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• Polyamines have been suggested to counteract oxidative damage in plants. Here, we present a detailed analysis of polyamine accumulation and its relationship to photosynthetic 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, whereas putrescine conjugated to soluble and cell-wall bound components accumulated predominantly in tissue undergoing cell death in Bel W3 plants. Accumulation was caused by a redirection of the conjugation pathway, as well as by a transient increase in arginine decarboxylase and ornithine decarboxylase specific activity. This increase seemed to be regulated at post-transcriptional level.
• Measurements of chlorophyll content and fluorescence showed that, in addition to visible necrotic lesions, Bel W3 plants suffered considerable photosynthetic damage in other parts of the leaf.
• Accumulation of conjugated putrescine is part of the ozone-induced programmed cell death response in Bel W3 plants. Ozone-induced synthesis of free putrescine is not correlated with ozone-resistance in Bel B plants, which are apparently impaired in 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 of defense mechanisms without activating hypersensitive cell death.
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Ozone in the troposphere is formed by the interaction of nitrogen oxides, hydrocarbons and UV-radiation and is considered the most phytotoxic among the major gaseous pollutants (Heagle, 1989). The effects of ozone on vegetation include acute damage, leading to cell death, as well as chronic damage like inhibition of growth, reduction of CO2 assimilation and premature senescence (Sandermann, 1996; Schraudner et al., 1997; Miller et al., 1999). It is widely reported that different cultivars of plants vary greatly in their susceptibility to ozone (Heggestad, 1991). In many cases susceptibility is quantified by the extension of visual damage as a result of cell death. However, reduction in plant growth and productivity is mainly caused by long-term ozone exposure at lower than critical concentrations (chronic exposure) that induces changes in biochemical and physiological processes in the absence of visual damage (Heath & Taylor, 1997; Pell et al., 1997).
In Arabidopsis thaliana, ozone-induced cell death seems to occur via two distinct mechanisms (Rao & Davis, 1999). Plants that failed to accumulate salicylic acid (SA) upon ozone exposure were unable to activate antioxidant defense responses and displayed toxic cell death. On the other hand, in mutant plants in which ozone induces hyperaccumulation of SA, ozone exposure leads to an increased production of reactive oxygen species (ROS) which triggers a programmed cell death pathway similar to the hypersensitive response (HR) observed in incompatible plant–pathogen interactions. In fact, many typical plant responses to pathogen infection like an oxidative burst, SA and ethylene biosynthesis, cell wall modifications or the accumulation of phenolic compounds and several pathogenesis-related (PR) proteins, are elicited by ozone, suggesting that pathogen attack and ozone exposure activate 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 cells and essential for normal growth and development (Slocum & Flores, 1991). In plants, polyamines are proposed to play an important role in embryogenesis, senescence, flowering, as well as in defense responses to abiotic and biotic stress (Bagni, 1989). Studies on ozone-sensitive and -tolerant Nicotiana tabacum (tobacco) cultivars have shown a rapid increase of putrescine, both free and conjugated to hydroxycinnamic acids, in the ozone tolerant cultivar Bel B (Langebartels et al., 1991). In the hypersensitive cultivar Bel W3, however, only a small increase was observed at a later stage when necrotic lesions have already been formed. It was suggested that polyamines might protect the plant from cellular oxidative stress by inhibition of membrane lipid peroxidation (Kidata et al., 1979) or playing as radical scavengers in the form of hydroxycinnamic acid conjugates (Bors et al., 1989). This hypothesis is supported by the observation that feeding exogenous polyamines to tomato and tobacco plants resulted in a significant suppression of ozone-induced leaf injury. Furthermore, it has been demonstrated that putrescine conjugated to caffeic, ferulic or ρ-coumaric acid has a high capacity for radical scavenging (Bors et al., 1989). Conjugated polyamines might also contribute to the modulation of AOS levels in the cell, an important factor in regulating hypersensitive cell death (Rao et al., 2000).
