Barley polyamine oxidase isoforms 1 and 2, a peculiar case of gene duplication Manuela Cervelli 1 , Marzia Bianchi 1 , Alessandra Cona 1 , Cristina Crosatti 2 , Michele Stanca 2 , Riccardo Angelini 1 , Rodolfo Federico 1 and Paolo Mariottini 1 1 Dipartimento di Biologia, Universita ` ‘Roma Tre’, Rome, Italy 2 Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d’Arda (PC), Italy Plant polyamine oxidase (PAO), a flavin adenine dinu- cleotide-containing enzyme, catalyzes the oxidation of spermidine (Spd) and spermine (Spm) to 4-aminobut- anal and N-(3-aminopropyl)-4-aminobutanal, respect- ively, plus 1,3-diaminopropane and H 2 O 2 [1–3]. Because these compounds cannot be converted directly to other polyamines, plant PAO is considered to be involved in the terminal catabolism of polyamines. Two barley (Hordeum vulgare) paralogous PAO genes (HvPAO1 and HvPAO2, formerly BPAO1 and BPAO2) code for two protein isoforms which share 73% amino acid identity [4]. In particular, HvPAO2 isoform has been purified, characterized and compared with the maize (Zea mays) counterpart PAO, ZmPAO, Keywords biochemical characterization; enzyme isoform; gene duplication; polyamine oxidase; tissue specificity Correspondence P. Mariottini, Dipartimento di Biologia, Universita ` degli Studi ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Roma, Italy Fax: +39 06 55176321 Tel: +39 06 55176359 E-mail: mariotpa@bio.uniroma3.it (Received 09 May 2006, revised 23 June 2006, accepted 30 June 2006) doi:10.1111/j.1742-4658.2006.05402.x Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 iso- form in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfer- tilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spike- lets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and compar- able K m values for both substrates, i.e. 0.89 · 10 )5 m and 0.5 · 10 )5 m for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activ- ity significantly differs in substrate specificity and pH optimum when com- pared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles. Abbreviations HvPAO1, barley polyamine oxidase 1; HvPAO2, barley polyamine oxidase 2; PAO, polyamine oxidase; Spd, spermidine; Spm, spermine; Ubi, ubiquitin; ZmPAO1, maize polyamine oxidase 1; ZmPAO2, maize polyamine oxidase 2. 3990 FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS the best characterized plant member of this class of enzymes [2,5–8]. The maize PAO gene family is repre- sented by a small number of copies; three genes enco- ding polyamine oxidase (ZmPAO1, ZmPAO2 and ZmPAO3, formerly MPAO1, MPAO2 and MPAO3) and their upstream regions have been previously char- acterized [9]. They show a highly conserved gene organization and almost identical amino acid sequences, indicating that they originated from dupli- cation events. Molecular modeling of HvPAO2 shows the same global fold of ZmPAO, but the two proteins have different catalytic properties [4]. Both precursor enzymes include a cleavable N-terminal leader; more- over, HvPAO2 has an eight-residue-long carboxyl extension (DELKAEAK) that directs this protein to the vacuole [10]. Thus this C-terminus is responsible for the different subcellular localization observed in leaf tissues between the two enzymes, as HvPAO2 is symplastic in mesophyll cells, at variance with ZmPAO, which is apoplastic in the same tissue [4,10,11]. While HvPAO2 transcript is the major form detectable in all barley plant tissues analyzed so far, HvPAO1 gene transcription is tissue specific, being observed by RT-PCR analysis only in the ear [4]. The presence of the N-terminal signal peptide in HvPAO1 indicates the transit of this protein in the secretory pathway, possibly targeting this protein to extracellular compartment, as in the case of ZmPAO protein. The amino acid identity shared by these two enzymes is 84% higher than the one shared by HvPAO2 and ZmPAO (73%), indicating that HvPAO1 and ZmPAO1-3 genes are orthologous [4]. The physiologi- cal roles ascribed to ZmPAO relate mainly to poly- amine homeostasis, as well as hydrogen peroxide biosynthesis in the apoplast [3]. The latter functional implication arises from the analysis of several experi- mental results concerning: (i) the high specific activity in extracellular fluids [12]; (ii) the correlation of PAO expression with photomorphogenic growth regulation and the hypersensitive response [13–15]; (iii) the inhibi- tion of hydrogen peroxide release by PAO activity