Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

Physiologia Plantarum 133: 140–156. 2008 Copyright ª Physiologia Plantarum 2008, ISSN 0031-9317

Transgenic tobacco plants overexpressing polyamineoxidase are not able to cope with oxidative burstgenerated by abiotic factorsPanagiotis N. Moschou, Ioannis D. Delis, Konstantinos A. Paschalidis andKalliopi A. Roubelakis-Angelakis*

Department of Biology, University of Crete, PO Box 2280, 71409 Heraklion Crete, Greece

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 17 September 2007; revised

29 November 2007

doi: 10.1111/j.1399-3054.2008.01049.x

The molecular and biochemical mechanism(s) of polyamine (PA) action

remain largely unknown. Transgenic tobacco plants overexpressing polyamine

oxidase (PAO) from Zea mays exhibited dramatically increased expressionlevels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of

spermidine and spermine decreased significantly in the transgenic lines.

Although Dap was concomitantly generated with H2O2 by PAO, the latter was

below the detection limits. To show the mode(s) of H2O2 scavenging, the

antioxidant machinery of the transgenics was examined. Specific isoforms of

peroxidase, superoxide dismutase and catalase were induced in the trans-

genics but not in the wild-type (WT), along with increase in activities of

additional enzymes contributing to redox homeostasis. Onewould expect thatbecause the antioxidant machinery was activated, the transgenics would be

able to cope with increased H2O2 generated by abiotic stimuli. However,

despite the enhanced antioxidant machinery, further increase in the

intracellular reactive oxygen species (ROS) by exogenous H2O2, or addition

of methylviologen or menadione to transgenic leaf discs, resulted in oxidative

stress as evidenced by the lower quantum yield of PSII, the higher ion leakage,

lipid peroxidation and induction of programmed cell death (PCD). These

detrimental effects of oxidative burst were as a result of the inability oftransgenic cells to further respond as did the WT in which induction of

antioxidant enzymes was evident soon following the treatments. Thus,

although the higher levels of H2O2 generated by overexpression of Mpao in

the transgenics, with altered PA homeostasis, were successfully controlled by

the concomitant activation of the antioxidant machinery, further increase in

ROS was detrimental to cellular functions and induced the PCD syndrome.

Abbreviations – ADC, Arg decarboxylase; APX, ascorbate peroxidase; ASA, ascorbate; CAT, catalase; DAB, diamino benzidine;

DAO, diamine oxidase; Dap, 1,3-diaminopropane; DCFH-DA, 2#,7#-dichlorodihydrofluorescein diacetate; DHA, dehydroascorbate;

DHAR, dehydroascorbate reductase; ICF, intercellular washing fluid; MDHAR, monodehydroascorbate reductase; MV, methylviologen;

NBT, nitroblue tetrazolium; ODC, Orn decarboxylase; PA, polyamine; PAO, polyamine oxidase; PCD, programmed cell death; PCR,

polymerase chain reaction; PH-PA, pellet hydrolyzed polyamine; POX, peroxidase; Put, putrescine; ROS, reactive oxygen species;

S-PA, soluble polyamine; SAM, S-adenosyl-L-methionine; SAMDC, S-adenosyl-L-methionine decarboxylase; SH-PA, soluble

hydrolyzed polyamine; SOD, superoxide dismutase; Spd, spermidine; SPDS, Spd synthase; Spm, spermine; WT, wild-type.

140 Physiol. Plant. 133, 2008

Introduction

The main cellular polyamines (PAs), the diamine putres-

cine (Put) and the tri- and tetra-amines spermidine (Spd)and spermine (Spm), respectively, are small, ubiquitous

cationic molecules at cytosolic pH. During the past

decade, it has been increasingly accepted that PA

homeostasis affects growth and development and also

stress responses in eukaryotic cells (Arias et al. 2005, Fos

et al. 2003, Kusano et al. 2007 and references therein,

Kuznetsov et al. 2007, Papadakis and Roubelakis-

Angelakis 2005, Paschalidis and Roubelakis-Angelakis2005a, 2005b, Perez-Amador et al. 2002, Takahashi et al.

2004, Tiburcio et al. 1994,Vuosku et al. 2006, Yamaguchi

et al. 2006).

The pathway of PA biosynthesis is ubiquitous in living

organisms. It is a relatively short pathway in terms of

participating enzymes, most of which have been charac-

terized and the corresponding genes/cDNAs cloned from

different sources (Kaur-Sawhney et al. 2003, Kusano et al.2007). Orn decarboxylase (ODC, EC 4.1.1.19) and Arg

decarboxylase (ADC, EC 4.1.1.19) catalyze the removal

of the carboxyl group from Orn and Arg, respectively, to

yield Put. S-adenosyl-L-methionine decarboxylase

(SAMDC, EC 4.1.1.50) introduces S-adenosyl-L-methio-

nine (SAM) into the pathway, which is then used in its

decarboxylated form (dcSAM) as an aminopropyl donor

in the conversion of Put to Spd and Spm (Roje 2006). Theactual transfer of the aminopropyl moiety is catalyzed by

the enzymes Spd synthase (SPDS, EC 2.5.1.16) and Spm

synthase (EC 2.5.1.22) (Hashimoto et al. 1998, Panicot

et al. 2002). The catabolic phase involves deamination of

Put, Spd and Spm by the action of amine oxidases. The

flavoprotein polyamine oxidase (PAO, EC 1.5.3.3)

oxidizes Spd and Spm at their secondary amino groups.

PAO yields D1-pyrroline and 1,5-diazabicyclononanefrom Spd and Spm, respectively, along with 1,3-diami-

nopropane (Dap) and H2O2. In contrast, Put is N-

methylated by Put N-methyltransferase, which catalyzes

the first committed steps in the biosynthesis of nicotine

and tropane alkaloids (Sato et al. 2001).

