expression was restricted to the vascular bundles (Fig. 8, Ab and Ae), and SPMS expression was distributed widely throughout the cortex, xylem, and pith regions (Fig. 8, Ac and Af). In the transgenics expressing AtPAO5 promoter-GFP::AtPAO5, GFP-tagged AtPAO5 was expressed in the protophloems and metaphloems of roots (Fig. 8, Ba and Bb) and of hypocotyls (Fig. 8Bc; Bonke et al., 2003). It is likely that AtPAO5 expression is associated with actively dividing tissues. In silico data available from the University of Toronto support our observations (Supplemental Fig. S10). Hierarchical clustering and heat map analyses of tissue-specific PA metabolic gene expression profiles revealed that AtPAO5 belongs to the same branch as ACL5, whereas SPMS was closer to AtPAO2 and AtPAO3 (Supplemental Fig. S11). These results demonstrate that the two Arabidopsis tetraamine-producing enzymes, ACL5 and SPMS, have different tissue-distribution patterns, and that the locations of AtPAO5 expression overlap with those of ACL5 expression. This evidence can explain, at least in part, the AtPAO5 specificity for T-Spm in planta.

Figure 8.

Spatial expression of AtPAO5, ACL5, and SPMS and subcellular localization of AtPAO5. A, Cross sections of inflorescence stems derived from 10-cm-long shoots (Aa−Αc) and leaf petioles of 7-week-old plants (Ad−Αf). Ad, AtPAO5 promoter constructs; Ae, ACL5 promoter constructs; Af, SPMS promoter constructs. B, Confocal images of AtPAO5 promoter-GFP::AtPAO5 fusion construct expression. Ba, Root of a 10-d-old seedling. Samples were stained with propidium iodide. Bb, Magnification of Ba. Bc, Stem section of a 10-d-old seedling. C, Representative GFP images of root protoplasts prepared from the 10-d-old wild type and AtPAO5 promoter-GFP::AtPAO5 fusion construct expression. Ca, Protoplast from wild-type plant roots; >1,000 root-derived protoplasts were observed, but no GFP fluorescence was detected. Cb, Protoplast prepared from roots expressing AtPAO5 promoter-GFP::AtPAO5 fusion construct. In both cases, protoplasts were stained with DAPI and then observed by confocal microscopy. DAPI, 4′,6-Diamino-phenylindole; DIC, differential interference contrast. Bar = 100 µm in Ba to Bc; bar = 20 µm in Ca to Cb.

Subcellular Localization of AtPAO5

A magnified view (Fig. 8Bb) of the GFP image (Fig. 8Ba) obtained for AtPAO5 promoter-GFP::AtPAO5 expression suggested that GFP-tagged AtPAO5 localized in the cytoplasm. To confirm this, we prepared protoplasts from wild-type roots and transgenic plants, and monitored GFP using confocal microscopy. No GFP fluorescence was observed from wild-type protoplasts (Fig. 8Ca), whereas GFP fluorescence was detected in the cytoplasm of protoplasts derived from transformants expressing GFP::AtPAO5 (Fig. 8Cb). Moreover, when AtPAO5 was fused to GFP at both N termini and C termini and this construct was expressed in onion (Allium cepa) epidermal cells, GFP fluorescence was observed in the cytoplasm (Supplemental Fig. S12).