inhibitors [16]; and (iv) the histochemical and ultra- structural studies that demonstrated the association of this enzyme with the cell wall [16,17]. Many of these studies highlighted the physiological implications of PAO-mediated hydrogen peroxide syn- thesis in the apoplast related to peroxidase-catalyzed reactions or as a compound triggering signal transduc- tion leading to hypersensitive response. However, the vacuolar localization of HvPAO2 and the predicted apoplastic localization of HvPAO1 draw a new scen- ario to the possible role multiplicity of this enzyme family. In fact, even in maize, this multiplicity may have been underestimated. A recent immunogold ultra- structural study has in fact shown that ZmPAO could be detected in the cytoplasm of differentiating xylem and rhizodermal cells of young root tissues [17]. This localization has been correlated with two possible additional functions, reactive oxygen species-induced programmed cell death of xylem elements [17] and hydrogen peroxide-dependent cross-linking of polysac- charides within the secretory pathway [13,17]. In par- ticular, the early cross-linking of hemicellulose and pectin which occurs in young cells or tissues could result in the formation of large coagula that would have a loosening effect within the cell wall due to their scarce interactions with the cellulose microfibrils, this being diverse from apoplastic polymer cross-linking, which is thought to strengthen the cell wall inasmuch as it occurs after the hemicelluloses and pectins have already bonded to cellulose microfibrils [18]. Under this view, it is reasonable to hypothesize that, in bar- ley, different PAO isoforms, specifically expressed dur- ing development in different tissues and organs, could play different physiological roles. This article describes a detailed analysis of HvPAO1 and HvPAO2 gene expression in barley ear and the characterization of the main biochemical features of purified and in vitro synthesized HvPAO1 enzyme. RT-PCR analysis was also carried out on different ear tissues and internodes. Constructs containing HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2 promoter sequences [4,9], fused to the b-glucuronidase gene (GUS), were transiently expressed in roots, leaves and ears of barley, and in roots and leaves of maize with the aid of a biolistic delivery system. In this study, we present evidence that barley HvPAO1 and HvPAO2 genes represent an interesting evolutionary case of gene duplication, since the orthologous HvPAO1 coding sequence corresponds to ZmPAO1-3 genes, while the paralogous HvPAO2 coding sequence could be consid- ered as a more recently evolved gene with a different physiological role. Results Accumulation of HvPAO1 and HvPAO2 mRNAs in different barley tissues The transcription level of HvPAO1 and HvPAO2 mRNAs has been examined in different stem inter- nodes and whole ear at 30-days postfertilization and in various ear tissues by RT-PCR analysis (Fig. 1). PCR-amplified mRNAs have been probed with primer-pairs specific for HvPAO1 and HvPAO2 isoforms and within the linear range of PCR M. Cervelli et al. Barley polyamine oxidase isoform 1 gene expression FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS 3991 amplification conditions. In details, the HvPAO1 transcript is accumulated only in the ear (Fig. 1A, left panel); even with saturating conditions there were no detectable PCR amplified products in any stem internodes (not shown), thus confirming previ- ous results reported by Cervelli et al. [4]. On the contrary, HvPAO2 mRNA is accumulated in all the tissues examined, including ear. A further RT-PCR analysis has been carried out on different ear tissues (Fig. 1A, right panel) and interestingly the accumula- tion pattern shown by HvPAO1 transcript indicates that this gene is expressed in basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets, at variance with the HvPAO2 gene, which is expressed at a comparable level only in sterile spikelets (Fig. 1A, right panel). The transcription accumulation pattern of the ribosomal protein S12 (rp-S12) mRNA has been also analyzed as a control housekeeping gene for RNA stability and the quan- tity processed for each sample. As HvPAO2 promo- ter was capable of driving GUS expression in barley developing caryopsis, exclusively in the embryo (see section below on HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2::GUS expression in different plants and