Ozone-response mechanisms in tobacco: implications
of polyamine metabolism
Marianne Louise van Buuren
1
, Lucia Guidi
2
, Silvia Fornalè
1
, Francesca Ghetti
1
, Marina Franceschetti
1
, Gian Franco Soldatini
2
and Nello Bagni
1
1
Dipartimento di Biologia Evoluzionistica Sperimentale e Centro Interdipartimentale per le Biotecnologie, Università di Bologna, via Irnerio 42, 40126
Bologna, Italy;
2
Dipartimento di Chimica e Biotecnologie Agrarie, Università di Pisa, 56124 Pisa, Italy
Summary
• Polyamines have been suggested to counteract oxidative damage in plants. Here,we present a detailed analysis of polyamine accumulation and its relationship tophotosynthetic parameters in two tobacco (
Nicotiana tabacum
) cultivars (ozone-sensitive Bel W3 and ozone-tolerant Bel B) after a single ozone pulse and after a 1-month exposure in the open air.• Free putrescine accumulated in undamaged tissue of both cultivars, whereasputrescine conjugated to soluble and cell-wall bound components accumulated pre-dominantly in tissue undergoing cell death in Bel W3 plants. Accumulation wascaused by a redirection of the conjugation pathway, as well as by a transient increasein arginine decarboxylase and ornithine decarboxylase specific activity. This increaseseemed to be regulated at post-transcriptional level.• Measurements of chlorophyll content and fluorescence showed that, in additionto visible necrotic lesions, Bel W3 plants suffered considerable photosynthetic dam-age in other parts of the leaf.• Accumulation of conjugated putrescine is part of the ozone-induced programmedcell death response in Bel W3 plants. Ozone-induced synthesis of free putrescine isnot correlated with ozone-resistance in Bel B plants, which are apparently impairedin signal transduction pathways that are necessary to control the cellular redox state.However, Bel B plants are able to perceive ozone stress and to induce a series ofdefense mechanisms without activating hypersensitive cell death.
Ozone in the troposphere is formed by the interaction ofnitrogen oxides, hydrocarbons and UV-radiation and isconsidered the most phytotoxic among the major gaseouspollutants (Heagle, 1989). The effects of ozone on vegetationinclude acute damage, leading to cell death, as well as chronicdamage like inhibition of growth, reduction of CO
2
assimilation and premature senescence (Sandermann, 1996;Schraudner
et al
., 1997; Miller
et al
., 1999). It is widelyreported that different cultivars of plants vary greatly in theirsusceptibility to ozone (Heggestad, 1991). In many casessusceptibility is quantified by the extension of visual damage
as a result of cell death. However, reduction in plant growthand productivity is mainly caused by long-term ozoneexposure at lower than critical concentrations (chronicexposure) that induces changes in biochemical andphysiological processes in the absence of visual damage(Heath & Taylor, 1997; Pell
et al
., 1997).In
Arabidopsis thaliana
, ozone-induced cell death seems tooccur via two distinct mechanisms (Rao & Davis, 1999).Plants that failed to accumulate salicylic acid (SA) upon ozoneexposure were unable to activate antioxidant defenseresponses and displayed toxic cell death. On the other hand,in mutant plants in which ozone induces hyperaccumulationof SA, ozone exposure leads to an increased production of
NPH_539.fm Page 389 Thursday, November 7, 2002 4:29 PM
reactive oxygen species (ROS) which triggers a programmedcell death pathway similar to the hypersensitive response(HR) observed in incompatible plant–pathogen interactions.In fact, many typical plant responses to pathogen infectionlike an oxidative burst, SA and ethylene biosynthesis, cell wallmodifications or the accumulation of phenolic compoundsand several pathogenesis-related (PR) proteins, are elicited byozone, suggesting that pathogen attack and ozone exposureactivate the same or overlapping signal transduction pathways(Sandermann
et al
., 1998; Rao
et al
., 2000).Polyamines have received considerable interest as possible
protectants against ozone stress. The polyamines putrescine,spermidine and spermine are ubiquitous in all eukaryotic cellsand essential for normal growth and development (Slocum &Flores, 1991). In plants, polyamines are proposed to play animportant role in embryogenesis, senescence, flowering, aswell as in defense responses to abiotic and biotic stress (Bagni,1989). Studies on ozone-sensitive and -tolerant
Nicotianatabacum
(tobacco) cultivars have shown a rapid increase ofputrescine, both free and conjugated to hydroxycinnamicacids, in the ozone tolerant cultivar Bel B (Langebartels
et al
.,1991). In the hypersensitive cultivar Bel W3, however, only asmall increase was observed at a later stage when necroticlesions have already been formed. It was suggested thatpolyamines might protect the plant from cellular oxidativestress by inhibition of membrane lipid peroxidation (Kidata