DISCUSSION

AtPAO5 Oxidizes T-Spm to Produce Spd

Two allelic AtPAO5 knockout mutants, pao5-1 and pao5-2, contained approximately 2-fold higher T-Spm, but similar levels of Put, Spd, and Spm compared with those in the wild-type plants (Fig. 1; Supplemental Fig. S3). Higher T-Spm accumulation was not observed in other pao loss-of-function Arabidopsis mutants (Supplemental Fig. S1). The pao5 mutants had more rosette leaves and fewer and shorter inflorescence stems at 7 and 8 weeks old compared with those of the wild type (Figs. 2 and 3; Supplemental Fig. S4), suggesting that the transition from vegetative to reproductive growth is delayed in the mutants. Aerial growth inhibition of pao5 seedlings was induced by 5 μm T-Spm but not 100 μm Spm, 1 mm Put, or 1 mm Spd (Fig. 4; Supplemental Fig. S6). The pao5-2 mutant phenotype was rescued by complementation with the wild-type genomic AtPAO5. These results indicate a high probability that the observed pao5 mutant phenotypes result from AtPAO5 dysfunction. The pao5-2 mutant phenotype was also recovered by introduction of the AtPAO1 ORF under the control of the AtPAO5 promoter. Fincato et al. (2012) reported that the expression of AtPAO1 was restricted to the transition region between the meristematic and elongation zones of roots and anther tapetum. We also reported that AtPAO1 was specifically expressed in anthers (Takahashi et al., 2010). According to the results of quantitative RT-PCR performed at various stages of growth development, AtPAO1 showed low expression levels, whereas AtPAO5 showed higher expression levels with a peak at 3 weeks after sowing (Supplemental Fig. S7). This difference in expression between AtPAO1 and AtPAO5 may explain why pao1-2 plant did not display T-Spm-induced growth inhibition (Fig. 4A). Clear differences in PA contents among the wild type, pao5-2, and complemented and noncomplemented pao5-2 lines were observed for T-Spm and Put (Fig. 5D). Growth inhibition was induced by low T-Spm concentrations, but not by higher concentrations (1 mM) of Put (Supplemental Fig. S6). These results suggest that AtPAO5 functions as a T-Spm oxidase in Arabidopsis. A recent article reported that AtPAO5 is an SMO/dehydrogenase (Ahou et al., 2014). This conclusion is based on the following results. First, the recombinant AtPAO5 that was partially purified from Cauliflower mosaic virus CaMV35S::AtPAO5-6×His Arabidopsis transgenic plants prefer the substrates Spm, T-Spm, and N¹-acetylSpm but not Spd. Second, the levels of Spm, N¹-acetylSpm, and T-Spm were decreased in the above Arabidopsis transgenics compared with those of wild-type plants. This indicates that all three PAs (Spm, N¹-acetylSpm, and T-Spm) are in vivo substrates of AtPAO5. This discrepancy with our observations may be explained by the constitutive expression of AtPAO5 using the CaMV35S promoter. The ectopically produced AtPAO5 may be able to access and metabolize the Spm substrate in CaMV35S::AtPAO5 transgenic plants. Recombinant AtPAO5 catalyzes the BC of T-Spm (or Spm) to Spd (Figs. 6 and 7; Table I; Supplemental Fig. S9). This conclusion is consistent with a result presented by Ahou et al. (2014).

Why does AtPAO5 oxidatively deaminate T-Spm but not Spm? This may be explained, at least in part, by the spatiotemporal expression of ACL5 (T-Spm synthase gene) and AtPAO5 (Fig. 8), and supported with additional in silico data (Supplemental Figs. S10 and S11). SPMS expression is associated with SPDS1 and SPDS2 expression (both encode Spd synthase; Panicot et al., 2002), and AtPAO2 and AtPAO3 (Supplemental Fig. S10). We propose that AtPAO5 catalyzes the BC of T-Spm to Spd in Arabidopsis (Fig. 9).

Figure 9.

Model describing the importance of T-Spm metabolism in Arabidopsis. In the wild type, both T-Spm synthesis, catalyzed by ACL5, and T-Spm BC, catalyzed by PAO5, are functional, so plants grow well. In acl5, which is T-Spm deficient, stem growth is significantly disrupted (Hanzawa et al., 2000). The pao5 mutant, which lacks T-Spm catabolism, similarly shows stem growth retardation due to delayed transition from vegetative to reproductive growth. WT, Wild type.

Comparison of PAOs in Arabidopsis and Rice

The rice genome contains seven PAO genes, OsPAO1 to OsPAO7 (Ono et al., 2012). OsPAO1, OsPAO3, OsPAO4, and OsPAO5 catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a). The latter three enzymes localize in peroxisomes (Ono et al., 2012), whereas OsPAO1 localizes in cytoplasm (Liu et al., 2014a). OsPAO7 contains a putative signal peptide at its amino terminal, localizes in apoplast, and catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO6 is expected to catalyze a TC-type reaction and localize in apoplast because of strong similarity to OsPAO7. Therefore, rice PAOs localize in three cellular compartments (cytoplasm, peroxisome, and apoplast) and catalyze BC- and TC-type reactions. Arabidopsis PAOs localize to two cellular compartments (cytoplasm and peroxisome) and catalyze only BC-type reactions. AtPAO1 and AtPAO5 localize in cytoplasm (Tavladoraki et al., 2006; Ahou et al., 2014; this study), and the remaining three AtPAOs localize in peroxisomes (Kamada-Nobusada et al., 2008; Moschou et al., 2008). The apparent absence of apoplast-localized PAOs and TC-type reactions in Arabidopsis remains to be understood.