In tobacco plants, PA titers and biosynthesis follow

a basipetal decrease along the plant axis and negatively

correlate with cell size. The shoot apical meristem is themain site of Spd and Spm biosynthesis, and the root

synthesizesmostly Put. High and low SPDS are correlated

with cell division and expansion, respectively. Put

biosynthetic pathways are differently regulated in hyper-

and hypogeous tobacco tissues: only ADC is responsible

for Put synthesis in old, hypergeous vascular tissues, and

ODC in hypogeous tissues. Furthermore, ODC expres-

sion coincides with early cell divisions in marginalsectors of the lamina, and SPDS strongly correlates with

later cell divisions in the vascular regions (Paschalidis and

Roubelakis-Angelakis 2005b). Expression of diamine

oxidase (DAO, EC 1.4.3.6) and PAO in developing

tobacco tissues precedes and overlaps with nascent

nuclear DNA and also with peroxidases (POXs) and

lignification. In mature and old tobacco tissues, expres-sion of PAO and POX coincides with G2 nuclear phase

and endoreplication. In young vs the older roots,

expression of PAO and POX decreases with parallel

inhibition of G2 advance and endoreplication. DNA

synthesis early in development and the advance in cell

cycle/endocycle are temporally and spatially related to

PA catabolism and vascular development (Paschalidis

and Roubelakis-Angelakis 2005a).PAO, in addition to reducing PAs, is also a H2O2-

producing enzyme, which has been proposed to partic-

ipate in developmental processes and responses to biotic

and abiotic stresses (Cona et al. 2005, 2006, Paschalidis

and Roubelakis-Angelakis 2005a, Paschalidis et al. 2001,

Rea et al. 2004, Yoda et al. 2003). Recently, the first

evidence of a direct DNA oxidative damage by murine

Spmoxidase activitieswas shown (Amendola et al. 2005),and Spm content was proposed to act as a DNA shielding

molecule and a scavenger against radical species (Lovaas

1997).

Plant response to oxidative stress is controlled by

a cascade of molecular networks, which may activate

stress-related mechanisms either to reestablish homeo-

stasis and to protect proteins and membranes leading to

cell survival or to induce susceptibility leading tooxidative damage, such as lipid peroxidation and ion

leakage leading to senescence and cell death (Alvarez

et al. 1998, Papadakis and Roubelakis-Angelakis 2005,

Sasaki-Sekimoto et al. 2005). Plant engineering strategies

for oxidative stress tolerance include the expression of

genes involved in signaling and regulatory pathways,

antioxidant genes (Laloi et al. 2004, Mittler 2006) or

genes encoding for enzymes mediating synthesis ofantioxidant molecules. Genetic engineering for

increased biosynthesis of specific PAs resulted, in several

cases, in plants with modified stress response. Over-

expression of adc induced a significant increment in Put

and a small increase in Spd and Spm levels, ultimately

protecting the plants from drought (Capell et al. 2004).

Overexpression of samdc from yeast in tomato resulted in

higher levels of Spm and Spd and lycopene in fruits,increasing the nutrient value of tomato juice (Mehta et al.

2002). Overexpression of spds cDNA from Cucurbita

ficifolia in Arabidopsis thaliana increased significantly

Spm levels and enhanced tolerance to various stresses

(Kasukabe et al. 2004). Rea et al. (2004) have developed

transgenics overexpressing Mpao. In these transgenics,

high expression levels of the transgene were detected in

Physiol. Plant. 133, 2008 141

the extracellular space and the amount of soluble

polyamines (S-PAs) was lower to that of the wild-type

(WT), while a small but significant change in reactive

oxygen species (ROS)-scavenging capacity was verified.

In this work, we also developed tobacco transgenics

overexpressing the maize pao gene (Fig. 1). Our Mpaooverexpressers were thoroughly analyzed for all fractions

of PAs [free, soluble conjugated and insoluble conjugated

PAs; S-PAs, soluble hydrolyzed polyamines (SH-PAs) and

pellet hydrolyzed polyamines (PH-PAs), respectively],

along with the expression of all genes involved in the

antioxidant machinery and the H2O2 fate, in an effort

to unravel their behavior under normal and oxidative

stress conditions. The transgenics contained significantly

reduced S-Spd and S-Spm, and the increased activity of

PAO resulted in the production of H2O2, which signaled

the expression of specific antioxidant isoenzymes. Thus,no intracellular H2O2 was allowed to be built up. On the

contrary, when exogenous H2O2 or elicitors of H2O2

were supplied, the transgenic cells with the impaired

PA homeostasis were not able to overcome and the

intracellular H2O2 level was beyond the required

Fig. 1. Transformation cassette and expression levels of the pao transgene in leaf tissues in WT (N. tabacum L. cv. Xanthi) and in threeMpao transgenic

lines 2.2, 4 and 6.5. (A) Map of the transformation cassette. (B) Northern blot using a P32-Mpao-derived probe. (C) EtBr staining in 1% (w/v) agarose and

densitometric analysis of the Northern blot. (D)Western blot (53 kDa) using an anti-MPAO polyclonal antibody. (E) Coomassie Blue staining in 7.5%PAGE

(top of E), rehybridization of the samemembraneswith anti-actin polyclonal antibody (center of E) and densitometric analysis of thewestern blot (bottom

of E). (F) Enzymatic activities of total extracted PAO. Data are the means of three independent experiments and bars represent �SE. Analysis of variance

showed that all data in transgenics presented statistically significant differences (P¼ 0.01) comparedwith data inWT. Higher exposure time (in B) showed

a transcript hybridization band also in WT plants. In (D), negative control (tissues of the shoot apex from WT) and positive control (tissues of the 25th

internode, i.e. base of the plant) inMPAO antibody were also used (data not shown), confirming previously described results (Paschalidis and Roubelakis-

Angelakis 2005a). CaMV, Cauliflower Mosaic Virus; EtBr, ethidium bromide; LB, left border, nptII, neomycin phosphotransferase; ocs, octopin synthase

terminator; RB, right border.


‘signature’ to signal further expression of the antioxidant

genes. Instead, the programmed cell death (PCD) syn-

drome was induced and executed.

Materials and methods

Plant material and growth conditions

Plants of tobacco (Nicotiana tabacum cv. Xanthi) were

grown in a growth chamber with an irradiance ofapproximately 50 mmol m22 s21, temperature of 25 �2�C and 16:8 h photoperiod. Cell suspension cultures

were generated from calli derived from shoot explants.

Localization of PAO: preparation of intercellularwashing fluid and fractions of soluble, extractableand tightly wall-bound PAO

Intercellular washing fluid (ICF) fraction was obtained as

described previously by Rea et al. (2004). After two

additional washings of the apoplastic compartment to

obtain residual intracellularwashing fluids, the tissuewas

grounded and the soluble, extractable and tightly wall-

bound PAO fractions were obtained according to Cona

et al. (2005). To obtain as pure fractions as possible, in the

washing procedures, three subfractions were obtained inwhich PAO activity was also estimated, giving gradually

declining PAO activity, with the third subfraction

exhibiting insignificant levels of PAO activity. PAO

activity in the fractions of cell suspension cultures was

measured as follows. Exponentially growing cells were

harvested and cells pelleted at 800 g at room temperature.