et al
., 1979) or playing as radical scavengers in the form ofhydroxycinnamic acid conjugates (Bors
et al
., 1989). Thishypothesis is supported by the observation that feeding exog-enous polyamines to tomato and tobacco plants resulted in asignificant suppression of ozone-induced leaf injury. Further-more, it has been demonstrated that putrescine conjugatedto caffeic, ferulic or
ρ
-coumaric acid has a high capacity forradical scavenging (Bors
et al
., 1989). Conjugated polyaminesmight also contribute to the modulation of AOS levels in thecell, an important factor in regulating hypersensitive celldeath (Rao
et al
., 2000).Two alternative pathways for putrescine synthesis exist in
plants: from ornithine by ornithine decarboxylase (ODC) orfrom arginine by arginine decarboxylase (ADC) through theintermediates agmatine and N-carbamoylputrescine (Bagni& Tassoni, 2001). Localization experiments in
Avena
havedemonstrated that ADC is associated with the thylakoidmembrane of the chloroplast (Borrell
et al
., 1995). In addi-tion, it has been shown that polyamines protect against chlo-rophyll degradation (Tassoni
et al
., 2000) and that polyamineconjugation by transglutaminase activity seems to have animportant role in protecting thylakoid and stromatal proteinsof antenna complexes, thereby preserving photosyntheticefficiency (Serafini-Fracassini
et al
., 1995). ADC activity wasreported to increase transiently in ozone-treated tobaccocultivar Bel B (ozone-tolerant) and this increase preceded theaccumulation of free and conjugated putrescine (Langebartels
et al
., 1991). The activity of ODC, however, remained
unchanged. cDNA corresponding to ADC and ODCgenes have been cloned from several plant species (Bell &Malmberg, 1990; Rastogi
et al
., 1993; Perez-Amador
et al
., 1995;Chang
et al
., 1996; Michael
et al
., 1996; Primikirios &Roubelakis-Angelakis, 1999); however, the expression ofthese genes during ozone stress has not been reported.
In this study, we present a detailed analysis of polyamineaccumulation and their relationship with photosyntheticparameters in tobacco cultivars Bel B and Bel W3 after a singleozone pulse and after a long exposure in open air for 1 month.Our results indicate that accumulation of conjugatedputrescine is part of the ozone-induced programmed celldeath response in Bel W3 plants and that ozone-induced syn-thesis of free putrescine is not correlated to ozone-resistance inBel B plants. Furthermore,
in vivo
measurements of chloro-phyll a fluorescence and chlorophyll content showed thatexposing Bel W3 plants to ozone causes, in addition to visiblenecrotic lesions, considerable photosynthetic damage in otherparts of the leaf.
Materials and Methods
Plant growth conditions
Tobacco plants cv. Bel W3 and Bel B were grown in a growthchamber with pollutant-free air under a 16-h photoperiod at20
°
C with 50–70% rh and 320 µmol photons m
−
2
s
−
1
.Experimental plants were approx. 2 months old and had sevento eight leaves. Leaves 4 and 5, counted from the bottom,were randomly collected from at least three different plantsand used for molecular and biochemical analyses. These leaveshad reached full expansion and consistently developed lesionsafter ozone exposure in the BelW3 cultivar. Plants utilized forexposure to tropospheric ozone in the urban area of Bolognawere cultivated in the same conditions as above described.
Ozone exposure and evaluation of visible injury
Plants were transferred to a growth chamber modified forozone fumigation and were acclimatized for 24 h before ozonetreatment. Growth chamber conditions were 25
°
C, 80% rh,350 ppm CO
2
, with a 14-h photoperiod averaging 350 µmolphotons m
−
2
s
−
1
at the top of the canopy. The plants were thentreated with a single pulse of ozone (130
±
22 nl l
−
1
for 7 h)or maintained in charcoal filtered air. Ozone was generatedwith an ozone generator Fisher (Mod. 500, Meckenheim,Germany) and the ozone concentration inside the chamberwas monitored continuously with a Monitor LaboratoriesAnalyser connected to a PC (Mod. 8810, Englewood, NJ,USA). For long-term exposure, plants were exposed for 1 month(September–October, 1996) to urban tropospheric ozonewhich resulted in a mean ozone concentration of 26 nl l
−
1
with a minimum of 2 nl l
−
1
and a maximum of 102 nl l
−
1
(Della Mea
et al
., 1998). The appearance of visible injury was
NPH_539.fm Page 390 Thursday, November 7, 2002 4:29 PM
evaluated at the end of the ozone exposure from the onset oflesion development. Each leaf was rated from 0 to 100 inincrements of 10, correlating with the percentage of visibleinjury covering the leaf. The ratings for all leaves on each plantwere summed to give one visible injury index for each plant.