organs), to detect the presence of any low level of HvPAO2 mRNA accumulation in the barley embryo, a further PCR analysis was carried out in the non- linear range of amplification (up to 35 cycles) using the same cDNA sample. The results are shown in Fig. 1(B); a PCR product of faint intensity specific for HvPAO2 transcript was visible only after 35 cycles, demonstrating the presence of the HvPAO2 mRNA in embryonic organs albeit in relatively small amounts. A B Fig. 1. HvPAO1 and HvPAO2 transcript detection by RT-PCR in different barley tis- sues. (A) Total RNA isolated from different stem internodes, whole ear and various ear tissues was analyzed by RT-PCR amplifica- tion procedure using specific primers for HvPAO1, HvPAO2 and, as a control, rp-S12 transcripts in the linear range of amplifica- tion (25 cycles). (B) cDNA from embryo was analyzed in saturating PCR condition (35 cycles). The PCR products were fractionated by 1.2% agarose gel electrophoresis. Expec- ted size of PCR fragments are indicated at right. Barley polyamine oxidase isoform 1 gene expression M. Cervelli et al. 3992 FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS Cis-elements search in upstream region of HvPAO2 gene The 5¢-flanking region of HvPAO2 gene was cloned by inverse PCR using specific oligonucleotides designed from HvPAO2 partial gene sequences [4] and analyzed by searching for putative cis-acting elements known in plants by using the PLACE cis-element database [19] (http://www.dna.affrc.go.jp/PLACE/). We found a well-conserved TATA-box, located 25 nucleotides upstream of the putative transcription start point, an I-box localized at )191 ⁄ )186, a G-box at )303 ⁄ )296 and a CCAAT-box at )405 ⁄ )409 (Fig. 2A). These putative light-response elements [20,21] are also present in the promoter regions of HvPAO1, ZmPAO1 and ZmPAO2 genes [4,9]. A sequence motif that is shared only by HvPAO2 and HvPAO1 upstream regions is the MYB1AT-box (consensus 5¢-WAACCA-3¢), located at )348 ⁄ )353 (Fig. 2A). This is a dehydration- stress response element present in Arabidopsis thaliana but not in rice [22]. To summarize, in spite of the fact that some sequence motifs are shared by HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2 genes, a comparat- ive sequence analysis did not show any evident com- mon cis-acting elements organization in their upstream regions. HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2::GUS expression in different plants and organs GUS expression was obtained in developing caryopses, roots and leaves of barley and in roots and leaves of maize by means of biolistic inoculations with the GUS gene under the control of different promoters. Figure 2(B) shows a schematic representation of the chimerical constructions used in these experiments. Twenty-four hours after bombardment, the different organs were assayed for GUS activity (Figs 3 and 4). A B C Fig. 2. Gene promoter::GUS constructs util- ized in transient GUS expression experi- ments. A. 5¢-flanking region of HvPAO2 gene. Upstream and exon nucleotide sequences are indicated with lower-case and upper-case letters (start translation codon in bold), respectively. Putative promo- ter sequence motifs are indicated with bold underlined letters and marked. The restric- tion site HindIII used in inverse PCR experi- ments is indicated with italics underlined letters. Exon sequence is numbered from the putative tsp (+ 1), upstream sequence is indicated by negative numbers. B. Schemes of pHTT515 and pHTT-PAOs construct vec- tors. C. Schematic representation of the constructs utilized in particle bombardment and detection (+) or absence (–) of their expression in different organs and plants. Numbering refers at the promoter sequences (open boxes) jointed to the UB intron region (grey boxes). M. Cervelli et al. Barley polyamine oxidase isoform 1 gene expression FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS 3993 Transient expression of HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2promoter::GUS constructs, using pHTT515 plasmid (UBpromoter::GUS) as a control (Fig. 2B), revealed that only barley immature caryopsis were competent for the HvPAO1 promoter driving GUS expression, especially in the embryo, aleurone layer and endosperm. On the contrary, it was inactive in roots and leaves of both barley and maize. Interest- ingly, HvPAO2 promoter was capable of driving GUS expression in barley roots and leaves, as expected, but also in