Báo cáo khoa học: Barley polyamine oxidase isoforms 1 and 2, a peculiar case of gene duplication docx

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].. While HvPAO2 transcrip

of gene duplication

Manuela Cervelli1, Marzia Bianchi1, Alessandra Cona1, Cristina Crosatti2, Michele Stanca2,

Riccardo Angelini1, Rodolfo Federico1and Paolo Mariottini1

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-aminobut4-aminobut-anal,

respect-ively, plus 1,3-diaminopropane and H2O2 [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 periso-formed 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 Kmvalues for both substrates, i.e 0.89· 10)5m and 0.5· 10)5m for Spd and Spm, respectively It has been found that HvPAO1 enzyme activ-ity significantly differs in substrate specificactiv-ity 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.

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

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 indicaExpec-ted at right.

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).

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.

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 Km 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 Km values

of 0.89· 10)5m and of 0.50· 10)5m for Spd and Spm substrates, respectively (Fig 6; Table 2) More-over, the Vmax 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.

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 Km 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 Vmax[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)1Æprotein)

Purification fold

Recovery (%)

(NH 4 ) 2 SO 4 70% sat precipitation

(fraction 2)

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 Km 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%.

a Present work; b Cervelli et al [4].

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)3U) Kmvalues were determined at

25 C, with Spd and Spm as substrates at

the respective pH optimum and then

calcu-lated from Lineweaver-Burk plots.

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 spatitempor-ally 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 FerFer-mentas, 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 (InvitroInvitro-gen, 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(N6) 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)

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.

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 20C 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 20C 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¢,

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 vitroprocedures 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,

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