Chlorophyll
a
fluorescence measurement
Chlorophyll
a
fluorescence analyses were carried out atthe end of the fumigation (time 0) and 24 h after ozonefumigation (time 24) using a modulated fluorometer(PAM2000, Heinz Walz, Effeltrich, Germany). Plants weredark-adapted for 40 min and the measurements were carriedout at room temperature. The minimal fluorescence yield (
F
0
)was obtained upon excitation with a weak measuring beamfrom a pulse light-emitting diode. Maximal fluorescence yield(
F
m
) was determined after exposure to a 600-ms saturationpulse of white light to close all reaction centres. Variablefluorescence (
F
v
) was calculated as difference between
F
m
and
F
0
and the maximal apparent efficiency of PSII (
F
v
/
F
m
)was measured. Subsequently, actinic white light (approx.300 µmol m
−
2
s
−
1
) was switched on and saturating pulseswere applied automatically at 60 s intervals for periodicdetermination of maximal fluorescence yield during actinicillumination (
F
m
′
), the level of modulated fluorescence duringa brief interruption of actinic illumination in the presence offar-red light (
F
0
′
) and the Chl fluorescence yield duringactinic illumination (
F
t
). Nonphotochemical fluorescencequenching (NPQ) was estimated from the Stern-Volmerparameter, that represents a relative measurement of thermaldissipation at the PSII level, calculated according to theequation NPQ =
F
m
/
F
m
′
−
1 (Bilger & Bjorkman, 1991).The coefficient of photochemical quenching, q
P
, was calculatedas (
F
m
′
–
F
t
)/(
F
m
′
–
F
0
′
) (Schreiber
et al
., 1986). Excitationpressure on PSII reflects the proportion of the primary stablequinone acceptor Q
A
in the reduced state; it is calculated as1 – q
P
. The quantum efficiency of excitation capture by oxidizedreaction centres of PSII was calculated from the equation
Φ
exc
= (
F
m
′
–
F
0
′
)/
F
m
′
and the quantum efficiency of PSIIphotochemistry,
Φ
PSII
, was estimated from (
F
m
′
–
F
t
)/
F
m
′
(Genty
et al
., 1989). All measurements were repeated fivetimes. For comparison of the means, the ANOVA followed bythe least significant difference (LSD) test was used.
Polyamine analysis by HPLC
Approximately 0.2 g f. wt of tobacco tissue were extracted in10 volumes of 4% (w/v) cold perchloric acid (PCA) andcentrifuged at 20 000
g
for 30 min at 4
°
C. The pellet wasresuspended in the original volume of PCA. Triplicates of thissuspension and of the supernatant were hydrolyzed with 6 NHCl in flame-sealed vials at 110
°
C for 20 h in order to releasepolyamines from their conjugates. Aliquots (0.2 ml) ofsupernatant (free polyamines), hydrolyzed supernatant
(soluble conjugated polyamines) and hydrolyzed pellet(insoluble conjugated polyamines) were dansylated accordingto Smith & Davies (1985) with minor modifications anddansyl-polyamines being extracted with toluene. Standardpolyamines were subjected to the same procedure. Dansyl-polyamines were analyzed by HPLC ( Jasco, Großumstad,Germany) with a reverse phase C18 column (SpherisorbODS2, 5 µm particle size, 4.6
×
250 mm, Phase Sep,1 ml min
−
1
flow rate) as described by Torrigiani
et al
. (1995)but using the following modified gradient: 0 min acetonitrile/water (50/50 v/v), 2 min acetonitrile/water (70/30 v/v),7 min acetonitrile/water (75/25 v/v), 12 min acetonitrile/water (100/0 v/v), 15 min acetonitrile/water (50/50 v/v).
ADC and ODC enzyme activities
The activities of ADC and ODC were determined by aradiochemical method as described by Tassoni
et al
. (2000).In preliminary assays the optimum pH was determined forboth enzyme activities. Tobacco tissues (0.4 g f. wt) weremacerated in an ice-cold mortar with five volumes of the assaybuffer (100 mM Tris-HCl pH 8.5, 50 µM pyridoxalphosphate) and centrifuged at 20 000
g
for 30 min at 4
°
C.0.2 ml of both supernatant and resuspended pellet were usedto determine (0.5 ml final assay volume) ADC and ODCactivities. The assays were performed by measuring the
14
CO
2
evolution from 7.4 kBq (approx. 1.3 µM) of L-[U-
14
C]-arginine (specific activity 11.7 GBq mmol
−
1
, Amersham, UK)or from 7.4 kBq (about 7.3 µM) of D,L-[1–
14
C]-ornithine(specific activity 2.11 GBq mmol
−
1
, Amersham, UK).10 mM unlabelled ornithine and 10 mM unlabelled argininewere added to the enzyme assay for ADC and ODC,respectively, to verify the arginase and ornithine trans-carbamoylase activities of the samples. Protein content wasmeasured using the method of Lowry
et al
. (1951) and bovineserum albumine as standard.
