Polyamine metabolism is upregulated in response to tobacco mosaic virus in hypersensitive, but not in susceptible, tobacco

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Blackwell Science Ltd

Polyamine metabolism is upregulated in response to tobacco mosaic virus in hypersensitive, but not in

susceptible, tobacco

Francesca Marini

1

, Lucietta Betti

1

, Sonia Scaramagli

2

, Stefania Biondi

2

and Patrizia Torrigiani

2

1

UCI-STAA – Istituto di Patologia Vegetale, Via F. Re 8, Università di Bologna, Italy;

2

Dipartimento BES, Via Irnerio 42, Università di Bologna, 40126

Bologna, Italy

Summary

• Change is reported in the biosynthetic and oxidative activity of hypersensitive(NN) and susceptible (nn) tobacco (

Nicotiana tabacum

) plants in response totobacco mosaic virus (TMV).• Mature leaves of nn and NN tobacco were collected over 0–72 h as uninoculatedcontrols or after inoculation with TMV or phosphate buffer (mock-inoculation).The polyamine response to inoculation was analysed by measuring activity andgene expression of S-adenosylmethionine decarboxylase (SAMDC), arginine-(ADC) and ornithine decarboxylases (ODC); incorporation of labelled putrescine;and activity of diamine oxidase (DAO).• In NN leaves SAMDC activity and transcript levels, and DAO activity increased inthe TMV-inoculated plants but not in the other treatments; a two-fold increase inDAO activity was seen after 72 h. Both ADC and ODC activity increased in NNleaves at 72 h in TMV-inoculated plants; ADC mRNA increased with activity. Theincrease in SAMDC mRNA (24 h) preceded the rise in activity (72 h). [

3

H]putrescineadded to NN leaves led to enhanced label recovery and incorporation into spermidineand spermine in TMV-inoculated plants. No significant changes in biosynthetic oroxidative activity occurred in nn plants.• After TMV inoculation, NN, unlike nn, tobacco plants upgrade polyaminesynthesis and oxidation; this leads to changes in cellular components which mightinduce programmed cell death.

Key words:

arginine decarboxylase (ADC), diamine oxidase (DAO), hypersensitiveresponse (HR),

Nicotiana tabacum

(tobacco), ornithine decarboxylase (ODC),polyamines, S-adenosylmethionine decarboxylase (SAMDC), tobacco mosaic virus(TMV).

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Author for correspondence:

P. Torrigiani Tel: +30 0512091291 Fax: +39 051242576 Email: [email protected]

Received:

2 May 2000

Accepted:

19 September 2000

Introduction

It has been purported for many years that the diamineputrescine and the polyamines spermidine and spermine,generally known as polyamines, are involved in the responseto pathogen attack in that both fungal and bacterial patho-gens elicit the production of their amide conjugates (Martin-Tanguy, 1985). The most studied function of free polyamines,which are polycationic components of prokaryotic and eukar-

yotic cells and also part of virus particles (Heby & Persson,1990; Rabiti

et al.

, 1994; Cohen, 1998), is that of plantgrowth regulators (Bagni & Torrigiani, 1992) due to theircomplex interactions mainly with nucleic acids, but alsowith phospholipids, proteins (Feuerstein & Marton, 1989)and cell wall anionic polysaccharides (Messiaen & Van Cutsem,1999). In fact, they seem to cover a large spectrum of actionin that they participate in the homeostatic adjustment ofcells relative to environmental changes (Flores, 1990). In

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addition, the endogenous polyamine pool in pathogenscan be modulated using analogues and this may representa useful tool in controlling plant diseases (Walters &Mackintosh, 1997).

Among the various host-pathogen systems, the tobacco–TMV interaction offers advantages in that much is knownabout the molecular markers of the defence response, suchas salicylic acid (SA), ethylene, jasmonate and pathogenesis-related (PR) proteins. Moreover, the N gene confers resist-ance to TMV in the hypersensitive (NN genotype) plantswhich develop a hypersensitive response (HR), while theabsence of that gene (nn genotype) confers susceptibility toTMV and development of the systemic infection (Whitham

et al.