developing caryopses, albeit exclusively in the embryo, as well as in roots and leaves of maize. Fur- thermore, ZmPAO1, ZmPAO2 and UB promoters transpired to be active in all analyzed organs of barley and maize. Results obtained in the transient GUS expression experiments are summarized in Fig. 2(C). Fig. 3. Histochemical localization of glucoronidase (GUS) activity in different barley tissues after gene bombardment. HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2promoter::GUS (pHTT-PAOs) and UBpromoter::GUS (pHTT515) expression vectors were biolistically delivered to bar- ley roots, leaves and ears. Organs were histochemically reacted with X-Glu and examined for blue staining assessment with a Zeiss stereo- microscope and photographed. Longitudinal section of ears is enlarged at right. Photographs are representative of three different experiments each performed in triplicate. Barley polyamine oxidase isoform 1 gene expression M. Cervelli et al. 3994 FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS Purification of the HvPAO1 protein from developing caryopses HvPAO1 was extracted from immature caryopses with a high ionic strength salt solution. The enzyme was then partially purified from supernatant obtained after centrifugation of the crude homogenate through a fractionation in 70% saturated ammonium sulfate and two chromatographic steps (hydroxylapatite and SP-sepharose columns). By this procedure, a 58-fold purification of the enzyme was achieved (Table 1). No detectable HvPAO2 activity was revealed overall dur- ing the entire procedure of HvPAO1 purification, nei- ther in the purification steps reported in Table 1 (1–4 fractions), nor in hydroxylapatite and SP-sepharose flow-through and washing fractions. In western blot analysis, the SP-sepharose eluate (fraction 4; Table 1) showed a band of 53 kDa molecular mass, corres- ponding to the HvPAO1 expected mass (Fig. 5) when probed against polyclonal anti-ZmPAO antibodies; it has already been demonstrated that these cross-react with the less conserved HvPAO2 protein [4]. In vitro HvPAO1 protein synthesis In order to confirm that the PAO activity present in the SP-sepharose eluate (fraction 4; Table 1) could be ascribed to the HvPAO1 isoform, we carried out the in vitro synthesis of HvPAO1 protein utilizing the pET17b-HvPAO1 plasmid as a template in three different cell-free translation systems and precisely RTS-100 Roche (Roche Diagnostics, Monza, Italy), Escherichia coli T7 S30 Extract System for circular DNA (Promega Italia, Milano, Italy) and Pure-System Classic (Post Genome Institute, Tokyo, Japan), as des- cribed in the Methods section. The highest in vitro syn- thesized HvPAO1 protein yield (0.1 U) was obtained with the RTS-100 Roche translation system, as detec- ted by enzymatic assay. Moreover, western blot analy- sis of the in vitro translated product probed against polyclonal anti-ZmPAO antibodies, showed a band of 53 kDa molecular mass, thus confirming the presence of HvPAO1 protein (Fig. 5). ZmPAO, HvPAO2 and HvPAO1 protein catalytic properties ZmPAO and HvPAO2 showed pH optima and K m val- ues for Spd and Spm oxidation (Table 2) identical to those previously reported by Cervelli et al. [4]. Cata- lytic properties of the HvPAO1 enzyme purified from immature caryopsis resulted identical to those exhib- ited by the in vitro synthesized HvPAO1 recombinant enzyme for both Spd and Spm substrates. In partic- ular, HvPAO1 enzymatic activity showed an optimum at pH 7.0 for Spm and Spd oxidation and K m values of 0.89 · 10 )5 m and of 0.50 · 10 )5 m for Spd and Spm substrates, respectively (Fig. 6; Table 2). More- over, the V max ratio for Spd and Spm at pH 7.0 was Fig. 4. Histochemical localization of glucoronidase (GUS) activity in different maize tissues after gene bombardment. HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2promoter::GUS (pHTT-PAOs) and UBpromoter::GUS (pHTT515) expression vectors were biolistically delivered to maize roots and leaves. Organs were histochemically reacted with X-Glu and examined for blue staining assessment with a Zeiss stereomicroscope and photographed. Photographs are representative of three different experiments each performed in triplicate. M. Cervelli et al. Barley polyamine oxidase isoform 1 gene expression FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS 3995 found to be 1.3. The HvPAO1 biochemical features exhibited by the in vitro synthesized recombinant