RNA extraction and northern blot analysis
RNA was extracted from approx. 200 mg f. wt of leaftissue using the RNA FAST kit (Molecular System, SanDiego, CA, USA). Total RNA 15 µg was separated byelectrophoresis in formaldehyde gels and blotted toHybond
TM
nylon membrane (Amersham, Little Chalfont,UK) by standard methods (Sambrook
et al
., 1989).
32
P-CTPlabeled probes were prepared from inserts of clones by oligo-labelling (Ready-To-Go DNA labelling Beads, PharmaciaBiotech, Uppsala, Sweden) and hybridized to blots in20 mM Pipes (pH 6.8), 0.6 M NaCl, 4 mM EDTA,0.2% (w/v) gelatin, 0.2% (w/v) Ficoll 400, 0.2% (w/v)polyvenylpyrrolidone, 1% SDS, 0.5% sodium pyroph-osphate containing 500 µg sheared salmon sperm or herringtestis DNA at 65°C. Hybridized membranes were washedtwice in 2 × SSC, 0.5% (w/v) SDS and twice in 0.5 × SSC,
NPH_539.fm Page 391 Thursday, November 7, 2002 4:29 PM
0.5% (w/v) SDS at 65°C for 15 min each. Membranes weresubsequently exposed to KODAK X-OMAT film at −70°Cfor 3–5 days as well as to phosphor imager screens (MolecularDynamics, Sunnyvale, CA, USA). Quantitative analysis ofhybridizing bands was performed using Quantity Onesoftware (Bio-Rad, Hercules, CA, USA) and the valueobtained for each band was corrected for loading differencesby values obtained by rehybridizing blots with a 18Sribosomal gene probe from Medicago truncatula.
Results
Lesion development
Ozone-sensitive Bel W3 tobacco plants displayed visiblesymptoms of injury typical of acute ozone exposure at 10–24 hafter the start of a single 7 h ozone exposure at 120–150 nl l−1.The surfaces of middle-aged leaves were covered from 20 to50% with necrotic lesions on the following day, while youngerleaves showed less injury (0–10% of leaf area). Bel B plantsdid not show any visible symptoms of damage.
After 1 month exposure to urban tropospheric ozone, 20–50% of the leaf surface had developed necrotic lesions in theozone-sensitive Bel W3 cultivar, while no visible damagecould be observed in Bel B plants.
Chlorophyll a fluorescence in O3-treated leaves
Before the ozone treatment the two tobacco cultivarsexhibited different patterns of photosynthetic parameters.The strongest difference between the two cultivars main-tained in filtered air was the almost two times highervalue of NPQ measured in Bel B plants (Table 1). The valuesof Fv : Fm ratio were 0.777 and 0.781 for Bel W3 and Bel Bcultivars, respectively, which were considerably lower thanthose reported by Bjorkman & Demmig (1987) fordicotyledonous species measured at 77K (0.843).
In Bel W3 plants, ground fluorescence (F0), maximal fluo-rescence (Fm) and Fv : Fm ratio did not change when measured
at the end of fumigation (time 0). The parameters derivedfrom the quenching analysis also remained unchanged withthe exception of the NPQ, which strongly increased com-pared with untreated plants. When the measurements werecarried out 24 h after fumigation (time 24 h) significant dif-ferences in some parameters of fluorescence were detected.Indeed, the Fm value and the Fv : Fm ratio strongly decreasedin ozone-treated plants. In addition, the state of reduction ofthe primary acceptor of PSII, QA, as indicated by (1 – qP), aswell as the nonphotochemical quenching coefficient increasedsignificantly. A strong decrease in the quantum yield of PSII(ΦPSII) and the efficiency of excitation capture (Φexc) wasmeasured.
In the Bel B cultivar a decrease in Fv : Fm ratio was detectedin ozone-treated leaves at the end fumigation (time 0). Thisdecrease was caused by the increased values of F0. However,at 24 h after fumigation (time 24 h) the Fv : Fm ratio hasrecovered to about control levels (Table 1). The reduction stateof the QA and the ΦPSII did not change in ozone-treatedBel B plants during the entire period. An increase in NPQwas observed in this cultivar at time 0 but at time 24 h NPQ waslower compared with control plants. A similar pattern wasobserved also for the Φexc.