The supernatant corresponds to the ICF fraction. The

pellet was washed with culture medium until no measur-able activity in the supernatant after the centrifugation

step could be obtained. The supernatant derived from the

last wash was concentrated 20 times before the measure-

ment of PAO activity. Approximately 10 washes were

enough to completely eliminate residual PAO activity in

the supernatant. Pellet fractions (soluble, extractable and

tightly wall bound) were obtained as described above for

the leaf.

Vector construction and plant transformation

The maize pao cDNA (Tavladoraki et al. 1998) wassubcloned in pART 7 plasmid vector using EcoRI. The

pART 7 was digested with NotI and the excised fragment

subcloned into the pART27 binary vector, which was used

to transform Agrobacterium tumefaciens strain LBA4404.

Stable transformation was performed using tobacco

leaf discs and transformants were selected against 150

mg l21 kanamycin in Murashige and Skoog medium.

Screening of transgenics

Polymerase chain reaction (PCR) was performed usingspecific primers for the nptII gene with the sequence 5#-GGT TCT CCGGCCGCT TGG-3# for the forward primer

and 5#-TCGGGAGCGGCGATACCG-3# for the reverseprimer. Southern blotting was performed using the

restriction enzyme NotI. DNAwas transferred to a mem-

brane and hybridized to a cDNA maize pao-derived32P-labeled probe.

PA analysis, protein extraction and enzyme assays

Soluble, soluble conjugated and insoluble conjugated PAfractions were determined as previously described, using

an HP 1100 High Performance Liquid Chromatographer

(Hewlett-Packard, Waldbronn, Germany) (Kotzabasis

et al. 1993), and a standard curve for Dap was also

determined.

Total proteins were extracted as already described in

Papadakis and Roubelakis-Angelakis (2005). For PAO

and DAO assays, a spectrophotometric method develo-ped by Federico et al. (1985) was used with minor modi-

fications. Briefly for PAO, tissue was ground to a fine

powder in liquid nitrogen with a mortar and pestle and

proteins were extracted after the addition of 5 volumes

g21 FW of ice-cold K-phosphate buffer pH 6.5, supple-

mented with 10 mM dithiothreitol and 10 mM pyridoxal

phosphate. Samples were left on ice for 20 min and

centrifuged at 10 000 g at 4�C for 20 min. Aliquots of thesupernatant were added in the reaction buffer containing

K-phosphate buffer pH 6.5 and 10mM Spd-3HCl (Sigma-

Aldrich, Greece) to a final volume of 500 ml and incub-

ated for 1 h. The reaction was stopped with the addition

of 63 ml 20% TCA, and subsequently, 1.25 ml 1 M

diaminobenzidine was added. Samples were incubated

for 30min at 4�C, and the absorbancewas read at 460 nm

after a brief centrifugation. One unit of enzyme repre-sents the amount of enzyme that catalyzes the oxidation

of 1 mmol of substrate per minute. A radiometric method

was also used for PAO and DAO assays according to

Paschalidis and Roubelakis-Angelakis (2005a).

Spectrophotometric assays were used for POX enzyme

activities according to Paschalidis and Roubelakis-

Angelakis (2005a). Total superoxide dismutase (SOD)

activity was quantified according to the photochemicalmethod of Beauchamp and Fridovitch (1971). Mono-

dehydroascorbate reductase (MDHAR) and dehydroas-

corbate reductase (DHAR) activities were determined

according to Serrano et al. (1994) and Asada (1984),

respectively. For the determination of ascorbate peroxi-

dase (APX), total proteins were similarly extracted by

further adding 1 mM ascorbate (ASA). APX activity was


determined according to Nakano and Asada (1981).

Catalase (CAT) activity was determined by measuring the

initial rate of H2O2 decomposition at 240 nm (e: 0.036mM21 cm21). One milliliter of the reaction mixture con-

sisted of 50 mM K-phosphate, pH 7.0, and 15 mMH2O2.

Native IEF, activity staining, determination of ASAand protease assay

Native IEF PAGE was used to detect the isoenzymes of

POX by activity staining of protein extracts separated in

gel over a pH gradient 3.0–10.0. Isoenzymes were

visualized by incubating native IEF gels in solution

containing 50 mM K-phosphate buffer, pH 7.4, 0.1 mgml21 4-chloro-1-napthol and 0.16% H2O2.

For the activity staining of APX, 10mMASAwas added

in the IEF electrophoresis buffer, and 10% gels were

prerun for 30 min at 20 mA (Rao et al. 1995). Sub-

sequently, the gelswere incubated in thedark in a solution

containing 50 mM K-phosphate, pH 7.0, and 2 mM ASA;

the gels were incubated in the dark for another 30 min in

50 mM K-phosphate, pH 7.0, 4 mM ASA and 2 mMH2O2.Bands were visualized after the incubation of gels in

coloring solution [50 mM K-phosphate, pH 7.8, 14 mM

tetramethylethylenediamine (TEMED) and 1.2 mM nitro-

blue tetrazolium (NBT)]. For the activity staining of SOD,

a method described in Beauchamp and Fridovitch (1971)

was used. Native gels were stained for CAT activity ac-

cording to Clare et al. (1984).

For the determination of ASA content in leaves,a previously described method of Yasui and Hayashi

(1991) was used. Tissues (1 g FW) were homogenized in

3 ml of cold 5% (w/v) metaphosphoric acid (Yamamoto

et al. 2005). After centrifugation at 12 000 g for 20 min,

the supernatant was used for the determination of ASA

and dehydroascorbate (DHA). ASA and DHA were

separated in anHPLC system (HP1100;Hewlett-Packard)

using 2 mM perchloric acid aqueous solution anddetected by absorbance at 300 nm after reaction with

100 mM NaOH containing 100 mM NaBH4.

For protease activity assay, 20 mg of total protein was

separated in 10% acrylamide gel containing 1 mg ml21

gelatin. After electrophoresis, the resolving gel was

incubated overnight at 37�C in 40 mM Tris–HCl, pH

8.6, and 0.2% Triton X100. The gel was stained in

Coomassie Blue R-250, destained and the proteaseactivity appeared as white bands.

In situ H2O2 and O�22 and epifluorescent detection

of ROS

For in situ detection of H2O2, a modified previously

described method (Yoda et al. 2003) was used. Spd (10

mM) was infiltrated in leaves with a 1-ml syringe without

a needle, and they were incubated at 25�C under con-

tinuous light for the indicated time points. At the end

of incubation time, DAB (1 mg ml21, pH 3.8) was in-

filtrated and leaves were incubated for 1 h. Deposits

formed were visualized after discoloring leaves in boiledabsolute ethanol. For the in situ detection of O�2

2 , the

same procedure as described above for leaf discs was

followed, except that 0.5 mg ml21 NBT in K-phosphate

buffer pH 7.8 was infiltrated in untreated tissues and 0.1

mg ml21 for treated ones.