, 1994). Previous studies were aimed at understand-ing the response to TMV in hypersensitive and susceptibletobacco (

Nicotiana tabacum

cv. Samsun) plants in termsof polyamine metabolism. An early positive polyamineresponse in TMV-inoculated leaves from NN plants is detect-able already at 5 h, while not in nn plants (Rabiti

et al.

,1998). As far as the spatial and temporal patterns of poly-amine response are concerned, in NN plants an increasingconcentration gradient in free and conjugated putrescineand spermidine towards the centre of the hypersensitivelesion was reported in parallel to putrescine biosyntheticactivity gradients at 3 and 5 d after inoculation (Torrigiani

et al.

, 1997); opposite or no changes were observed in nnplants.

Recently, in TMV-inoculated tobacco leaves, a specificincrease in free spermine in intercellular fluids (but not inthe whole leaf tissue) was reported 4 d after inoculation(Yamakawa

et al.

, 1998). The authors suggest a role forexogenous spermine in the salicylate-independent inductionof acidic PR proteins.

Concerning the mechanism of action of free polyamines,it may be based on one or more of the following: (a) ligandsfor specific plasma membrane polyamine-binding pro-teins (Tassoni

et al.

, 1998); (b) modulators of membranepermeability (Roberts

et al.

, 1986); (c) precursors of hydro-xycinnamoyl acid amides (HCA; Martin-Tanguy, 1985) and(d) producers of hydrogen peroxide through cell wall-locateddi-(DAO) and polyamine oxidases (PAO) (Federico & Angelini,1991). The latter are involved in signalling programmed celldeath (PCD, Møller & McPherson, 1998) which also mediatesthe HR (Mittler

et al.

, 1997).In this paper we further examined the time course of

the polyamine response to TMV in NN and nn tobaccoplants with respect to biosynthesis by analysing: activityand gene expression of S-adenosylmethionine decarboxylase(SAMDC), which leads to spermidine and spermine syn-thesis through the production of decarboxylated SAM(decaSAM) and the activity of specific synthases; incorpora-tion of the labelled precursor putrescine into spermidine andspermine as an additional indication of biosynthetic activ-ities; and activity and gene expression of arginine-(ADC) and

ornithine decarboxylases (ODC) which lead to putrescinebiosynthesis, the former indirectly, the latter directly. On theother hand, putrescine catabolism was evaluated through theactivity of DAO which oxidatively deaminates diamines.These enzymes are involved in the modulation of diamineand polyamine levels both in the cytoplasm and cell wall.Results show that, upon inoculation with TMV, hypersens-itive plants react in an opposite manner in comparison tosusceptible ones by upregulating polyamine synthesis andoxidation.

Materials and methods

Plant material and TMV inoculation

TMV-susceptible (nn) and TMV-resistant (NN) plants of

Nicotiana tabacum

(L.) cv. Samsun were grown in theglasshouse with a photoperiod of 8 h (1.87 W m

–2

) at 25

°

Cup to the vegetative stage of 15

±

1 leaves (

c

. 6 wk).The third-fifth mature leaf counting from the bottom of

5 resistant and susceptible tobacco plants was inoculated bytreatment with carborundum (400 mesh size) and 200

µ

lpurified TMV suspension (0.1 mg ml

–1

0.01 M sodiumphosphate buffer, pH 7; Torrigiani

et al.

, 1995). An equalnumber of plants were mock-inoculated by treating eachleaf with carborundum and 200

µ

l phosphate buffer. Non-inoculated plants were considered as healthy controls. Leaves(5 per treatment) from inoculated-, mock-inoculated and con-trol plants were collected, frozen in liquid nitrogen immedi-ately (0 h) or at 5, 24 and 72 h after inoculation, and storedat

−

80

°

C until use.

Enzyme activity assays

All enzyme procedures were carried out in an ice bath. ForSAMDC (EC 4.1.1.21) activity tissue (200–300 mg f.wt) was homogenised with 3 volumes of 100 mM Tris-HCl buffer, pH 7.6, containing 50

µ

M EDTA and 25

µ

Mpyridoxal phosphate. The homogenate was centrifuged at20 000

g

for 30 min at 4

°

C and the supernatant was usedfor the enzyme assay. SAMDC activity was determined byincubating 0.2 ml aliquots of the supernatant with 3.7 kBqS-adenosyl-L-[carboxyl-

14

C]-methionine (2.22 TBq mol

−

1

,Amersham Pharmacia Biotech Italia, Milano, Italy) andmeasuring the rate of

14

CO

2

evolution from the labelledsubstrate during a 2-h incubation at 37

°

C as previouslydescribed (Scaramagli

et al.