Accumulation of polyamines
Ozone-treated Bel W3 plants showed a 4.5 fold increase infree putrescine content compared with control plantsimmediately after the end of ozone exposure (Fig. 1b) whilethe level of conjugated putrescine in both the soluble andinsoluble fraction was lower with respect to controls. At thistime point the levels of free and conjugated putrescine werenot significantly altered in ozone-treated Bel B plants(Fig. 1a). At 24 h after the end of ozone treatment, Bel B andBel W3 cultivars contained 2.5 and 3.3 fold, respectively,higher levels of free putrescine in plant exposed to ozonecompared with control plants (Fig. 1c,d). Ozone-treatedBelW3 plants also contained higher levels of soluble andinsoluble conjugated putrescine compared with controls
Bel W3 F0 Fm Fv/Fm 1 – qP NPQ ΦPSII Φexc
Control 92a 416a 0.777a 0.195b 0.353c 0.580a 0.719a
O3 time 0 105a 441a 0.761a 0.198b 0.557b 0.512a 0.636a
O3 time 24 h 94a 270b 0.624b 0.652a 0.891a 0.231b 0.333b
O3 time 0 105a 466a 0.774c 0.290a 1.002a 0.427a 0.602c
O3 time 24 h 96b 477a 0.792a 0.283a 0.505c 0.497a 0.694a
Results are presented as mean for five replicates. All measurements were done on fully expanded leaves. For each parameter and for each cultivar, means flanked by the same letters are not significantly different (P = 0.05) following the ANOVA test. Nonphotochemical fluorescence quenching (NPQ).
Table 1 Chlorophyll a fluorescence parameters determined on Bel W3 and Bel B tobacco cultivars before (control), at the end (time 0) and 24 h (time 24 h) after ozone fumigation
NPH_539.fm Page 392 Thursday, November 7, 2002 4:29 PM
whereas no differences were found between control andozone-treated Bel B plants. The levels of the other poly-amines, spermidine, spermine and 1,3-diamino-propane,a product of polyamine oxidation as well as spermidinebreakdown through acetylation (Bagni & Tassoni, 2001),were also measured but except for a slight increase in 1,3-diamino-propane in ozone-treated Bel W3 plants no signi-ficant changes were found (data not shown).
To determine the distribution of polyamines within ozone-damaged leaves, different leaf areas were collected from2.5 months Bel W3 plants that had been exposed for 1 monthat urban tropospheric ozone and the results compared withBel B plants exposed to the same conditions. During thisperiod (September–October, 1996) the mean ozone concen-tration was 26 nl l−1 with a minimum of 2 nl l−1 and amaximum of 102 nl l−1 (Della Mea et al., 1998).
Before exposure to urban tropospheric ozone the leafpolyamine content displayed a different pattern in the twocultivars. The total polyamine content was higher in theozone-sensitive Bel W3 cultivar compared with the Bel Bcultivar (Fig. 2a,b). After 1 month exposure to urban tropo-spheric ozone, there was an increase in the concentration ofdiamino-propane (data not shown), putrescine and spermi-dine in both cultivars due to growth increment (Figs 3 and 4),whereas the spermine content did not show significantchanges (data not shown). In Bel B plants (Fig. 3), very fewconjugated polyamines were present in the insoluble fraction(8–10 nmol g−1 f. wt for putrescine and spermidine and about1 nmol g−1 f. wt for spermine). On the other hand, freepolyamine content was of the same order of magnitude asin Bel W3 plants. In Bel W3 plants, conjugated putrescinehad increased about four times after exposure to urbantropospheric ozone within the necrotic lesions (Fig. 4a). The
concentration of free putrescine and spermidine (Fig. 4a,b),on the other hand, was low in necrotic tissue and increasedmoving towards healthy, undamaged tissue.
Activity of polyamine biosynthetic enzymes
ADC and ODC activities were determined in both solubleand particulate leaf fractions of control and ozone-treated
Fig. 1 Putrescine content in leaves of Bel B and Bel W3 tobacco plants at time 0 h and time 24 h after ozone exposure (open columns, filtered air; closed columns, ozone). The assay was repeated on three sets of plants with analogous results; results presented are representative of one experiment. Values are the mean ± SD of three different determinations.