In situ localization of H2O2 was performed using

the highly sensitive, cell-permeable probe 2#,7#-dichlo-rodihydrofluorescein diacetate (DCFH-DA) (MolecularProbes, Eugene, OR). Cells were harvested, after centri-

fugation at 800 g, and were incubated in 1 ml buffer [20

mM K-phosphate, pH 6.0, supplemented with 50 mM

DCFH-DA and 3mgml21 horseradish peroxidase (Sigma-

Aldrich)] for 10 min at 25�C in darkness. An aliquot of

cells (0.1 ml) was removed, washed in the same buffer

and visualized immediately. Pictures were taken with an

epifluorescence microscope (Nikon Eclipse E800 1, Tokyo,Japan) with excitation filter EX 450-490 and emission

filter BA 520 using a SONY655 SONYDXC-950P (Tokyo,

Japan) camera.

Western blotting, RNA extraction and northernblotting

Total protein extracts were electrophoretically resolved,transferred to membranes and hybridized against an anti-

PAOmaize polyclonal antibody (Angelini et al. 1995), an

anti-CAT tomato polyclonal antibody, an anti-SOD-2 and

SOD-4 polyclonal antibody and an anti-actin polyclonal

antibody (Sigma-Aldrich). Total RNA was extracted with

the optimized hot phenol method according to Iandolino

et al. (2004), transferred to amembrane and hybridized to

a pao cDNA-derived 32P-labeled probe.

Chl a fluorescence, ion leakage, lipid peroxidationand DNA fragmentation

Chl a fluorescence emission was measured with a Handy

PEA instrument (Hansatech Instruments, Norfolk, UK).

Samples were dark adapted for 15 min and illuminated

afterward with continuous light (650 nm peak wavelength1800 mmol photons m22 s21 maximum light intensity) for

5 s provided by an array of three light-emitting diodes

focusedonacircleof 5mmdiameter of the sample surface.

Ion leakage was measured as conductivity using a K610

conductivity meter (mV/cm; Consort, Turnhout, Belgium).

Malondialdehyde contentwas used as indicator of lipid

peroxidation because of increased ROS generation and


was determined by a color reaction with thiobarbituric

acid (Heath and Packer 1968).

DNA fragmentation assay was performed as stated

in Papadakis and Roubelakis-Angelakis (2005). For the

detection of nuclear DNA fragmentation (Tunel assay), cell

cultureswere treatedwith 10mMH2O2 (DNase Iwas usedas a positive control). Untreated cells were the controls.

After an 18-h treatment, the cells were fixed in 4% (w/v)

paraformaldehyde in PBS buffer (pH 7.4), as described

by Asai et al. (2000). Nuclei were stained with 4#,6-diamidino-2-phenylindole (1mgml21) and the free 3#-OH

groups of fragmented DNAmolecules were labeled by the

Tunel (TdT-mediated dUTP nick-end labeling) method,

using the InSituCellDeathDetectionKit (Promega,Madison,WI), as instructed by the manufacturer. DNA was

visualized using a NIKON 800 fluorescence microscope.

Results

Production and screening of Mpao tobaccotransgenic lines: overexpression of Mpao generesulted in increased PAO protein and specificactivity

N. tabacum L. cv. Xanthi plants were transformed via

Agrobacterium-mediated transformation, using the

pART7/pART27 system, carrying the full-length maize

pao cDNAclone, downstreamof the constitutive promoter

35S originated by the Cauliflower Mosaic Virus (Fig. 1A).

Several transgenic lines were obtained by selecting trans-formants on kanamycin. Furthermore, PCR analysis for

the kanamycin gene (nptII) and Southern blotting were

performed to verify the stable transgene insertion into the

plants’ nuclear DNA. Both transcript expression and PAO

protein activity levels were determined in T0, T1 and T2

generations (Fig. 1) with similar results. The transgenics

overexpressing Mpao showed an approximately 20%

decrease in height and internode length (Table 1). Threetransgenic lines 2.2, 4 and 6.5 were used for further

analysis. Leaves (Fig. 1), stems and roots (data not shown)

were molecularly and biochemically characterized. In all

transgenic tissues, the levels of Mpao transcripts were

higher, comparedwithWT (Fig. 1B, C) and correlatedwith

both higher PAO protein levels in the soluble (Fig. 1D, E)

and the insoluble (data not shown) fraction andwith higher

enzymatic activities (Fig. 1F).

PAO enzymatic activity is localized mainlyextracellularly

The native MPAO protein is mainly localized in the

apoplasticcompartment (Conaetal. 2005). Toexamine the

localization of the recombinant enzyme in the transgenics,

different fractions of ICF were collected from leaf tissues(Fig. 2). Also, the leaf extract was separated in three main

fractions, namely soluble (S), extractable (E) and tightly

wall bound (T) (Fig. 2). Thehighest PAOenzymatic activity

and immunoreactive protein in the transgenics was

localized in the ICF fraction, followed by the S fraction

(Fig. 2A, B). A similar trend was found in the WT plants.

TheE fractionwasobtainedby increasing the ionic strength

of thebuffer toextract the ionically cellwall-boundPAO. Inmature tissues, the E fraction was higher (pellet fraction;

Paschalidis and Roubelakis-Angelakis 2005a), and in

leaves of transgenics, the E fraction accounted for app-

roximately 15–25% of the total PAO activity. In addition,

the T fraction of PAO (obtained after the addition of Triton

X-100 to the pellet) accounted for no more than 8% of the

total PAO activity in transgenics but was not detectable in

the leaves of WT plants.