OsPAO1 belongs to the same clade as AtPAO5 and catalyzes the BC of T-Spm (or Spm) to Spd (Liu et al., 2014a). OsPAO1 expression was induced by exogenous application of T-Spm and Spm to rice plants (Liu et al., 2014a). It will be important to determine whether OsPAO1 functions as a T-Spm oxidase in rice as AtPAO5 does in Arabidopsis.

T-Spm Content Is Fine Tuned in Arabidopsis

The mutant genes acl5 (Hanzawa et al., 2000) and bushy and dwarf2 (bud2; Ge et al., 2006) were identified in PA biosynthesis pathways. ACL5 encoded T-Spm synthase (Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010; Takano et al., 2012), and its loss-of-function mutant (acl5) displayed a stem growth defect (Fig. 9; Hanzawa et al., 2000). The ACL5 allelic mutant tkv developed thicker veins in its leaves and inflorescence stems. ACL5 was specifically expressed in xylem vessel elements, and gene dysfunction resulted in morphological changes in the xylem vessel elements (Muñiz et al., 2008). The bud2 mutant resulted from loss of function of one member (SAMDC4) of a small gene family encoding SAMDC (Ge et al., 2006; Cui et al., 2010). A T-DNA insertion mutant (SALK_007279) of SAMDC4 contains null levels of T-Spm (K. Watanabe and T. Kusano, unpublished data), and T-Spm suppressed SAMDC4 and ACL5 expression (Kakehi et al., 2010). This evidence clearly supports a role for SAMDC4 in T-Spm synthesis. The in silico data suggest that SAMDC4 expression in various tissues is quite similar to that of ACL5 (Supplemental Fig. S11), and indicate that normal growth and development is markedly disturbed if T-Spm synthesis is blocked (Fig. 9).

Our study demonstrated that Arabidopsis pao5 mutants were blocked in T-Spm oxidation and showed a delay in the transition from vegetative to reproductive growth. At the seedling stage, externally supplied low-dose T-Spm inhibited the aerial growth of pao5-1 and pao5-2 mutants (Fig. 4; Supplemental Fig. S6). The T-Spm-induced growth inhibition of pao5 plants was recovered when plants contained lower endogenous T-Spm concentrations than those of the pao5 mutants, suggesting that Arabidopsis has an upper threshold of optimum T-Spm content. When the T-Spm content exceeds the upper limit, growth of Arabidopsis aerial tissues is specifically inhibited (see the model in Fig. 9). In the presence of low doses of T-Spm, the pao5-2 and noncomplemented transgenic pao5-2 plants exhibited increased vein thickness compared with the wild type and complemented transgenic pao5-2 plants (Fig. 5C; Supplemental Fig. S8). This suggests that, if endogenous T-Spm content reaches an upper limit, vascular development in aerial tissues such as petioles and leaves is disturbed by a yet unknown mechanism.

Our results strongly suggest that the T-Spm oxidase AtPAO5 controls the T-Spm concentration in a low and narrow range. The T-Spm content has to be fine-tuned in Arabidopsis because zero T-Spm content causes growth defects, as observed in the acl5 (tkv) and bud2 mutants, and levels beyond the upper limit also result in growth defects, as observed in pao5 (Fig. 9). This fine-tuning of T-Spm is clearly distinct from that of the other major PAs, Put, Spd, and Spm (Figs. 4 and 5; Supplemental Fig. S6).