Fig. 2 Polyamine content in leaves of (a) Bel B and (b) Bel W3 tobacco plants before the start of urban tropospheric ozone exposure. Data are the mean ± SD of three different determinations. (black columns, putrescine; open columns, spermidine; grey columns, spermine)
Fig. 3 Polyamine content in leaves of Bel B plants after 1 month of urban tropospheric ozone exposure. Data are the mean ± SD of three different determinations. (open columns, putrescine; closed columns, spermidine)
NPH_539.fm Page 393 Thursday, November 7, 2002 4:29 PM
plants (Fig. 5). The results are expressed as pmol g−1 f. wt butsimilar results were obtained when data were expressed on amg protein basis. Arginase and ornithine transcarbamoylaseactivities in the soluble fraction were 36% and 29%, respectively.In the particulate fraction ornithine transcarbamoylase
activity accounted for 23% of the ODC activity measuredwhile no arginase activity could be recovered. The values inFig. 5 were corrected accordingly. ODC and ADC weredetermined using the substrates in trace amount in order toevaluate the physiological activities. ODC was the majorenzyme for putrescine biosynthesis in both cultivars and itsactivity was approx. five- and 8–10-fold higher with respect toADC in Bel B and Bel W3 plants, respectively. Most activityof each enzyme was concentrated in the soluble fractionexcept in ozone-treated Bel W3 plants at 24 h (Fig. 5c,d).
Immediately after the end of ozone treatment (time 0), solubleADC activity was approx. 50% higher in ozone-exposed plantscompared with controls in both Bel B and Bel W3 cultivars(Fig. 5a). At 24 h, however, the levels in ozone treated plants haddropped to values similar or inferior to those of control plants(Fig. 5c). A very similar trend was observed for ODC activityfollowing ozone treatment in both cultivars (Fig. 5b,d).
Expression of polyamine biosynthetic genes and pathogenesis related protein PR1
The mRNA level of the putrescine biosynthetic enzyme ADCwas measured by Northern blot analysis (Fig. 6). In both BelB and Bel W3 cultivars the ADC transcript level was lower inozone-exposed plants compared with control plants at time 0while at time 24 h a small increase could be observed. Analmost threefold reduction in ADC transcript level was foundin Bel B and Bel W3 control plants at time 24 h comparedwith time 0. Analysis of PR1 gene expression showed a four–fivefold induction right after ozone treatment in both Bel Band Bel W3 plants although the mRNA level was approx.threefold higher in Bel W3 plants. At 24 h after ozonetreatment PR1 mRNA levels had further increased and
Fig. 4 (a) Putrescine and (b) spermidine content in different parts (healthy, around necrotic and necrotic) of Bel W3 tobacco leaves exposed for 1 month to urban tropospheric ozone. Values are the mean of three different determinations. SD (< 5%) was omitted.
Fig. 5 Arginine decarboxylase (ADC) (a, c) and ornithine decarboxylase (ODC) (b, d) enzyme activities in leaves of ozone-treated Bel B and Bel W3 tobacco plants at time 0 h (a, b) and time 24 h (c, d) after ozone exposure. (Open columns, soluble fraction; closed columns, particulate fraction). Values are the mean ± SD of three different determinations.
NPH_539.fm Page 394 Thursday, November 7, 2002 4:29 PM
showed a more than 10-fold induction in both cultivars.Expression of the ODC gene was too low to be detected byNorthern blot analysis (data not shown).
Discussion
Effect of ozone on polyamine metabolism in Bel B and Bel W3 tobacco cultivars
Induction of polyamine biosynthesis has been described asa major physiological switch in the development of ozonetolerance in the Bel B tobacco cultivar (Langebartels et al.,1991). To study the effect of ozone on putrescine accumula-tion, we analyzed the enzyme activity and mRNA accumula-tion of its biosynthetic enzymes ADC and ODC.
Putrescine was the main polyamine accumulating in leavesof ozone-treated Bel B and Bel W3 tobacco cultivars. Whilein Bel W3 plants free putrescine levels increased immediatelyafter ozone treatment (time 0), no changes occurred in Bel B.Both ADC and ODC activities increased in ozone-treated BelB plants at this timepoint, anticipating the putrescine accu-mulation observed at 24 h. In ozone-treated Bel W3 plantsthe increase in free putrescine coincides with a decrease inboth soluble and insoluble putrescine conjugates, suggestinga redirection of the conjugation processes. At 24 h after ozonetreatment, Bel B and Bel W3 showed a similar increase in freeputrescine in comparison to control plants while ADC andODC enzyme activities had dropped to below control levelsindicating that de novo synthesis of putrescine occurred withinthe 24 h following the end of ozone fumigation.