Overexpression of Mpao reduced significantly theendogenous Spd and Spm levels with concomitanthigh production of Dap

S-PA, SH-PA and PH-PA titers were determined in the first

fully developed leaf ofWTand transgenic lines (Fig. 3). A

general decrease in the titers of soluble higher PAs, andespecially of Spd, was observed in the transgenic lines,

whereas the SH-PA and PH-PA fractions did not decrease

substantially. Put was the more abundant PA and it also

decreased in the transgenics, except in line 4 (Fig. 3). The

decrease in Put levels of transgenic lines 2.2 and 6.5

could be attributed to increase in SAMDC-specific

activities in the leaves, which were 0.58 � 0.03 nmol

CO2mg21 h21, 0.92� 0.06 nmol CO2mg21 h21, 0.57�0.04 nmol CO2 mg21 h21 and 0.81 � 0.04 nmol CO2

mg21 h21 protein in WT, S2.2, S4 and S6.5 lines,

respectively. To exclude the possibility that PAO

could act on Put, or alternatively DAO was upregulated,

the Put oxidizing activity was measured; the transgenics

did not exhibit significant increase in DAO activity (data

not shown). In addition, free Dap levels increased

significantly in all transgenic lines, especially in theleaves (Fig. 3).

Table 1. Biometric characters including height (H), internode length (IL)

and distance of the first fully developed leaf from shoot apex (A) inWTand

transgenic lines 2.2, 4 and 6.5. Values are expressed in mm, and data are

the means of 10 independent plants � SE. Asterisks indicate statistically

significant differences at P � 0.001.

Characteristics WT 2.2 4 6.5

H 62.0 � 1.8 52.6 � 4.3* 57.6 � 2.9 56.2 � 2.4*

IL 11.6 � 0.7 8.5 � 1.1* 8.0 � 0.6* 8.0 � 0.6*

A 21.4 � 2.2 13.0 � 0.6* 17.0 � 1.6 13.2 � 0.6*


O�22 and H2O2 were transiently detectable in

transgenic lines overexpressing Mpao

Hydrogen peroxide is generated as a product of the PAO-

catalyzed reaction. Furthermore, our previous results

have shown that Spd and Spm inhibit NAD(P)Hoxidase in

plant protoplasts (Papadakis and Roubelakis-Angelakis

2005). Therefore, it was of interest to determine the levels

of superoxides and H2O2 in WT and transgenic lines.

Although increased PAO resulted in higher H2O2

generation in the transgenic lines, as the levels of Dap

suggested (equalmoles ofDap andH2O2 are producedby

the PAO reaction), no increase in the levels of ROS using

epifluorescence microscopy in cell suspensions (Fig. 4A)

or the luminol/lucigenin chemiluminescence method in

both leaves and cell suspensions could be detected (data

not shown). The results were further confirmed by in situ

detection of H2O2 using the DAB infiltration method.

Incubation of WT leaves with 10 mM Spd resulted in thegeneration of a reddish-brownproduct, whichwas higher

after 1 h (before DAB infiltration; Fig. 4B). Incubation of

transgenic leaves with 10 mM Spd, however, resulted in

a dramatic generation of a reddish-brown product in the

first 2 min compared with WT, but when the incubation

time increased to 1 h, this colored product was

diminished (Fig. 4B). The reddish-brown color started to

decrease in the transgenic lines as early as 5 minfollowing incubation with Spd (data not shown). Thus,

Spd was quickly oxidized by the overexpressed Mpao in

the transgenics and was completely depleted within 1 h.

The antioxidant machinery was highly induced inthe transgenics

As mentioned before, the H2O2 generated by high PAOactivity in the transgenic tobacco plants, as the increased

Fig. 2. PAO localization in WT and transgenic line 2.2. (A) Western blot

analysis in line 2.2 using an anti-MPAO-specific antibody against protein

extracts, loaded on the basis of the protein content of each sample (10

mg/well), obtained from ICF, soluble (S), extractable (E) and tightly wall-

bound (T) fractions. (B) PAO activity in ICF, S, E and T fractions isolated

frommesophyll of WTand 2.2. (C) PAO activity in ICF, S, E and T fractions

isolated from cell suspension cultures of WTand 2.2. Data are the means

of three independent experiments and vertical bars represent �SE.

Fig. 3. S-, SH- and PH-PA fractions and S-Dap fraction in leaf tissues of

tobacco WT and transgenic lines 2.2, 4, and 6.5. Data are the means of

10 independent experiments and vertical bars represent �SE. Asterisks

indicate statistically significant differences (P ¼ 0.01) from WT.


Dap levels suggested (Fig. 3), could not be detected by

in vitro measurements or by in situ staining but was only

detected when DAB and Spd were infiltrated simulta-

neously or within a 2-min interval (Fig. 4). These results

suggested that the antioxidant machinery should have

been induced promptly in the transgenics. Thus, the

antioxidant enzyme activities were monitored. Increasedspecific activities of PAOwas accompanied by increased

POX (EC 1.11.1.7) activity in the overexpressing lines

(Fig. 5B) and correlated well with the PAO levels (Fig. 1).

The abundance of themost anodic POX isoform 6 and the

cathodic isoform 1 was greater in all transgenic lines and

that of isoforms 4 and 5 was lower, whereas isoforms 2

and 3 were unaffected toMpao overexpression (Fig. 5A).

Isoform 1 could be implicated in ROS detoxification incells, whereas isoform 6 could participate in the cell wall

cross-linking, although a dual role for both POXs cannot

be ruled out. Anodic POX isoforms are considered to be

localized in the cell wall contributing to developmental

processes, such as the regulation of the balance between

cell wall expansion and stiffening (Boeuf et al. 1999).

PAs have been reported to inhibit NADPH oxidase in

both animals and plants (Ogata et al. 1996, Papadakis andRoubelakis-Angelakis 2005) and to exert a protective role

via this inhibition. Therefore, a decrease in PA titers might

have resulted in higher NADPH oxidase activity and O�22

levels, the latter being scavenged by SOD (EC 1.15.1.1).

The specific activity of SOD increased by 28, 44 and 30%

in the transgenic lines 2.2, 4 and 6.5, respectively,

compared withWT (Fig. 6B). Three isoenzymes were de-

tected: the slower migrating in native PAGE MnSODmit,followed by Cu/ZnSODcyt and the faster migrating

FeSODchl (Fig. 6A). The increase in SOD activity was

mainly because of the increased cytosolic isoform, as

indicated by the isoenzymic analysis and immunoreac-

tive SOD protein (Fig. 6C).

In parallel to SOD activity, CAT (EC 1.11.1.6)-specific

activity increased with two main isoforms being induced

(Fig. 6E,D), as did CATprotein levels in the overexpressing

Fig. 4. In vivo ROS epifluorescence in suspension cells and in situ DAB detection of H2O2 (in leaves) inWT tobacco and transgenic lines 2.2, 4 and 6.5. (A)

Epifluorescence ROS detection inWT, 2.2, 4 and 6.5 cell suspension cultures. (B) H2O2 detection using the DAB infiltrationmethod. Leaveswere infiltrated

with 10 mM Spd and incubated for 0 min (syringe spot at the bottom right of the leaf), 1 min (top left), 2 min (bottom left) and 1 h (top right) before the

infiltration of DAB and decoloration, as described in Materials and methods.