, 1999a). The liberated

14

CO

2

was trapped in 1 M KOH and analysed by scintillationcounting in a Beckman LS 6500 counter.

Putrescine oxidising (DAO, EC 1.4.3.6) activity wasassayed by a radiometric method based on the productionof [

14

C]

∆

1

-pyrroline from labelled putrescine as previouslydescribed (Scaramagli

et al.

, 1999a). Samples were homo-genised in 100 mM potassium phosphate buffer, pH 8,


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containing 2 mM dithiothreitol, and centrifuged at 20 000

g

for 30 min at 4

°

C. Aliquots (0.2 ml) of supernatant orresuspended pellet were incubated at 37

°

C for 30 min in thepresence of 7.4 kBq [1,4–

14

C]putrescine (4.03 GBq mmol

–1

,NEN, Boston, MA, USA) in the absence or in the presenceof 100

µ

M unlabelled putrescine.After adding 2% (w/v) sodium carbonate to stop the re-

action, the [

14

C]pyrroline was immediately extracted in 1 mltoluene and 500

µ

l of the lipophilic phase were withdrawn,added to 2 ml scintillation liquid (Ultima Gold, BeckmanAnalytical, Milano, Italy) and the radioactivity counted. Inpreliminary experiments enzyme activity was measured inrelation to putrescine concentration (0–1 mM) and the leafhomogenate or 20 000

g

pellet were subjected to sonicationwith an MSE 150 W ultrasonic disintegrator for 3 cycles of30 s each as described in Torrigiani

et al

. (1995).ADC (EC 4.1.1.19) and ODC (EC 4.1.1.17) activities

were measured as previously described by Rabiti

et al.

(1998).Samples were extracted in 5 volumes of ice-cold 0.1 MTris-HCl buffer, pH 8.5, containing 50

µ

M pyridoxal5-phosphate, and centrifuged at 20 000

g

for 30 min at4

°

C. Enzyme activity assays were performed by measur-ing the

14

CO

2

evolution from 7.4 kBq L-[1–

14

C]ornithine(2.11 GBq mmol

–1

, Amersham Pharmacia Biotech, Italia) orDL-[U-

14

C] arginine (11 GBq mmol

–1

, Amersham PharmaciaBiotech Italia), for ODC and ADC, respectively, in thepresence of 2 mM unlabelled substrate during a 2-h incuba-tion at 37

°

C. CO

2

was entrapped in KOH and radioactivitycounted.

In all cases protein content was measured according toBradford’s method (Bradford, 1976), using bovine serumalbumin as standard. Data represent the means

±

SD of 2–3experiments (3 replicates each); differences between meanswere analysed by Student’s

t

-test (significance level at 0.001,0.01 or 0.05, as specified).

Polyamine analysis

Samples of 0.2–0.5 g were analysed for free and conjugatedpolyamines by homogenizing them in three volumes ofcold 5% (w/v) trichloroacetic acid (TCA) and centrifugingfor 10 min at 20 000

g

. Replicate aliquots (0.3 ml) of thesupernatant were placed in glass ampoules with the samevolume of 12 N HCl; the ampoules were then flame sealedand incubated at 110

°

C for 18 h to allow the hydrolysis ofcovalent linkages between polyamines and other molecules.Standard polyamines were also subjected to the same pro-cedure. The hydrolysates were taken to dryness and thenresuspended in 0.3 ml TCA. Aliquots (0.1 ml) of the super-natant (free polyamines) and hydrolysed supernatant (con-jugated polyamines) were dansylated, dansyl-polyaminesextracted in benzene described by Torrigiani

et al.

(1987)and separated by thin layer chromatography (TLC) plates.Spots were visualised under UV light, scraped off the TLC

plate, eluted in acetone, their fluorescence measured using aJasco FP-770 (Tokyo, Japan) spectrofluorometer (excitation360 nm, emission 506 nm) and compared with standardpolyamines. Data represent the means

±

SD of 2 experiments(3 replicates each).