pro- tein and the native partially purified enzyme from bar- ley ears are congruent, demonstrating that the PAO activity detected in the SP-sepharose eluate (fraction 4; Table 1) could be reasonably ascribed to the exclusive presence of HvPAO1 enzyme with respect to HvPAO2 in developing caryopses. In fact, the presence of any detectable HvPAO2 activity in the SP-sepharose eluate (Table 1) would result in different PAO catalytic parameters, with K m and pH optimum values for Spd and Spm intermediate between those of purified HvPAO2 and in vitro synthesized HvPAO1. HvPAO1 catalytic properties were very similar to those of ZmPAO (Table 2), moreover HvPAO1 showed com- parable affinity and identical pH optima values for both Spd and Spm substrates (pH 7.0); analogously ZmPAO showed comparable affinity and identical pH optima values for both substrates (pH 6.5). On the contrary, HvPAO1 enzymatic features differ from the ones of HvPAO2 that preferentially oxidizes spermine at pH 5.5 and spermidine at pH 8.0 with a ten-fold lower V max [4]. Discussion Our results definitely support the identification of the HvPAO1 enzyme as the major product of HvPAO gene expression in barley ear (Fig. 1A, left panel). In fact, HvPAO1 mRNA was detectable by standard RT-PCR analysis only in the ear ([4] and this work), indicating that HvPAO1 gene expression is ear-specific. Further ear-tissue dissection demonstrated that in all the samples examined (basal lemma, rachis, awn, embryo-deprived caryopsis and embryo) by RT-PCR, the HvPAO1 gene is expressed at comparable levels (Fig. 1A, right panel). The only tissue where it was possible to detect HvPAO2 gene expression with stand- ard PCR conditions (25–30 cycles) resulted to be the Table 1. Purification of the HvPAO1 protein from barley developing caryopses. HvPAO1 purification was performed from developing caryop- ses at 30 days postfertilization, as described in the Methods section. The enzyme was partially purified from supernatant obtained after cen- trifugation of the crude homogenate (fraction 1), through a fractionation in 70% saturated ammonium sulfate (fraction 2) and two chromatographic steps (fraction 3 and 4). Purification step Total volume (mL) Protein (mgÆmL )1 ) Total protein (mg) Activity (UÆmL )1 ) Total activity (U) Specific activity (UÆmg )1 Æprotein) Purification fold Recovery (%) Crude extract (fraction 1) 236 0.94 221.84 0.002 0.472 0.002 1.0 100.0 (NH 4 ) 2 SO 4 70% sat. precipitation (fraction 2) 104 1.24 128.96 0.004 0.416 0.003 1.5 88.1 Hydroxylapatite eluate (fraction 3) 44 0.24 10.56 0.005 0.220 0.021 10.5 46.6 SP eluate (fraction 4) 15 0.043 0.644 0.005 0.075 0.116 58.0 15.9 Fig. 5. Western blot analysis of ZmPAO, HvPAO2 and HvPAO1. ZmPAO and HvPAO2 were purified as previously described [4,5]. HvPAO1 was purified and in vitro synthesized as described in Experimental procedures. Analysis was performed running: 0.1 U of ZmPAO and HvPAO2; 0.002 U of ear purified and RTS-100 Roche TS produced HvPAO1. Proteins were reacted, after deglyco- sylation, with anti-ZmPAO polyclonal antibodies [4]. M, protein molecular weight marker (Fermentas). Table 2. K m values for ZmPAO, HvPAO2 and HvPAO1. For the determination of the K m values, ZmPAO and HvPAO2 were purified as previously described [4,5]. HvPAO1 was synthesized in vitro as described in Experimental procedures. Data were obtained at 25 °C with Spd and Spm as substrates at the specific pH optimum. K m values concerning Spd and Spm oxidation by ZmPAO and HvPAO2 were within the standard error of the values previously reported by Cervelli et al. [4]. Ear purified and RTS-100 Roche TS produced HvPAO1 showed identical K m values. All K m values, calculated from Lineweaver-Burk plots, are means of three different experiments, each performed in duplicate. SD was 8%. Enzyme pH Substrate K m (M) ZmPAO 6.5 Spd 1.0 · 10 )5 a,b ZmPAO 6.5 Spm 2.7 · 10 )5a,b HvPAO2 8.0 Spd 56.0 · 10 )5a,b HvPAO2 5.5 Spm 0.48 · 10 )5a,b HvPAO1 7.0 Spd 0.89 · 10 )5a HvPAO1 7.0 Spm 0.50 · 10 )5a a Present work; b Cervelli et al. [4]. Barley polyamine oxidase isoform 1 gene expression M. Cervelli et al. 3996 FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS sterile spikelets. Using a higher number of PCR cycles, we were also able to detect a very small amount of the HvPAO2 transcript in the embryo tissue (Fig. 1B). Thus, the HvPAO2 gene is also transcriptionally active in the embryo, albeit at a very low level, probably