Two alternative pathways for putrescine synthesis exist in plants: from ornithine by ornithine decarboxylase (ODC) or from arginine by arginine decarboxylase (ADC) through the intermediates agmatine and N-carbamoylputrescine (Bagni & Tassoni, 2001). Localization experiments in Avena have demonstrated that ADC is associated with the thylakoid membrane of the chloroplast (Borrell et al., 1995). In addition, it has been shown that polyamines protect against chlorophyll degradation (Tassoni et al., 2000) and that polyamine conjugation by transglutaminase activity seems to have an important role in protecting thylakoid and stromatal proteins of antenna complexes, thereby preserving photosynthetic efficiency (Serafini-Fracassini et al., 1995). ADC activity was reported to increase transiently in ozone-treated tobacco cultivar Bel B (ozone-tolerant) and this increase preceded the accumulation of free and conjugated putrescine (Langebartels et al., 1991). The activity of ODC, however, remained unchanged. cDNA corresponding to ADC and ODC genes 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 of these genes during ozone stress has not been reported.
In this study, we present a detailed analysis of polyamine accumulation and their relationship with photosynthetic parameters in tobacco cultivars Bel B and Bel W3 after a single ozone pulse and after a long exposure in open air for 1 month. Our results indicate that accumulation of conjugated putrescine is part of the ozone-induced programmed cell death response in Bel W3 plants and that ozone-induced synthesis of free putrescine is not correlated to ozone-resistance in Bel B plants. Furthermore, in vivo measurements of chlorophyll a fluorescence and chlorophyll content showed that exposing Bel W3 plants to ozone causes, in addition to visible necrotic lesions, considerable photosynthetic damage in other parts of the leaf.
Materials and Methods
Plant growth conditions
Tobacco plants cv. Bel W3 and Bel B were grown in a growth chamber with pollutant-free air under a 16-h photoperiod at 20°C with 50–70% rh and 320 µmol photons m−2 s−1. Experimental plants were approx. 2 months old and had seven to eight leaves. Leaves 4 and 5, counted from the bottom, were randomly collected from at least three different plants and used for molecular and biochemical analyses. These leaves had reached full expansion and consistently developed lesions after ozone exposure in the BelW3 cultivar. Plants utilized for exposure to tropospheric ozone in the urban area of Bologna were cultivated in the same conditions as above described.
Ozone exposure and evaluation of visible injury
Plants were transferred to a growth chamber modified for ozone fumigation and were acclimatized for 24 h before ozone treatment. Growth chamber conditions were 25°C, 80% rh, 350 ppm CO2, with a 14-h photoperiod averaging 350 µmol photons m−2 s−1 at the top of the canopy. The plants were then treated with a single pulse of ozone (130 ± 22 nl l−1 for 7 h) or maintained in charcoal filtered air. Ozone was generated with an ozone generator Fisher (Mod. 500, Meckenheim, Germany) and the ozone concentration inside the chamber was monitored continuously with a Monitor Laboratories Analyser 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 ozone which 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 evaluated at the end of the ozone exposure from the onset of lesion development. Each leaf was rated from 0 to 100 in increments of 10, correlating with the percentage of visible injury covering the leaf. The ratings for all leaves on each plant were summed to give one visible injury index for each plant.