Labelling procedure

For labelling experiments, [1,4(

n

)-

3

H]putrescine (0.37 MBqin 10

µ

l, specific activity 0.85 TBq nmol

–1

, AmershamPharmacia Biotech Italia) was injected with a 1-ml-syringeinto the main vein of control NN tobacco leaves and ofleaves immediately after TMV-inoculation. Equal amountsof labelled putrescine were re-injected 6 and 24 h after thefirst injection. Radioactivity distribution was monitoredduring the first 6 h in control plants by homogenising theleaf tissue in 0.1 M Tris-HCl buffer, pH 7.6, and countingseparately the various zones of the leaf after 2, 4 and 6 hfrom injection. For labelled di- and polyamine analysisleaves were collected after 6 and 72 h from control and virus-inoculated plants and stored at

−

80

°

C until use. Labelledfree and conjugated polyamines were separated by TLCas described above and spots dissolved in acetone; the latterwas placed in vials with scintillation cocktail and countedfor radioactivity.

RNA extraction and northern blot

Total RNA was extracted from approx. 200–300 mg freshweight explants using an RNeasy Plant Mini Kit (Qiagen,Hilden, Germany) according to the manufacturer’s instruc-tions. RNA (15

µ

g per track) was size-fractionated on a1.2% agarose formaldehyde gel and transferred in 10

×

SSC(20

×

SSC: 0.3 M sodium citrate, 3.0 M NaCl, pH 7) ontonylon membranes (Hybond-N, Amersham) overnight accordingto standard methods (Sambrook

et al.

, 1989). RNA wascross-linked to the membrane by exposure to UV at 312 nm(Vilber Lourmat, Marné La Vallée, France) for 4 min.

For the tobacco probes (kindly supplied by A. J. Michael,Institute of Food Research, Norwich, UK), RNA isolation,PCR conditions, cloning of the PCR products and sequen-cing are described in Michael

et al

. (1996). RNA blots wereprehybridised at 42

°

C for 2 h and hybridised at 42

°

C for18–20 h with [

32

P]dCTP-labelled PCR fragments (randompriming with a Rediprime DNA labelling Kit, Amersham)of SAMDC, ADC and ODC as described in Scaramagli

et al

. (1999a); they were amplified from tobacco by usingspecific oligonucleotide primers homologous to the 5

′

and 3

′

ends of the ORFs of the respective genes. The followingprimers were used to obtain approx. 1.0 kb, 2.0 kb and 1.2 kbPCR products of SAMDC, ADC and ODC, respectively:5

′

-CTAATGGATTCGGCCTTGCCTGTC-3

′

(sense) and5

′

-CACAGCCCTCAAGACACTACTCC-3

′

(antisense) forSAMDC; 5

′

-ATGCCGGCCTTAGGTTGTTGTGTAG-3

′



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(sense) and 5

′

-ACAACTTCAAGCGGTGCAATAGGACCA-3

′

(antisense) for ADC; 5

′

-GGATGGCCGGCCAGACAGTCA-TCG-3

′

(sense) and 5

′

-TAGAGGTGGTTCATCAGCTTGG-3

′

(antisense) for ODC.Following hybridization, membranes were washed as

previously described by Scaramagli

et al.

(1999a) and thenexposed to X-ray film at

−

80

°

C for 24 h (ADC, SAMDC)or 7 d (ODC) with intensifying screen (DuPont, Wilmington,DE, USA). Equal loading of RNA on gels was verified byethidium bromide staining. Band densities were quantifiedin each sample using the image analysis Phoretix programme(Phoretix International Ltd, Newcastle upon Tyne, UK) anddata are shown as relative intensity, normalised to the loadingcontrols.

Results

Diamine oxidizing activity

Since diamine oxidizing enzymes (DAO) are mainly localisedin the cell wall (Federico & Angelini, 1991) the DAO activityassay was checked for yield after sonication. Results showthat sonication of the leaf homogenate or pellet did notaffect the recovery of enzyme activity in those same fractionsor in the derived supernatant and pellet (data not shown);the same activity measured in the homogenate, sonicated ornot, was recovered in the supernatant fraction. Since part ofthe activity remained associated to the pellet fraction furtherexperiments were performed using both supernatant (solubleactivity) and pellet fractions (compartmented activity).