rep- resenting a basal transcriptional activity. This is in line with the transient GUS expression experiments that confirmed the specific and strong expression of the HvPAO1 gene in barley ear, at variance with the weak expression of the HvPAO2 gene, exclusively localized in the embryo (Figs 2C and 3). As expected, the HvPAO1 gene is silent in roots and leaves of maize (Figs 2C and 3). Interestingly HvPAO2, ZmPAO1 and ZmPAO2 promoters exhibit the same transcription pattern, being able to drive GUS expression in all the organs and plants analyzed in this study (Figs 2C, 3 and 4). Moreover, the ZmPAO1-2 promoters are also active in barley embryo, albeit at a very low level, like the HvPAO2 gene; it seems that the barley embryo is able to allow a basal transcriptional level of these promoter sequences. Sequence analysis of 5¢ flanking regions of HvPAO2 (Fig. 2A), ZmPAO1 and ZmPAO2 genes albeit sharing some potential cis-acting elements, do not show any obvious common promoter architec- ture as expected by their identical transcription pattern [4,9]. Furthermore, there are no evident sequence fea- tures in the HvPAO1 and HvPAO2 promoters that could explain their different gene expression profiles. So, we are facing a puzzling gene duplication event that occurs in barley, since the paralogous HvPAO2 and ZmPAO1-3 genes share a common tissue expres- sion, which is at variance with the orthologous HvPAO1 and ZmPAO1-3 genes that show a different tissue regulation. The very similar catalytic properties shown by HvPAO1 and ZmPAO could be ascribed to the closer phylogenetic relationship existing between them (84% identity), as compared with that between HvPAO2 and ZmPAO1 (73% identity) (Table 2). It is interesting to recall that, even if the global fold and the flavin adenine dinucleotide-binding pocket are well conserved in HvPAO1, HvPAO2 and ZmPAO, the substitution of Phe403 of ZmPAO by a tyrosine resi- due in HvPAO2 could probably play a relevant role in the different substrate specificity and kinetic parame- ters observed for this isoform [4]. Furthermore, HvPAO1 amino acid sequence shows a different C-ter- minus when compared with the HvPAO2 coding sequence, which has an extra eight-residue long tail (DELKAEAK) responsible for the symplastic localiza- tion of this isoform [10]. On the contrary, the higher similarity determined between HvPAO1 and ZmPAO, strongly suggests an apoplastic localization of the HvPAO1 isoform. Indeed, recent results have shown that ZmPAO is also present at high levels in the cyto- plasm, most probably in the secretory pathway of young tissues undergoing or destined to programmed cell death, such as developing xylem vessels and xylem parenchyma of both the root and mesocotyl as well as root cap cells [17]. Later, during cell maturation, ZmPAO is found mainly in the cell wall [17]. On the basis of these results, the authors hypothesized that ZmPAO could play a dual role in these tissues being involved either in programmed cell death or cell wall differentiation through the action of its reaction Fig. 6. HvPAO1 catalytic parameters for Spd and Spm oxidation. HvPAO1 was purified from developing caryopses at 30-day post- fertilization, as described in Experimental procedures. Data reported are the average of three different experiments, each with two replicates. SD was 8%. (A) HvPAO1 cat- alytic activity pH optima were determined at 25 °C, in 0.2 M sodium phosphate buffer (pH range 4.5–8.5) with Spd or Spm as sub- strates. PAO activity is expressed as per- cent of the maximum value. (B) HvPAO1 (1 · 10 )3 U) K m values were determined at 25 °C, with Spd and Spm as substrates at the respective pH optimum and then calcu- lated from Lineweaver-Burk plots. M. Cervelli et al. Barley polyamine oxidase isoform 1 gene expression FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS 3997 products, hydrogen peroxide and aminoaldehydes and ⁄ or modulation of polyamine levels [3,13,17]. One can hypothesize that in barley PAO tissue-specific functions are associated with the distinct isoforms HvPAO1 and HvPAO2, an event that arose in the course of evolution of C3 cereals. According to the tis- sue distribution of HvPAO1 and HvPAO2, the prefer- ential HvPAO2 substrate Spm has been detected at higher level than Spd in barley leaves [23], whereas in the developing grains a higher level of Spd than Spm has been found [24]. Figure 7 summarizes the evolu- tionary relationship among the cereal