The bud2 mutant displayed auxin hyposensitivity and cytokinin hypersensitivity (Cui et al., 2010), whereas the tkv mutant had reduced polar auxin transport (Clay and Nelson, 2005). Yoshimoto et al. (2012) recently demonstrated that T-Spm counteracted the effects of auxin, which stimulated xylem differentiation by repressing Monopreros/auxin responsive factor5. The same group demonstrated that T-Spm modulated auxin-related genes (Tong et al., 2014). Milhinhos et al. (2013) reported that overexpression of the ACL5 homolog in hybrid aspen (Populus temula × P. tremuloides) resulted in T-Spm accumulation and negatively affected indole-3-acetic acid (IAA) accumulation. The authors proposed an elegant negative-feedback-loop model; expression of the ACL5 homolog was enhanced by IAA via up-regulation of the class III homeodomain Leu zipper transcription factor genes, which leads to lower IAA accumulation. Milhinhos et al. (2013) further proposed that this negative loop functioned to maintain T-Spm levels. Populus spp. plants overexpressing an ACL5 homolog showed dwarf phenotypes similar to those of the Arabidopsis pao5 mutants. Future investigations using pao5 mutants will be performed to unravel the molecular mechanism for how excess T-Spm negatively regulates the transition from vegetative to reproductive growth or the aerial growth of pao5 mutants at the seedling stage.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild-type Col-0 plants and T-DNA insertion lines of AtPAO5 and other AtPAOs provided by the Arabidopsis Biological Resource Center (Ohio State University) were used in this work. All seeds were surface sterilized with 70% (v/v) ethanol for 1 min and then with a solution of 1% (v/v) sodium hypochloride and 0.1% (v/v) Tween 20 for 15 min, followed by extensive washing with sterile distilled water. Sterilized seeds were placed on one-half-strength MS 1.5% (w/v) agar plates (pH 5.6) containing 1% (w/v) Suc. Seeds were also placed onto soil mixed with a 1:1 ratio of Supermix-A (Sakata Seed) to vermiculite (Nittai). Growth conditions were 22°C with a 14-h-light/10-h-dark photocycle.

Chemicals

Put, Spd, and Spm were purchased from Nacalai-Tesque. T-Spm, NorSpm, NorSpd, and N¹-acetyl Spm were chemically synthesized (Samejima et al., 1984; Niitsu and Samejima, 1986). All other analytical grade chemicals were obtained from Sigma-Aldrich, Wako Pure Chemical Industries, and Nacalai-Tesque.

Establishment of AtPAOs T-DNA Insertion Mutants

The T-DNA insertion lines for AtPAO1 (SALK_028335 and SAIL_882_A11), AtPAO2 (SALK_046281), AtPAO3 (GK-209F07), AtPAO4 (SALK_133599), and AtPAO5 (SAIL_664_A11 and SALK_053110) were obtained from the Arabidopsis Biological Resource Center (Ohio State University). The T-DNA insertion sites were confirmed by PCR using genomic DNAs as the template and AtPAO gene-specific primers listed in Supplemental Table S1A. PCR products were analyzed by agarose gel electrophoresis and stained with ethidium bromide.

PAO Activity Assay

Recombinant AtPAO5 oxidation activities for Spm, T-Spm, Spd, N¹-acetyl Spm, and NorSpm were determined spectrophotometrically by following the formation of a pink adduct resulting from oxidation of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid catalyzed by horseradish peroxidase (Tavladoraki et al., 2006). To determine the optimum pH, 100 mm MES buffer (pH 4.0–5.5), 100 mm sodium phosphate buffer (pH 5.5–8.5), and 100 mm HEPES buffer (pH 8.5–10.0) were used. In a typical experiment, 0.5 to 3.0 µg of protein was added to a buffered solution containing 500 µm of each substrate, 100 µm 4-aminoantipyrine, 1 mm 3,5-dichloro-2-hydroxybenzenesulfonic acid, and 10 U mL⁻¹ horseradish peroxidase, and the increase in A₅₁₅ was measured using a multiplate reader (M200; Tecan) or spectrophotometer (U-2900; Hitachi).