Fig. 5. Native IEF of POX and enzymatic specific activity in the leaf soluble

fraction of tobaccoWTand transgenic lines 2.2, 4, and 6.5. (A) IEF at a pH

gradient 3–10. (B) Specific activity. Data are the means of three

independent experiments and bars represent�SE. POX activity presented

statistically significant differences (P¼ 0.01) in lines 4 and 6.5, compared

with WT.



lines (Fig. 6F), and followed similar pattern to PAO levels.

These results could support the posttranslational regula-

tion of CAT, possibly via the action of PAs, as Kim et al.

(2001) and Rhee et al. (2005) have already proposed.

APX (EC 1.11.1.11) is a H2O2-detoxifying enzymewith

low Km for its substrate, acting especially at low substrate

concentrations. At higher concentrations of H2O2, the

main detoxifying effect may be exerted by CAT. The main

cytosolic isoenzyme of APX (Fig. 6H) and APX (Fig. 6I)

activity increased, in a pattern similar to that of SOD and

CAT. It is of interest that line 4, one of the less Mpao

overexpressing lines with lower PAO levels (Fig. 1) and

higher PA levels (Fig. 3), showed the highest expression

levels of POX (Fig. 5), SOD (Fig. 6A, B, C) and APX

(Fig. 6H, I), compared with lines 2.2 and 6.5. Thus,

moderate overexpression of Mpao in line 4 (Fig. 1), and

PA levels (Fig. 3), resulted in stronger induction of the

above-mentioned antioxidant enzymes, whereas higher

Mpao expression levels in line 2.2 (and line 6.5), resulting

to even lower titers of PAs, was associated with lower

induction of the antioxidant enzymes, suggesting that

higherMpao overexpression beyond a specific threshold

results in higher accumulation of ROS and inability of the

antioxidant machinery.

In contrast to animal cells, plant cells synthesize high

amounts of ASA, an additional hydrophilic redox buffer

that provides robust protection against oxidative chal-

lenge (Laloi et al. 2004). The redox state of ASA is

controlled by, among others, two enzymes of Halliwell–

Asada pathway, MDHAR (EC 1.6.54) and DHAR (EC

1.8.5.1), which increased significantly in the transgenic

lines (Fig. 6G). In addition, ASA content decreased in 2.2

and 6.5 transgenic lines, whereas DHA increased

(Table 2). Antioxidants, such as ASA, may induce the

expression of genes associated with abiotic stress pro-

tection and provide information about the cellular redox

homeostasis (Foyer and Noctor 2005), which, in turn,

could result in appropriate induction of events linked to

development/acclimation processes or, alternatively,

execution of cell death programs.

Transgenic lines overexpressing Mpao weresusceptible to induced oxidative stress

Because the transgenic lines exhibited increased steady-

state antioxidant activity, one would expect that they

could also be more tolerant to additional ROS, induced

by abiotic agents. Leaf discs from the transgenics treated

with 100 mM H2O2 showed Chl loss and decreased

photochemical efficiency of PSII (Fv/Fm; Fig. 7A) accom-

panied by ion leakage (Fig. 7B), lipid peroxidation

(Fig. 7C), extensive tissue damage (Fig. 8C) and DNAfragmentation (data not shown). Similar results were

observed using 0.6 and 1.2 mM methylviologen (MV)

under normal photon flux conditions (50 mmol m22 s21)

(Fig. 7D, E).On the contrary, leaf discs from theWTplants

were significantly more tolerant. These data indicate that

major decline in PA levels, namely Spd and Spm, may

well result in increased susceptibility of the transgenics to

oxidative stress, expressed as instability of the photosyn-thetic apparatus and loss of cell membrane integrity.

To examine what was the reason of failure of the

overexpressers to efficiently scavenge the additional

H2O2, the antioxidant enzymes POX, CAT, SOD and

APX were studied after treatment with H2O2 in both WT

and transgenic lines; POX, CAT and SOD increased the

first 6–12 h only in the WT and declined to basal levels

thereafter. Moreover, two isoenzymes of CAT wereinduced during stress, whereas an increment of all SOD

isoenzymes could be observed inWT (Fig. 9B,D). Similar

results were obtained when plants or cell suspension

cultures were treated with MV or menadione, respec-

tively (data not shown). As APX is labile to high H2O2

concentrations, it was quickly inactivated after 6 h; the

inactivation occurred more rapidly in the transgenics and

the same trendwas observed for SOD,which is also labileto high H2O2 (Fig. 9F, G), suggesting that transgenic cells

accumulate higher amounts of ROS or, alternatively, PA

depletion may have a detrimental effect on enzymatic

activities. Moreover, higher lipid peroxidation and ROS

accumulation in the transgenicswere observedwhen leaf

discs or cells were treated with MV or menadione,

Fig. 6. Isoenzymic analyses and specific activities of SOD, CATand APX, and immunoreactive SOD andCAT protein in the soluble fraction of leaves inWT

and 2.2, 4 and 6.5 transgenic lines. (A) Detection of isoenzymes of SODby activity staining of protein extracts separated in native gel. (B) Specific activity of

SOD. (C) Western blot of the cytoplasmic SOD protein (top of C), Coomassie blue staining in 7.5% PAGE (second from top of C), rehybridization of the

same membrane with anti-actin polyclonal antibody (third from top of C) and densitometric analysis of the SOD protein (bottom of C). (D) Detection of

isoenzymes of CAT by activity staining of protein extracts separated in native gel. (E) Specific activity of CAT. (F) Western blot of CAT protein (top of F),

Coomassie Blue staining in 7.5% PAGE (second from top of F), rehybridization of the samemembrane with anti-actin polyclonal antibody (third from top

of F) and densitometric analysis of the CAT protein (bottom of F). (G) Specific activities of MDHAR and DHAR. (H) APX activity staining in protein extracts

separated in native gel. (I) Specific activity of APX. Numerical data are the means of three independent experiments and bars represent �SE. Analysis of

variance showed that data on SOD, CATand APX in transgenic lines were significantly different (P¼ 0.01), comparedwith the corresponding data inWT.