PAO genes stud- ied in this work. Is there any specific role for HvPAO1 in the ear tissues and in particular in embryonic tissues and aleurone, where the expression of HvPAO1 gene is prominent, if not exclusive, compared with HvPAO2? The available data suggest that HvPAO1 could have a specific role in the aleurone cells. This is a tissue that plays a key role during the germination of cereal seeds. Aleurone cells secrete, under the stimulus of embryo-synthesized gibberellin, amylase and other hydrolytic enzymes involved in endosperm reserve mobilization. Gibberellin also induces programmed cell death in these cells, a process that is mediated by hydrogen peroxide [25,26]. Thus the accumulation of HvPAO1 during the development of barley caryopsis could be functional to the production of hydrogen peroxide needed in the programmed cell death process taking place in the aleurone during germination. However it should be recalled that hydrogen peroxide production during germination could also have a general protective role against microbial pathogens. Alternatively, HvPAO1 could have a role in the regu- lation of polyamine levels in the aleurone cells and in the embryo as well. Indeed, it has been recently reported that DNA synthesis early in development and the advance in cell cycle ⁄ endocycle are tempor- ally and spatially related to polyamine catabolism and vascular development [27]. Moreover, polyamines are active in triggering the synthesis of nitric oxide in specific tissues of Arabidopsis thaliana seedlings [28]. This molecule is known to delay programmed cell death in aleurone cells and also to have pleiotropic effects on many facets of plant development and defense [29]. Experimental procedures Chemicals Restriction and DNA-modifying enzymes and protein molecular weight marker were purchased from MBI Fer- mentas (MBI Fermentas, St. Leon-Rot, Germany). Sper- midine (Spd) and spermine (Spm), horseradish peroxidase, 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulf- onic acid were purchased from Sigma-Aldrich-Fluka (Sigma, Milano, Italy). TRIZOL reagent was from Invitro- gen (Invitrogen, Milano, Italy). pGEM-Teasy vector and the E. coli T7 S30 Extract System for circular DNA were from Promega (Promega Italia, Milano, Italy). pET17b vector and E. coli BL21 DE3 competent cells were from Novagen (Novagen Inc., Madison, WI, USA). CHU(N 6 ) medium was from Duchefa (Duchefa Biochemie B. V., Haarlem, the Netherlands). Hydroxylapatite and gold par- ticles (1.0 lm in diameter) were from Bio-Rad (Bio-Rad, Milano, Italy). SP-sepharose was from Amersham Biosciences (Amersham Biosciences, Milano, Italy). Carb- oxymethylcellulose was from Whatman (Whatman, Maid- stone, UK). Peroxidase-conjugated goat antirabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The RTS-100 Roche translation system was from Roche (Roche Diagnostics, Monza, Italy). The Pure-System Clas- sic Mini Kit was from the Post Genome Institute (Post Genome Institute, Tokyo, Japan). Other chemicals came from Sigma-Aldrich-Fluka, Bio-Rad and J. T. Baker (Baker Italia, Milano, Italy). HvPAO2 HvPAO1 LEAVES ROOTS EARS ZmPAO1,2 Extracellular localization ? Orthologous gene Paralogous gene HvPAO1 promoter HvPAO2, ZmPAO1,2 promoters ? Fig. 7. Schematic representation of the evolutionary relationship among cereal PAO genes. Arrow width represents the promoter expression level according to both RT-PCR analysis and biolistic delivering experiments. The promoter boxes color reflects the tissue specific expression. Barley polyamine oxidase isoform 1 gene expression M. Cervelli et al. 3998 FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS Plant material Seedlings and adult plants of barley (H. vulgare) cultivar Aura [30] were grown at the ‘Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d’Arda, Italy’. Seeds of barley cultivar Aura and of maize (Z. mays) cultivar Corona (Monsanto Agricoltura, Italy) were soaked for 12 h in aerated tap water, germinated at 20 °C in the dark and grown aseptically in a growth chamber with a 16 : 8 h light–dark cycle on Magenta vessels (Sigma-Aldrich-Fluka, Milano, Italy) containing 0.8% agar. For ZmPAO and HvPAO2 purification, maize and barley seeds were germi- nated with 1 cm of fertile soil, at 20 °C in the dark. Two- day-old barley seedlings were exposed to natural light for 4 days before harvesting; maize seedlings were kept in the dark before protein extraction. Thirty-day postfertilization