HPLC Analysis of Enzyme Reaction Products

To determine the PA oxidation reaction products, purified recombinant AtPAO5 was incubated with 150 µm PA (Spm or T-Spm) in 100 mm phosphate buffer (pH 7.5, 6.5, or 6.0) at 37°C for various time periods. The reaction was stopped by addition of 9 volumes of 5% (v/v) perchloric acid. To each 1 mL of reaction products, 1 mL of 2 n NaOH was added, followed by 10 µL of benzoyl chloride, and the reaction was incubated at room temperature for 20 min. After addition of 2 mL of saturated sodium chloride and 2 mL of diethyl ether followed by vigorous mixing, the phases were separated by centrifugation for 5 min at 1,500g at 4°C. Aliquots of the organic solvent phase (1.5 mL each) were evaporated, and the residue was resuspended in 50 µL of methanol. The benzoylated PAs were analyzed with a programmable Hewlett Packard series 1100 liquid chromatograph using a reverse-phase column (4.6 × 250 mm; TSKgel ODS-80Ts; Tosoh Bioscience) and detected at 254 nm. One cycle of the run consisted of a total of 60 min at a flow rate of 1 mL min⁻¹ at 30°C; this included 42% acetonitrile for 25 min for PA separation, increased up to 100% acetonitrile during 3 min, then 100% acetonitrile for 20 min for washing, decreased down to 42% (v/v) acetonitrile during 3 min, and finally to 42% acetonitrile for 9 min.

Complementation Lines for the pao5 T-DNA Insertion Mutant

To generate the pao5-2 complementation lines, the AtPAO5 genomic fragment was obtained by PCR using the primer pair listed in Supplemental Table S1G. The KpnI- and SalI-double-digested fragment was subcloned into the corresponding sites of the binary vector pPZP2H-lac (Fuse et al., 2001), yielding pPZP2H-AtPAO5. The AtPAO5 ORF region in pPZP2H-AtPAO5 was replaced by fusion PCR with the respective ORFs of AtPAO1, AtPAO2, AtPAO3, or AtPAO4, resulting in constructs pPZP2H-AtPAO1 to pPZP2H-AtPAO4, respectively. The final constructs were introduced into Agrobacterium tumefaciens strain GV3101 and then transformed into pao5-2 plants using the floral-dip method (Clough and Bent, 1998). Transformants were selected on MS agar media containing 25 mg mL⁻¹ hygromycin (hyg) and 50 mg mL⁻¹ carbenicillin. T2 seeds were obtained from self-fertilization of primary transformants, surface sterilized, and grown on hyg-containing plates, and those showing a 3:1 (hygromycin resistant [hyg^R]:hygromycin sensitive) segregation ratio were selected to produce homozygous (hyg^R/hyg^R) T3 lines that were used for further study.

Preparation of Recombinant AtPAO5 Protein in Escherichia coli

The AtPAO5 coding region was amplified by RT-PCR from Arabidopsis total RNA using gene-specific primers (Supplemental Table S1G). The amplified PCR products were digested with the respective restriction enzymes and cloned in-frame with the 6×His tag of the pCold vector (Takara Bio), resulting in pCold-AtPAO5. After confirmation of the cloned fragments by DNA sequence analysis, the pCold-AtPAO5 was transformed into E. coli Rosetta2 (DE3) cells, and recombinant AtPAO5 protein tagged with 6×His at the N terminus was produced according to the manufacturer’s instructions (Takara Bio). In brief, an E. coli cell culture with an optical density at 600 nm of 1.4 was treated with 2% ethanol and heat shocked at 60°C for 2 min as previously described (Chen et al., 2002). The culture was then cooled to 15°C for 30 min before adding isopropylthio-β-galactoside to a final concentration of 0.5 mm and being further incubated for 24 h. E. coli cells were collected by centrifugation, resuspended in 50 mm sodium phosphate buffer (pH 8.0) containing 300 mm sodium chloride, 10 mm imidazole, and 1 mm phenylmethylsulfonyl fluoride, and disrupted by sonication. After centrifugation at 17,000g for 25 min at 4°C, the cleared supernatant was collected and applied to a nickel-nitrilotriacetic acid agarose column (Qiagen). The column was washed with washing buffer (50 mm sodium phosphate buffer [pH 8.0] containing 300 mm sodium chloride and 20 mm imidazole), and the bound proteins were eluted with elution buffer (50 mm sodium phosphate buffer [pH 8.0] containing 300 mm sodium chloride and 250 mm imidazole). The purified recombinant AtPAO5 protein was immediately dialyzed against 50 mm sodium phosphate buffer (pH 8.0) containing 300 mm sodium chloride.