Similarly, DHAR activity was also significantly different in lines 2.2 and 6.5 and MDHAR activity in line 2.2, compared with WT.


respectively (see also below; Figs. 7C and 8B), suggestingthe inability of the transgenic cells to control and retain

low levels of ROS. The reduced PA levels may account for

the higher ROS accumulation, either through their

protective role on the antioxidant mechanism or through

a positive effect on specific antioxidant activities. A direct

role for PAs on ROS scavenging cannot be ruled out. An

interesting finding was the differential induction of

protease activity between WT and transgenics duringstress conditions (Fig. 9H, I).

To examinewhether the detrimental effect ofH2O2was

due to induction of the PCD syndrome, nuclear DNA

fragmentation (Tunel) in cells (Fig. 8A) was performed.

Positive Tunel nuclei occurred to a great extent in

transgenic cells, suggesting that the resulting damages

were because of the execution of a PCD program,

possibly triggered by excessive ROS.Menadione was used as an elicitor of ROS (Skopelitis

et al. 2006) to test their differential accumulation in

transgenics during stress conditions. The accumulation of

ROS only in transgenic lines 2 h after the incubation with

10 or 20 mM menadione suggested that the antioxidant

machinery in transgenics is insufficient to sustain ROS

levels within a normal limit even 1 h after the incubation

with the elicitor. The simultaneous incubation of trans-genic cells with ASA reduced ROS levels to almost non-

detectable amounts (Fig. 8B; T 1ASA).

Discussion

Hydrogen peroxide at low concentrations can participate

in the signaling cascade, inducing the expression of

various genes, including the antioxidant ones and/orother stress-responsive genes, whereas at high concen-

trations, H2O2 participates in the induction of the PCD

syndrome (Papadakis and Roubelakis-Angelakis 2005,

Skopelitis et al. 2006, Vacca et al. 2004). Also, H2O2 is

the substrate for the POX-mediated cross-linking of cell

walls (Fry et al. 2000). Thus, the effect of PAs in plant

growth, development and stress responses could be

exerted either directly or via the H2O2 generated by theirPAO-mediated oxidation. Furthermore, Spd and to

a lesser extend Spm inhibit, at least in in vitro plantmodels such as plant protoplasts, NAD(P)H oxidase

which under stress conditions mediates superoxide

generation, whereas Put in the same in vitro model

prevents the execution of the PCD syndrome (Papadakis

and Roubelakis-Angelakis 2005). Therefore, PAs seem to

contribute to the homeostasis of ROS. They could act as

direct radical scavengers; Spm and other PAs have been

reported to act as antioxidants directly or through thechelation of Fe21 that catalyzes the generation of OH

radical via the Fenton reaction (Lovaas 1997) or by

inhibiting the NADPH oxidase mediating the production

of superoxide ions (Papadakis and Roubelakis-Angelakis

2005).

Recently, the studies aiming to show the molecular/

physiological roles of PAs have been facilitated by the

availability of transgenic plants, with altered expressionof the adc gene in rice (Capell et al. 2004), the spds gene

in Arabidopsis (Kasukabe et al. 2004), the samdc gene in

tomato (Mehta et al. 2002) and theMpao gene in tobacco

(Rea et al. 2004). These transgenics have mostly been

assessed for their stress response or phenotype alteration,

that is the adc overexpressors show improved tolerance to

drought (Capell et al. 2004) and the samdcoverexpressors

have greater lycopene content in tomato fruits (Mehtaet al. 2002). In parallel to work by Rea et al. (2004), we

developed tobacco transgenics overexpressing theMpao

gene (Fig. 1). Three transgenic lines were selected that

exhibited more than 10-fold greater PAO-specific activ-

ities compared with the WT, in agreement with the

respective abundance of the Mpao transcripts and PAO

protein (Fig. 1). A statistically significant reduction in the

endogenous Spd and Spmwas found, in analogy with thedramatic increase in the abundance of PAO activity, and

this reduction was accompanied by higher free Dap

content, which, along with H2O2, is a product of the PA

oxidation (Fig. 3).

Because H2O2 is the product of PA oxidation by PAO,

one would expect that there should be a parallel increase

in the endogenous ROS content in the transgenic cells.

Attempts to measure the endogenous H2O2 using thelucigenin/luminol reactions (data not shown) or to detect

Table 2. ASA and DHA contents, ASA pool and redox state. ASA and DHA content, ASA pool (ASA1 DHA) and redox state (ASA/DHA) determined in

tobacco WTand transgenics. Data are the means of two independent experiments � SE. Asterisks indicate statistically significant differences (P ¼ 0.01)

from WT.

Line ASA (mmol g21 FW) DHA (mmol g21 FW) ASA 1 DHA (mmol g21 FW) ASA/DHA

WT 0.898 � 0.014 0.122 � 0.005 1.020 � 0.022 7.33 � 0.08

2.2 0.845 � 0.011* 0.138 � 0.006* 0.982 � 0.019 6.12 � 0.04*

4 0.899 � 0.016 0.127 � 0.011 1.025 � 0.024 7.09 � 0.12

6.5 0.841 � 0.012* 0.154 � 0.012* 0.995 � 0.028 5.47 � 0.14*


in situ ROS epifluorescence in transgenic suspension

cells or by DAB in leaves indicated that ROS were below

the detection level (Fig. 4). Thus, it was evident that under

normal conditions, overexpression of PAO did not result

in increased intracellular ROS (Fig. 4), although H2O2

was generated concomitantly with Dap (Fig. 3). That

H2O2 is a product of PA oxidation by PAO was tested by

infiltrating Spd into intact transgenic leaves. In theseleaves, H2O2 was detected as early as 2 min following

Spd injection (Fig. 4B) but disappeared soon thereafter.

The lack of intracellular accumulation of H2O2 in the

PAO overexpressing cells (Fig. 4B) but not in the WTwas

explained by the increased expression of the antioxidant

genes and the high specific activities of POX (Fig. 5B),

CAT, APX, MDHAR and DHAR, which were significantly

greater in the transgenic cells, when compared with WT,concomitantly with the abundance of the respective

proteins/transcripts (Fig. 6). Not all isoforms of the

antioxidant enzymes were similarly expressed in the

transgenic cells. Indeed, only the most anodic (isoform 6)

and the most cathodic (isoform 1) of POX and the

cytosolic isoform of APX were mostly expressed (Figs. 5

and 6). The fact that SOD was also induced in the

transgenic cells (Fig. 6) reinforces the view that the

reduced intracellular Spm and Spd resulted in increased

activity of NAD(P)H oxidase (Papadakis and Roubelakis-

Angelakis 2005). The generated superoxide ions were

thereafter disproportionated by the induced SOD.Because endogenous H2O2 was scavenged by the

activated antioxidant machinery, one would further

expect that the transgenics would be more tolerant to

oxidative stress. To test this hypothesis, leaf discs from the

transgenic lines and the WT plants were treated with

100 mM H2O2. These transgenic leaf discs showed

extensive tissue damage accompanied by increased Chl

loss, lower photochemical efficiency of PSII and DNAfragmentation (Figs. 7A and 8C), when compared with