The analysis of DAO activity as a function of putrescineconcentration and the Lineweaver–Burke plot (data notshown) yielded an apparent K

m

of 237 and 287

µ

M, respect-ively, in the soluble and pellet fractions. DAO activitywas then analysed in NN and nn tobacco leaves at 0, 5, 24and 72 h from treatment in control, mock- and TMV-inoculated samples in order to discriminate its involvementin mechanical stress due to carborundum from that inpathogen-induced stress.

In hypersensitive plants, DAO activity was present in thesupernatant (Fig. 1a) and pellet (Fig. 1b) at comparablelevels in all the treatments and did not change significantlyin control and mock-inoculated leaves during the 72-h periodin either fraction. In virus-inoculated leaves, DAO specificactivity after 72 h was significantly (

P

< 0.001 for the solublefraction and

P

< 0.01 for the particulate fraction) higher(two–three-fold) than in mock-inoculated and controlsamples.

In nn plants, DAO activity was slightly lower in thesupernatant (Fig. 1c) than in the pellet (Fig. 1d), althoughits magnitude was comparable with that of NN plants.No significant differences in enzyme activity were detectedbetween control, mock- and TMV-inoculated samples atany time.

Fig. 1 Soluble (a, c) and compartmented (b, d) diamine oxidizing enzyme (DAO) activity in control (C, closed columns), mock-inoculated (M, open columns) and TMV-inoculated (V, hatched columns) leaves from hypersensitive (a, b) and susceptible (c, d) tobacco plants at different times after inoculation. Values are the mean ± SD (n = 9). Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001) with respect to controls.


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SAMDC activity and gene expression

The activity of SAMDC, whose localization is reported tobe cytosolic (Cohen, 1998), was determined in NN and nnleaves at different times after inoculation. No significantdifferences were detected in NN samples at 0, 5, and 24 hbetween control, mock- or virus-inoculated tissues, whileenzyme activity was significantly (P < 0.001) enhanced(about five-fold) after 72 h in TMV compared with mock-inoculated and control leaves (Fig. 2b). In nn plants SAMDCactivity levels were comparable to those of NN plants anddid not show any significant difference in terms of timecourse or treatment (Fig. 2c).

Since no changes were observed in SAMDC activityuntil 72 h, northern analysis of SAMDC was performedon control, mock- and virus-inoculated samples from NNplants at 24 and 72 h (Fig. 2a). At 24 h a transcript wasdetected whose signal intensity was comparable and higherin mock- and TMV-inoculated samples relative to controls.Two days later (72 h), SAMDC mRNA levels were similar incontrols and mock-inoculated samples. In TMV-inoculatedleaves transcript levels were strongly enhanced relative tocontrols and to 24-h inoculated samples (Fig. 2a).

Putrescine biosynthesis and free polyamine levels

Putrescine biosynthesis was evaluated in NN plants throughthe analysis of ODC and ADC transcript levels and activitiesin the three treatments at 24 and 72 h. As for SAMDC,ADC (Fig. 3b) and ODC (Fig. 3c) activities were notsignificantly different at 24 h in the three treatments whileat 72 h in TMV-inoculated leaves, both activities wereabout double (significance P < 0.01) compared with controlor mock-inoculated ones. Northern analysis of the samesamples revealed that ADC mRNA levels were higher at24 than at 72 h (Fig. 3a); at both times the signal wasmore intense in TMV-inoculated leaves relative to the othertreatments. Under the same experimental conditions noODC transcript was detectable even with a 7-d exposure.

Free putrescine and spermidine, but only traces ofspermine, were detected at 72 h in control, mock- andTMV-inoculated leaves both from hypersensitive (Fig. 4a)and susceptible (Fig. 4b) plants. In the former, putrescineaccumulated approx. two-fold in TMV-compared to mock-inoculated tissues and approx. three-fold compared withthe control ones. Minor increases (c. 30%) in spermidinecontent relative to mock-inoculated tissues were observed.The putrescine-to-spermidine ratio in NN leaves was closeto the unit except for TMV-inoculated samples whereputrescine accumulated almost two-fold relative to sper-midine (Fig. 4a). TCA-soluble conjugated putrescine andspermidine were detected at 72 h (168.2 and 26.2 nmol g–1

f. wt, respectively) in TMV-inoculated leaves; in the othertreatments these conjugates were one order of magnitude

less (10.1 and 2.3 nmol g–1 f. wt, respectively, for mock-inoculated; 8.2 and 1.4, respectively, for controls). In nnplants, no changes were detected in either putrescine orspermidine levels and the putrescine-to-spermidine ratio was1 in all the treatments (Fig. 4b).