RT-PCR and Quantitative Real-Time RT-PCR Analyses

Total RNA was extracted from whole seedlings or leaves using Sepasol-RNA I Super (Nacalai-Tesque). First-strand complementary DNA was synthesized with ReverTra Ace (Toyobo) using oligo(dT) primers. RT-PCR analysis was performed as previously described (Zhu et al., 2012) using the primers listed in Supplemental Table S1F. Quantitative real-time RT-PCR was performed using Fast-Start Universal SYBR Green Master (ROX; Roche Applied Science) on a StepOne real-time PCR system (Life Technologies Japan). Specific primer sets were designed and are listed in Supplemental Table S1E. The sizes of the amplified fragments are 75 bp (AtPAO1), 95 bp (AtPAO2), 87 bp (AtPAO3), 85 bp (AtPAO4), and 85 bp (AtPAO5). The amount of target mRNA was normalized using the housekeeping gene (CBP20) encoding the cap binding protein20, which was amplified with the primer pair listed in Supplemental Table S1E.

Construction of the AtPAO5 Promoter-GUS and AtPAO5 Promoter-GFP::AtPAO5-ORF Binary Plasmids and Generation of Transgenic Lines

Transgenic plants expressing GUS under the control of the AtPAO5 promoter were generated. The AtPAO5 promoter fragment (3.1 kb of the AtPAO5 upstream region) was amplified by PCR using AtPAO5-specific primers (Supplemental Table S1C). The amplified fragment sequence was verified and cloned into the pBI101 vector, resulting in an AtPAO5 promoter-GUS construct. The ACL5 promoter::GUS (Clay and Nelson, 2005) and SPMS promoter::GUS (Sagor et al., 2011) transgenic plants were used as reference controls. Transgenic plants expressing GFP::AtPAO5-ORF under the control of the AtPAO5 promoter were also generated. The 5,759-bp fragment sandwiched with KpnI and SalI, which covers 3.1 kb of AtPAO5 promoter region followed by AtPAO5 5′-untranslated region, GFP coding region fused to AtPAO5 ORF and AtPAO5 3′-untranslated region, were generated by a fusion PCR using the respective primers listed in Supplemental Table S1G. After verifying the DNA sequence, it was cloned into pPZP2H-lac (Fuse et al., 2001). The recombinant plasmids were introduced into A. tumefaciens strain GV3101, and then transformed into Arabidopsis (Col-0) plants using the above-described floral-dip method.

Histochemical GUS Activity Assays

Histochemical localization of GUS activity in plant tissues was assayed according to the method of Jefferson (1987). Tissue samples were collected, fixed in 90% acetone for 15 min, rinsed in 100 mm phosphate buffer, and incubated overnight with GUS staining solution (0.5 mg mL⁻¹ 5-bromo-4-chloro-3-indolyl β-d-glucuronide, 10 mm sodium phosphate buffer [pH 7.0], 0.5 mm potassium ferricyanide, 0.5 mm potassium ferrocyanide, 0.1% Triton X-100, 10 mm EDTA) at 37°C in the dark. After incubation, stained plant tissues were cleared by 70% ethanol to remove chlorophyll. GUS-stained sections were cleared in a solution consisting of 8 g of chloral hydrate, 1 mL of glycerol, and 2 mL of distilled water (Koizumi et al., 2009). Plant tissue samples were sliced to a thickness of 60 µm using a Vibratome (Hyrax VT1000A; Zeiss) and were stained, cleared, observed using an Olympus stereo-microscope, and photographed.

Measurement of Chlorophyll and Anthocyanin Contents

Chlorophyll content was determined using a SPAD-502 m (Konica Minolta) as described by Ling et al. (2011). Anthocyanin content was determined according to the procedure described by Teng et al. (2005). Frozen, homogenized tissues were extracted in 1 mL of 1% (v/v) hydrochloric acid in methanol for 1 d at 4°C. After centrifugation at 10,000g for 15 min, the absorbance of the cleared lysates was measured at 530 and 657 nm. Relative anthocyanin content was calculated by the formula provided in Teng et al. (2005).