WT leaf discs, suggesting that the already activated

antioxidant activity (Figs. 5 and 6) was not sufficient to

cope with extra ROS, beyond to that generated by the

Fig. 7. Susceptibility of transgenics overexpressing MPAO under oxidative stress conditions. Differences in maximum quantum yield of PSII, ion leakage

rate and malondialdehyde (MDA) content of WTand transgenic line 2.2 following treatment with H2O2 andMV. (A) Maximum quantum yield of PSII (Fv/

Fm) during treatment of leaf discs fromWTand transgenic lineswith 100mMH2O2. (B) Ion leakage rate following treatmentwith 100mMH2O2. (C)MDA

content after a 12-h treatment with 0.6 and 1.2 mM MV. (D) Ion leakage rate following treatment with 0.6 mM MV. (E) Ion leakage rate following

treatment with 1.2 mM MV. Data are the means of three independent experiments with three replicates each and vertical bars represent �SE. Asterisks



increased PAO activity. On the contrary, the WT cells

were able to better cope with the increased intracellularROS and responded to oxidative stress, exhibiting

increased SOD, CAT and APX activities soon after the

stress induction (Fig. 9).

Similar results were obtained when transgenics or

transgenic suspension cells were treated with either MV

or menadione, which are known to induce ROS genera-

tion (Criddle et al. 2006) and mimic the ROS generated

as stress response (Skopelitis et al. 2006). Again, thetransgenic cells showed higher lipid peroxidation,

photosynthetic imbalance, ion leakage, ROS accu-

mulation and PCD syndrome, reinforcing the results

obtained by the addition of H2O2 (Figs. 7 and 8). That

these detrimental effects were because of increased

intracellular ROS was supported by their prevention

when ASAwas added (Fig. 8B and data not shown).

PAs stabilize the DNA–chromatin complexes, andmodifications of chromatin and DNA structure have

been shown in PA-depleted cells (Davidson et al. 1999).

PA depletion halted mammalian cells in G0/G1, withthis checkpoint necessary to execute the apoptosis

program, related to impaired oxidative mechanism

(Dypbukt et al. 1994). In contrast, H2O2 generated by

PA oxidation in mammals, as well as other PA catabolic

products (Thomas and Thomas 2001) and nitric oxide

(Tun et al. 2006), was effective in inducing PCD (Ha

et al. 1997). In tissues undergoing lignification, PCD

and senescence, PA levels were at a minimum, whereasPA catabolism was at a maximum (Paschalidis and

Roubelakis-Angelakis 2005a). Such a correlation

between increased PA catabolism, PA depletion and

PCD was obvious under abiotic stress conditions in

transgenics overexpressing PAO. On the contrary, under

normal conditions, increased PA catabolism and gener-

ated H2O2 in these transgenics were not sufficient to

signal execution of a PCD program, although PAcatabolic products have been considered to be

Fig. 8. Detection of PCD and ROS on treatment with H2O2 and menadione in WT transgenics. (A) Tunel assay (PCD detection) in WT and transgenic

suspension cells at 18 h following treatmentwith 10mMH2O2. Cell nuclei were stainedwith 4#,6-diamidino-2-phenylindole (red, for better distinctness),

and Tunel-positive nuclei (green yellowish) exhibited DNA fragmentation. (B) ROS epifluorescence detection after 20 mMmenadione treatment for 2 h in

cell suspension cultures of WTand transgenics. Controls are indicated as ‘2m’ (minus menadione) and treatments ‘1m’ (plus menadione) Furthermore,

negative controls are also indicated as ‘no cells’ (no cells were added) and ‘1ASA’ (ASA was added in cells as a ROS quencher), in which no cells were

added or ASA added, respectively. (C) Phenotypic differences of WTand transgenic leaf discs after treatment with 100 mM H2O2 for 24 h. Experiments

were performed twice in triplicates. Transgenic lines presented statistically significant differences (P ¼ 0.05), compared with WT.


pro-apoptotic signals (Thomas and Thomas 2001). High

PA levels have been correlated with cell wall formation,

high redox state and protoplast regeneration (Papadakis

et al. 2005), whereas Spd- and Spm-treated protoplasts

accumulated high levels of intracellular H2O2,

generated by PAO, whose activity increased along withCAT activity (Papadakis and Roubelakis-Angelakis

2005).

As H2O2 has a high diffusion rate inside the cell, a dual

role for H2O2, dependent on specific concentrations, can

be proposed: (1) low augmentation in H2O2 concen-

trations (that may remain in cell walls), caused by PAO

overexpression, successfully acted as signal for the

induction of the antioxidant machinery, leading to cellsurvival/adaptation and normal plant growth and (2)

higher H2O2 concentration (that may also diffuse in the

secretory pathway), caused not only by PAO over-

expression but also by other mechanisms such as

NAD(P)H oxidase/SOD reactions or by exogenous

addition, induced susceptibility and PCD. H2O2 concen-

trations beyond a specific threshold shifted the response

from normal growth to susceptibility and induced thePCD syndrome in the transgenics. This is of interest

because it is now widely accepted that H2O2 is a nodal

point in the signal transduction pathway, which, depend-

ing on its concentration (‘signature’), may target different

subsets of genes. When the antioxidant machinery has

been induced, further increase of ROS do not act as a new

signal for the expression of the antioxidant genes, and this

antioxidantmachinery is no longer active and sufficient toprevent oxidative damage. Instead, the higher ROS levels

induce expression of the PCD syndrome. Whether this

inability for further antioxidant activity is the result and/or

linked to the reduced PA levels and if the homeostasis of

the endogenous PAs contributes to the regulation of the

antioxidant machinery of cells and in doing that

a threshold of endogenous concentration should be

maintained remains an open question.

Acknowledgements – The authors are grateful to Dr P.

Tavladoraki (University Roma Tre, Italy) for the generous gifts

of anti-MPAO polyclonal antibody andMpao cDNA and to Dr

G. Inamine (United States Department of Agriculture, Belts-

ville, MD) for providing the antibody against CAT. The project

was co-funded by the European Social Fund and National

resources, projects Herakleitos and Pythagoras, and was

implemented in the frame of COST858.

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Edited by C. H. Foyer


Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

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