Fig. 2 S-adenosylmethionine decarboxylase (SAMDC) transcript levels and activity in control (C, closed columns), mock-inoculated (M, open columns) and TMV-inoculated (V, hatched columns) leaves from tobacco plants at different times from inoculation. (a) Upper panel: northern analysis of SAMDC mRNA levels in hypersensitive plants. Each track was loaded with 15 µg total RNA and after size-fractionation transferred to a nitrocellulose membrane and hybridized with 32P-labelled PCR-derived fragment of tobacco SAMDC. Middle panel: RNA loading control; the gel was stained with ethidium bromide. Lower panel: densitometric analysis of respective band intensities normalised to ethidium bromide-stained loading control. (b) SAMDC activity in hypersensitive plants. (c) SAMDC activity in susceptible plants. Values are the mean ± SD (n = 6). Asterisks indicate significant differences (***, P < 0.001) with respect to controls.


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Labelled putrescine incorporation into spermidine/spermine

In order to further evaluate spermidine and spermine syn-thesis, radioactive putrescine was injected into the leaf mainvein in NN plants. Preliminary experiments conducted on

control plants showed that the labelled precursor diffusedthroughout the lamina and formed a concentration gradientwhich decreased from the middle towards the apical andbasal parts of the leaf (data not shown). Although most ofthe radioactivity remained confined to the main vein or inits immediate neighbourhood, part of it moved to the rest ofthe lamina within 2 h. Total label amount decreased withtime probably due to transport to the rest of the plant.

After 6 h, label recovery in the various fractions (Fig. 5a)was higher in TMV-inoculated than in control samplesalthough reciprocal ratios were comparable; it was higherin the TCA-supernatant (containing free and conjugatedpolyamines) than in the pellet (TCA-insoluble fraction).The radioactivity found in the supernatant was entirelyrecovered in the benzene fraction containing dansylated freepolyamines. At 72 h label in virus-inoculated tissues wasapprox. seven- and fourfold higher in the acid soluble andinsoluble fractions, respectively, than in controls (Fig. 5b).Labelled free putrescine recovery was higher in virus-inoculated (fourfold at 6 h and 10-fold at 72 h) than incontrol samples (Fig. 5a,b); putrescine incorporation into sper-midine and spermine (even though endogenous levels of thelatter were detectable only in trace amounts) was also higherin TMV-inoculated than in control samples (1.5-fold at 6 h,and ninefold and fourfold at 72 h for spermidine and sper-mine, respectively). Most of the label (approx. 70%) of thebenzene fraction was represented by polyamines. Probablydue to dilution exerted by endogenous conjugated putrescine,that exceeded 17-fold the free form, only trace amounts oflabel were found in conjugated forms.

Discussion

Present results show that in NN tobacco plants, differentlyfrom nn plants, the response to TMV implies a substantial

Fig. 3 Arginine decarboxylase (ADC) transcript levels and activity and ODC activity in control (C, closed columns), mock-inoculated (M, open columns) and TMV-inoculated (V, hatched columns) leaves from hypersensitive tobacco plants at 24 and 72 h from inoculation. (a) Upper panel: northern analysis of ADC mRNA. Each track was loaded with 15 µg total RNA and after size-fractionation transferred to a nitrocellulose membrane and hybridized with 32P-labelled PCR-derived fragment of tobacco ADC. Middle panel: RNA loading control; the gel was stained with ethidium bromide. Lower panel: densitometric analysis of respective band intensities normalized to ethidium bromide-stained loading control. (b) ADC activity. (c) ODC activity. Values are the mean ± SD (n = 6). Asterisks indicate significant differences (**, P < 0.01) with respect to controls.

Fig. 4 Free putrescine (Pu, closed columns) and spermidine (Sd, hatched columns) levels in control (C), mock-inoculated (M) and TMV-inoculated (V) leaves from hypersensitive (a) and susceptible (b) tobacco plants 72 h after inoculation. Values are the mean ± SD (n = 6).