The mRNA levels of the ADC and ODC genes could notbe correlated with their enzyme activities. However, becauseADC and ODC assays were determined at non-saturatingconditions a change in enzyme activity does not necessarilyreflect a change in protein level. In addition, a transientincrease in mRNA levels might have occurred before time 0.ADC seems to be mainly regulated at the post-transcriptionallevel in tobacco (Burtin & Michael, 1997) as well as inseveral other plant species (Rastogi et al., 1993; Watson &Malmberg, 1996; Primikirios & Roubelakis-Angelakis, 2001).Our mRNA data are consistent with a post-transcriptionalregulation of ADC during ozone stress in tobacco. Similarly,the low ODC mRNA accumulation may be explained by thecomplicated and tight regulation of this enzyme, whichoccurs at multiple levels (Canellakis et al., 1981).
The accumulation of free and conjugated putrescine in theBel W3 cultivar is apparently in contrast with the dataobtained by Langebartels et al. (1991) even if care was takento use plants of the same age, to analyze the same leaf numbersand to use similar ozone dosage and exposure time. Part of thediscrepancy might be explained by our finding that freeputrescine accumulates mainly in undamaged tissues, whileconjugated putrescine is predominant in necrotic tissues ofBel W3 plants. Biochemical analysis by Langebartels et al.(1991) was carried out on leaf discs taken with a cork borerwhich is difficult when performed on fragile and chlorotictissue. Therefore, the tissue analyzed might have beenpredominantly taken from undamaged parts of the leaves,thereby underestimating the level of conjugated putrescine. Inaddition, insoluble conjugated polyamines (bound to the cell
Fig. 6 Arginine decarboxylase (ADC) and PR1 mRNA accumulation in ozone-treated Bel B and Bel W3 plants at time 0 h and time 24 h. The amount of hybridizing radioactivity was corrected for loading differences by subtracting values obtained with a 18S-rRNA probe and expressed as percentage of maximum hybridization. (open columns, nontreated plants; closed columns, ozone-treated plants)
NPH_539.fm Page 395 Thursday, November 7, 2002 4:29 PM
wall components), which constitute the majority of ozone-induced polyamines (Bagni et al., 2000), were not included intheir analysis.
Several lines of evidence indicate that ozone induces a HR-like programmed cell death in Bel W3 plants, for example, thenduction of a biphasic oxidative burst, the biosynthesis of SAand ethylene, the induction of pathogenesis-related and anti-oxidant genes (Yalpani et al., 1994; Sanderman et al., 1998;Schraudner et al., 1998; Langebartels et al., 1991 Ernst et al.,1992). According to our results, the distribution of free andconjugated putrescine in ozone-damaged Bel W3 leaves issimilar to that seen in tobacco mosaic virus (TMV) infectedtobacco leaves that display HR cell death (Torrigiani et al.,1997), confirming that ozone-induced lesions are similar, ifnot identical, to those induced by an incompatible plant–pathogen interaction.
Due to its scavenging capabilities, soluble conjugatedputrescine that accumulates in necrotic lesions and surround-ing zones might play a role in attenuating ROS productionthereby limiting lesion spread. Alternatively, conjugatedpolyamines could play a role in plant defense mechanismsas demonstrated for example by the specific accumulationof a phenolic conjugate, p-coumaroyl-hydroxyagmatine withantifungal activity in a broad spectrum resistance reactioncontrolled by the mlo resistance alleles in barley (vonRopenack et al., 1998). Free putrescine, on the other hand,accumulates equally in ozone-sensitive (Bel W3) and ozone-tolerant (Bel B) cultivars making it unlikely that putrescineplays a role in conferring tolerance in Bel B plants. However,free putrescine might be important in counteracting acceler-ated senescence during chronic ozone exposure by stabilizingchloroplast thylakoid membranes (Besford et al., 1993).
Effect of ozone on photosynthesis in Bel B and Bel W3
The cellular redox state plays a central role in the regulationof plant defense responses and in ozone-induced cell deathand is influenced by several interacting signal transductionpathways that involve ROS, SA, jasmonic acid and ethylene(Rao et al., 2000). A recent study on mastoparan-induced HRin isolated mesophyll cells suggested a complex relationshipbetween photosynthesis and cell death (Allen et al., 1999). Toelucidate the role of photosynthesis in ozone-induced celldeath we compared chlorophyll a fluorescence in ozone-sensitive Bel W3 and ozone-tolerant Bel B tobacco plants.