ears were utilized for RT-PCR experiments, transient expression assays and HvPAO1 purification. RT-PCR analysis of HvPAO1 and HvPAO2 gene expression in different tissues Total RNA was isolated from different barley tissues by TRIZOL reagent, according to the manufacturer’s instruc- tions. Oligonucleotides utilized as primers for specific amplification of HvPAO isoforms were: HvPAO-N, reverse 5¢-GTTATTACTTAGTACCTCTTAAT-3¢, HvPAO-O, for- ward 5¢-GACGGAGATCTCCCACTC-3 ¢ and HvPAO-P, reverse 5¢-GGTTGTCCGACTGCTGCTC-3¢ for HvPAO1; HvPAO-Q, reverse 5¢-CTCGTCGGCGCGGTCCAT-3¢, HvPAO-R, forward 5¢-GAGGGGAGAATTGAAGA GAG-3¢ and HvPAO-S, reverse 5¢-GTCGTAGAGGCC ACCGCT-3¢ for HvPAO2 as already described [4]. Oligo- nucleotides for the control barley ribosomal protein S12 were: RPS12-A, reverse 5¢-ATTCTTCACCATAGTCCT-3¢, RPS12-B, forward 5¢-GTGAGCCAATGGACTTGATG-3 ¢ and RPS12-C, reverse 5¢-ATGCAAGAGCAGCCTAC AAC-3¢ [4]. HvPAO2 promoter isolation Barley DNA was extracted and purified as described in Cervelli et al. [4]. To clone 5¢- and 3¢-flanking regions of HvPAO2 gene, amplified products were obtained by inverse PCR using specific oligonucleotides designed from HvPAO2 partial gene sequences [4], in particular, HvPAO2-A, reverse 5¢-TACTGTGTTAGCACTGCTAGC-3¢, and HvPAO2-B, forward 5¢-GAGGGGAGAATTGAAGA GAG-3¢, specific for the 5¢-end region. The gene-specific primer couples were utilized on different samples of purified barley total DNA, previously digested with HindIII and self-ligated. A direct PCR to obtain the corresponding HvPAO2 promoter sequence was performed on total DNA with the oligonucleotides HvPAO2-C, forward 5¢-AAAAAGCTTACCAAAACTTGTGTAAACTT-3¢, and HvPAO2-D, reverse 5¢-TTTAGATCTGCCCTGCTCTCC GGCCCTGT-3¢, containing the HindIII and the BglII sites, respectively. The PCR-amplified product was cloned in the pGEM-Teasy vector. The promoter gene sequence has been deposited in the EMBL database under EMBL accession number AM231701. DNA methodology and construction of expression plasmids The methods described by Sambrook et al. [31] were used for the manipulation of plasmid DNAs and general DNA in vitro procedures. In order to amplify HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2 promoter regions ([4,9] and present work), gene-specific oligonucleotides containing HindIII and the BglII sites were designed spanning from the 5¢ end of the promoter sequence down to the transcrip- tion start site [primer sequences used are available on request from the first author (MC)]. HvPAO2, ZmPAO1 and ZmPAO2 promoter PCR products were cloned into the expression vector pHTT515 utilizing the HindIII and the BglII sites and replacing the original ubiquitin (Ubi) promoter sequence. To clone HvPAO1 promoter sequence [4], we used a different procedure because of the presence of a BglII site within the gene sequence. A HvPAO1 pro- moter sequence subclone inserted in pGEM-Teasy vector was digested with EcoRI and filled in at its extremities, then cloned in pHTT515 previously cut with HindIII and BglII and blunt ended. The Ubipromoter::GUS expression pHTT515 vector was used as a control plasmid expres- sing the GUS reporter gene driven by the house-keeping promoter Ubi. The HvPAO1, HvPAO2, ZmPAO1 and ZmPAO2promoter::GUS expression plasmids were se- quenced on both strands using the automated fluorescent dye terminator technique (Perkin Elmer ABI model 373 A). In order to clone the HvPAO1 cDNA (GenBank accession number AJ298131) by PCR amplification, the full-length cDNA was generated possessing modified 5¢- and 3¢-ends. In particular, the two following synthetic oligonucleotides were used to introduce NdeI and XhoI restriction sites at the 5¢- and 3¢-ends of HvPAO1 cDNA: HvPAO1cdna-DIR, 5¢-CATATGGCCGGCCCCAGGGTCATCATC-3¢ and HvPAO1cdna-REV, 5¢-CTGGAACTCGAGCTAGTCAA ACTTGCCCGG-3¢ , respectively. The amplified PCR prod- uct was restricted by NdeI and XhoI and ligated with the restricted NdeI ⁄ XhoI pET17b vector, to obtain the genetic construct encoding the mature form of HvPAO1 protein, named pET17b-HvPAO1. The recombinant cDNA con- struct was resequenced to check the accuracy of the nucleo- tide sequence and then utilized to transform E. coli BL21 DE3 (Novagen, Madison, WI, USA) competent cells. M. Cervelli et al. Barley polyamine oxidase isoform 1 gene expression FEBS Journal 273 (2006) 3990–4002 ª 2006 The Authors Journal compilation ª 2006 FEBS 3999 [...]... 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