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increase in polyamine turnover: SAMDC activity and geneexpression sensibly increase and a two-fold rise in DAOactivity occurs, in addition to stimulation of ADC andODC. These events were detectable in TMV-inoculated leavesstarting from 24 h while labelled putrescine incorporationinto spermidine and spermine occurred earlier (6 h). Thesefindings fit with previous results (Rabiti et al., 1998), indicat-ing enhanced free putrescine and spermidine accumulation(5 h) and conjugation (24 h) following TMV inoculation inNN plants. In excised HR lesions the first changes in poly-amine metabolism occurred at 3 d, but the TMV-inoculumwas 20-fold lower (Torrigiani et al., 1997). On the contrary,in nn plants no changes were detected at any time confirm-ing that they may be ‘genetically’ incapable of reacting tovirus inoculation in terms of polyamine metabolism.

With the experimental protocol adopted here it is alsoshown that the only effect of mechanical stress imposed bycarborundum was a transient SAMDC transcript accumula-tion in mock-inoculated NN plants relative to controls. Thismay indicate that hypersensitive plants promptly recoverfrom a modest abiotic stress.

DAO activity, which is as crucial as the biosynthetic activ-ities in controlling cellular (Scoccianti et al., 1991) as well as

extracellular putrescine levels (Federico & Angelini, 1991),shows here an affinity for putrescine comparable with thatpreviously reported in tobacco (Walton & McLauchlan,1990). The ineffectiveness of sonication on the recovery ofenzyme activity suggests that DAO enzyme is not completelyreleased from the cell wall where it is probably tightly bound(Federico & Angelini, 1991). The increase in soluble and com-partmented DAO activity after 72 h in TMV-inoculatedNN plants may be explained on the basis of the processes,occurring at the cell wall level, such as polysaccharide-proteincross-linkings and lignification (Pellegrini et al., 1994), whichrequire hydrogen peroxide and are involved in the establish-ment of the HR. DAO activity, in fact, has been reported toincrease in the cell wall in response to wound stress (Scaletet al., 1991), and to fungal infection in chick-pea (Angeliniet al., 1993). Such increases are spatially and temporallycorrelated to that of peroxidase activities which use as sub-strate the hydrogen peroxide resulting from diamine oxidation(Møller & McPherson, 1998). By producing hydrogenperoxide, polyamine oxidation also induces the formation ofreactive oxygen species (ROS; Allan & Fluhr, 1997); hydrogenperoxide and ROS are key signalling factors in programmedcell death (PCD; Mittler et al., 1997), have a direct anti-microbial effect (Peng & Kuc, 1992) and contribute to protein-polysaccharide cross-linkings (Yang et al., 1997). In fact, DAOactivity, with the consequent reduction in polyamine titres,has been reported to initiate PCD-driven developmental pat-terns in Arabidopsis (Møller & McPherson, 1998). In animalcells ODC over-expression (Poulin et al., 1995) but also poly-amine depletion by analogues (Hu & Pegg, 1997) induceapoptosis, a form of PCD. Probably, the two findings arenot in contradiction and simply confirm the contention thatextremely regulated endogenous levels of polyamines are neededin all physiological processes; beyond these levels, cells prefer tocommit ‘suicide’ rather than repair the damage. In the tobacco–TMV interaction, despite enhanced DAO activity, free put-rescine prevails throughout (Torrigiani et al., 1997; Rabitiet al., 1998, present data), and thus increasing levels of freeand conjugated putrescine and spermidine are associatedwith PCD.

In virus-inoculated hypersensitive plants the about five-fold increase in SAMDC activity at 72 h, which is precededby transcript accumulation at 24 h, suggests that regulationis primarily at the transcriptional level. SAMDC activity, infact, appears to be regulated both developmentally (Tayloret al., 1992; Lee et al., 1997) and post-translationally bycleavage of the proenzyme (Xiong et al., 1997). SAMDCtranscript accumulation has been reported to be modulatedboth in dividing and differentiating tissues (Taylor et al.,1992; Lee et al., 1997; Scaramagli et al., 1999b) and inresponse to exogenous stimuli such as growth regulators orpolyamine biosynthesis inhibitors (Scaramagli et al., 1999a).Moreover, previous studies report a twofold increase inSAMDC activity in Chinese cabbage infected with turnip

Fig. 5 Label recovery in different fractions from control (C) and TMV-inoculated (V) leaves from hypersensitive tobacco after 6 (a) and 72 h (b) from [3H]putrescine administration. Sn, TCA-supernatant (open columns); Pt, TCA-pellet (shaded columns); B, benzene phase (hatched columns); Pu, putrescine (horizontal hatched columns); Sd, spermidine (dotted columns); Sm, spermine (closed columns). Values are the means ± SD (n = 6).