In the Bel W3 cultivar at the end of fumigation (time 0) noalteration in chlorophyll fluorescence parameters was visiblewith the exception of an increase in the NPQ value. However,24 h after the end of fumigation, the Fv : Fm ratio dramaticallydecreased because of a substantial decrease in the Fm value.The ΦPSII also underwent a significant decrease due essentiallyto the strong increase in 1 – qP parameter. Because 1 – qP is ameasure for the reduction state of the primary quinoneacceptor, these data indicate a less effective reoxidation of this
electron acceptor suggesting that a fraction of the PSII trapswas closed during actinic illumination. These closed traps,which are unable to undergo charge separation and to take partin linear photosynthetic electron transport, should lead todecreased quantum yield. The increase in NPQ value was notsufficient to eliminate the photosynthetic damage in thiscultivar. Allen et al. (1999) induced HR in isolated Asparagussprengeri using a G-protein mastoparan (MP). A large increasein NPQ of chl a fluorescence accompanied the initial stage ofthe oxidative burst while (1 – qP) did not change and a similarresponse was found in Bel W3 tobacco plants immediatelyafter the end of the O3 exposure. As the oxidative burst con-tinued, a large increase in (1 – qP) was observed like in Bel W3tobacco plants 24 h after the start of the fumigation. It shouldbe noted, that these measurements were done on cells under-going HR, whereas measurements on tobacco Bel W3 plantswere performed on parts of the leaf that did not show anyvisible damage.
The response of Bel B plants that did not show any visibledamage was different. Indeed, the decrease in Fv/Fm ratiomeasured in ozone-treated plants was due essentially to anincrease in F0 value at time 0, which is associated with photo-inhibitory damage. This may indicate an alteration of thereaction centres of PSII in energy utilization. On the otherhand, Φexc was also reduced by ozone and this reduction wascorrelated with a large increase in NPQ. The PSII reactioncentres were apparently not damaged but they were not ableto carry out electron transport. The decrease of Φexc indicatesalso an increased probability that a photon absorbed by thePSII antennae is being dissipated as heat and demonstrates theoccurrence of a stress-induced down-regulation of photo-synthesis. However, the fast recovery of the Fv : Fm ratio 24 hafter the end of the fumigation indicated that chronic photo-inhibition was not taking place. This is also shown by the factthat the ΦPSII and 1 – qP are not altered in ozonated plants incomparison to the controls. Together, these data are indicativeof an active mechanism of stress tolerance. Down-regulationof PSII reaction centres seems to dissipate excitation energywhen active centres are closed, and their slightly lower trap-ping efficiency is reflected in the increase in dark-adapted F0,which commonly precedes and is then sustained duringphotoinhibition (Critchley & Russel, 1994). It is important tounderline that Bel B plants showed a strong stomatal closureupon ozone exposure (data not shown, Heggestadt, 1991;Sandermann, 1996). Therefore, it is likely that photosynthesisin ozone-exposed plants was not limited by a decline in PSIIefficiency, this being rather a regulatory adjustment of PSIIefficiency to a decreased carbon availability. This determinesa minor demand for reducing equivalent (NADPH and ATP)in the Calvin cycle that, in turn, causes a diminution in theelectron transport rate. The complete recovery 24 h after theend of the fumigation is consistent with this hypothesis.
In conclusion, photosynthesis is affected differently byozone exposure in Bel B and Bel W3 plants. In ozone-tolerant
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Bel B a regulatory mechanism plays a key role in the ozone-response that includes stomata closure and a reduction inCO2 assimilation rate. This results in a decrease of PSIIphotochemistry efficiency that might be important in limitingoxidative stress. In addition, defense responses are induced asshown by the induction of putrescine biosynthesis and theexpression of PR1. Therefore, Bel B plants are able to perceiveozone stress and to induce a series of defense mechanismswithout activating hypersensitive cell death. Bel W3 plants,on the other hand, are apparently impaired in one or moresignal transduction pathways that are necessary to control thecellular redox state. In these plants ozone activates localizedcell death as well as photosynthetic damage in other parts ofthe leaf.
Acknowledgements
This work was partially supported by funds from theUniversity of Bologna for selected research topics, specialproject ‘Apoptosis’ (N.B.) and funds for Research TrainingGrant of European Commission (M.L.B., N.B.), and by thefinanced Project ‘Molecular mechanisms involved in plantresponses to ozone’ funds from MURST (Ministry ofUniversity and Scientific and Technological Research, G.F.S.).We thank Dr J. Ryals (formerly at Novartis) for providing thetobacco PR-1 clone, Dr A.J. Michael (Institute of FoodResearch, Norwich, UK) for providing the tobacco ADC andODC clones and Dr M.J. Harrison (The S.R. NobleFoundation, Ardmore, OK) for providing the 18S ribosomalclone from Medicago truncatula. Seeds of tobacco cultivars BelB and Bel W3 were kindly provided by Dr G. Lorenzini. We aregrateful to Mr G. Bugamelli for precious help in growing plants.
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