Protoplast Preparation

Protoplasts were prepared by following the procedure described by Yoo et al. (2007). In brief, roots of 2-week-old Arabidopsis were aseptically cut into 0.5- to 1-mm pieces and then incubated with enzyme solution (20 mm MES [pH 5.7] containing 1.5% [w/v] cellulose R10, 0.4% [w/v] macerozyme R10, 400 mm mannitol, 20 mm potassium chloride, 10 mm CaCl₂, and 0.1% bovine serum albumin). To accelerate digestion, vacuum infiltration was applied for 30 min in the dark using a desiccator, and samples were incubated for 3 h at room temperature with gentle shaking. The undigested tissues were removed by filtration through a 75-μm nylon mesh. After centrifugation of the flow-through at 100g for 1 to 2 min, protoplasts were washed with a washing solution (2 mm MES [pH 5.7] containing 154 mm sodium chloride, 125 mm CaCl₂, and 5 mm potassium chloride). The protoplasts were resuspended at 2 × 10⁵ mL⁻¹ in MMG solution (4 mm MES [pH 5.7] containing 40 mm mannitol and 15 mm MgCl₂).

Histology and Microscopy

Histological methods were performed as previously described (Yoshimoto et al., 2012). Rosette leaves were fixed in a 9:1 mixture of ethanol: acetic acid, cleared in a mixed solution of 8 g of chloral hydrate to 1 mL of glycerol to 2 mL of water, and observed under a light microscope equipped with Nomarski optics (BX61; Olympus). Confocal images were captured using a laser scanning microscope (LSM 710; Zeiss).

Statistical Analysis

Data analyses were performed using the statistical tools (Student’s t test) of Microsoft Excel software.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AtPAO1 (Ag5g13700), AtPAO2 (At2g43020), AtPAO3 (At3g59050), AtPAO4 (At1g65840), AtPAO5 (At4g29720), ACL5 (At5g19530), SPMS (At5g53120), CBP20 (At5g44200), and Actin (At3g18780).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Identification and characterization of T-DNA insertion mutants of AtPAO1, AtPAO2, AtPAO3, and AtPAO4.

• Supplemental Figure S2. Identification of T-DNA insertion sites in the pao mutants with genomic PCR analysis.

• Supplemental Figure S3. PA contents in stems of 7-week-old wild-type, pao5-1, and pao5-2 plants.

• Supplemental Figure S4. Leaf phenotypes of the wild type, pao5-1, and pao5-2 during growth development.

• Supplemental Figure S5. Distribution of petiole length of wild-type, pao5-1, and pao5-2 plants at different growth stages.

• Supplemental Figure S6. Specific inhibition of pao5-1 and pao5-2 growth by exogenously applied T-Spm.

• Supplemental Figure S7. AtPAO1 and AtPAO5 expression patterns during growth and development.

• Supplemental Figure S8. Vascular networks of representative rosette leaves of wild-type, pao5-2, and transgenic pao5-2 plants expressing empty vector and AtPAO-ORF constructs under the control of the AtPAO5 promoter.

• Supplemental Figure S9. HPLC analysis of reaction products generated by AtPAO5 oxidation of Spm and T-Spm.

• Supplemental Figure S10. Expression profiles of AtPAO5 (A and D), ACL5 (B and E), and SPMS (C and F) in Arabidopsis.

• Supplemental Figure S11. Hierarchical clustering analysis and heat map of tissue-specific expression profiles of PA metabolism genes based on available microarray data.

• Supplemental Figure S12. Subcellular localization of AtPAO5-GFP and GFP-AtPAO5 fusion proteins in onion epidermal cells.

• Supplemental Table S1. Primers used in this study.

Glossary

PA

polyamine

PAO

polyamine oxidase

ADC

Arg decarboxylase

SPDS

spermidine synthase

SAMDC

S-adenosyl-Met decarboxylase

SPMS

spermine synthase

SMO

spermine oxidase

BC

back-conversion

TC

terminal catabolism

T-DNA

transfer DNA

RT

reverse transcription

Col-0

ecotype Columbia-0 of Arabidopsis

MS

Murashige and Skoog

ORF

open reading frame

IAA

indole-3-acetic acid

hyg

hygromycin