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yellow mosaic virus (Cohen et al., 1981), enhanced spermidinesynthase activity and incorporation of the newly synthesisedspermidine into the virus particles (Cohen, 1998). The risein SAMDC gene expression and activity leads to decarboxy-lated S-adenosylmethionine (decaSAM) accumulation and thismight stimulate spermine/spermidine synthase activities, sincethey depend upon substrate availability (Heby & Persson,1990). At 72 h, however, under the present experimentalconditions, free spermidine titres increased only slightly invirus-inoculated samples. By contrast, conjugated sper-midine levels markedly increased (c. 10-fold), thus, depletingthe free form; spermidine conjugation probably accountsfor the modest free amine accumulation and the enhancedSAMDC activity. In fact, polyamine depletion by means ofbiosynthetic inhibitors has been reported to enhance therespective biosynthetic enzyme activity both in plant andanimal cells (Hiatt et al., 1986; Heby & Persson, 1990).Another possible explanation for the lack of free spermidineaccumulation is that it was oxidized. In legumes, in fact, theamine is also a substrate for DAO, although with a loweraffinity than putrescine (Federico & Angelini, 1991). Sper-midine oxidation has also been reported in cultured eggplanttissues (Scoccianti et al., 2000).

The observed rise in ADC and ODC activities can accountfor putrescine accumulation at 72 h in virus-inoculatedsamples. The latter results from the balance between synthesis,oxidation, conjugation and conversion to higher polyamines,with conjugation probably being the most active process. Bothenzyme activities are regulated post-translationally (Cohen,1998), and both respond to TMV but not to mechanicalstress. In the present model system no cell division or elonga-tion occur, but cell death and wall lignification do, processeswhich nevertheless require active cell metabolism. ADC couldbe implicated in the stress response (Flores, 1990) whileODC in hydroxycinnamoyl amide (HCA) synthesis (Burtinet al., 1989).

However, there is no positive relationship between thepreviously reported increase in free putrescine and spermidinelevels at 5–24 h, and biosynthetic activities at these times(Rabiti et al., 1998 and present data). Since labelling experi-ments confirm early spermidine and spermine synthesis, weshould consider the following: the enzyme activity assay isperformed in vitro in a cell-free system while polyaminelevels are measured in vivo; SAMDC does not directly leadto higher amine synthesis; endogenous decaSAM might besufficient to support synthase activities for the first 24 h; and,most probably, transport of polyamines, which has been welldocumented in plants (Antognoni et al., 1998), could occurtowards the injured leaf or the incipient HR lesion. In fact,labelling experiments also show that TMV-inoculated NNleaves retain more free putrescine (10-fold at 3 d) thancontrols indicating a kind of ‘sink’ effect for putrescine, inagreement with the fact that PCD, as well as HR, requiresactive metabolism (He et al., 1993).

In conclusion, in NN plants but not in nn plants, followingTMV-inoculation, SAMDC gene expression and activity aswell as DAO activity are upregulated and polyamine bio-synthesis is detectable well before changes in biosyntheticenzyme activities. This leads to changes in cellular com-ponents, such as polyamines and hydrogen peroxide, whichinduce PCD. Further work is needed in order to establishwhether polyamine transport occurs towards the HR lesionand which is the relative contribution of free and conjugatedpolyamines in the establishment of the HR.

Acknowledgements

We gratefully acknowledge Anthony J. Michael, Instituteof Food Research, Norwich (UK) for kindly supplying thetobacco SAMDC, ADC and ODC probes. Funds are fromthe University of Bologna, Special Project Molecular Sig-nalling in Differentiation (P. T.) and ex-60% MURST(L. B. and P. T.).

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Polyamine metabolism is upregulated in response to tobacco mosaic virus in hypersensitive, but not in susceptible, tobacco