Chlorophyll a fluorescence measurement
Chlorophyll a fluorescence analyses were carried out at the end of the fumigation (time 0) and 24 h after ozone fumigation (time 24) using a modulated fluorometer (PAM2000, Heinz Walz, Effeltrich, Germany). Plants were dark-adapted for 40 min and the measurements were carried out at room temperature. The minimal fluorescence yield (F0) was obtained upon excitation with a weak measuring beam from a pulse light-emitting diode. Maximal fluorescence yield (Fm) was determined after exposure to a 600-ms saturation pulse of white light to close all reaction centres. Variable fluorescence (Fv) was calculated as difference between Fm and F0 and the maximal apparent efficiency of PSII (Fv/Fm) was measured. Subsequently, actinic white light (approx. 300 µmol m−2 s−1) was switched on and saturating pulses were applied automatically at 60 s intervals for periodic determination of maximal fluorescence yield during actinic illumination (Fm′), the level of modulated fluorescence during a brief interruption of actinic illumination in the presence of far-red light (F0′) and the Chl fluorescence yield during actinic illumination (Ft). Nonphotochemical fluorescence quenching (NPQ) was estimated from the Stern-Volmer parameter, that represents a relative measurement of thermal dissipation at the PSII level, calculated according to the equation NPQ = Fm/Fm′ − 1 (Bilger & Bjorkman, 1991). The coefficient of photochemical quenching, qP, was calculated as (Fm′ – Ft)/(Fm′ – F0′) (Schreiber et al., 1986). Excitation pressure on PSII reflects the proportion of the primary stable quinone acceptor QA in the reduced state; it is calculated as 1 – qP. The quantum efficiency of excitation capture by oxidized reaction centres of PSII was calculated from the equation Φexc = (Fm′ – F0′)/Fm′ and the quantum efficiency of PSII photochemistry, ΦPSII, was estimated from (Fm′ – Ft)/Fm′ (Genty et al., 1989). All measurements were repeated five times. For comparison of the means, the ANOVA followed by the least significant difference (LSD) test was used.
Polyamine analysis by HPLC
Approximately 0.2 g f. wt of tobacco tissue were extracted in 10 volumes of 4% (w/v) cold perchloric acid (PCA) and centrifuged at 20 000 g for 30 min at 4°C. The pellet was resuspended in the original volume of PCA. Triplicates of this suspension and of the supernatant were hydrolyzed with 6 N HCl in flame-sealed vials at 110°C for 20 h in order to release polyamines from their conjugates. Aliquots (0.2 ml) of supernatant (free polyamines), hydrolyzed supernatant (soluble conjugated polyamines) and hydrolyzed pellet (insoluble conjugated polyamines) were dansylated according to Smith & Davies (1985) with minor modifications and dansyl-polyamines being extracted with toluene. Standard polyamines were subjected to the same procedure. Dansyl-polyamines were analyzed by HPLC (Jasco, Großumstad, Germany) with a reverse phase C18 column (Spherisorb ODS2, 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 a radiochemical method as described by Tassoni et al. (2000). In preliminary assays the optimum pH was determined for both enzyme activities. Tobacco tissues (0.4 g f. wt) were macerated in an ice-cold mortar with five volumes of the assay buffer (100 mM Tris-HCl pH 8.5, 50 µM pyridoxal phosphate) and centrifuged at 20 000 g for 30 min at 4°C. 0.2 ml of both supernatant and resuspended pellet were used to determine (0.5 ml final assay volume) ADC and ODC activities. The assays were performed by measuring the 14CO2 evolution from 7.4 kBq (approx. 1.3 µM) of L-[U-14C]-arginine (specific activity 11.7 GBq mmol−1, Amersham, UK) or from 7.4 kBq (about 7.3 µM) of D,L-[1–14C]-ornithine (specific activity 2.11 GBq mmol−1, Amersham, UK). 10 mM unlabelled ornithine and 10 mM unlabelled arginine were added to the enzyme assay for ADC and ODC, respectively, to verify the arginase and ornithine transcarbamoylase activities of the samples. Protein content was measured using the method of Lowry et al. (1951) and bovine serum albumine as standard.
RNA extraction and northern blot analysis
RNA was extracted from approx. 200 mg f. wt of leaf tissue using the RNA FAST kit (Molecular System, San Diego, CA, USA). Total RNA 15 µg was separated by electrophoresis in formaldehyde gels and blotted to HybondTM nylon membrane (Amersham, Little Chalfont, UK) by standard methods (Sambrook et al., 1989). 32P-CTP labeled probes were prepared from inserts of clones by oligo-labelling (Ready-To-Go DNA labelling Beads, Pharmacia Biotech, Uppsala, Sweden) and hybridized to blots in 20 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 pyrophosphate containing 500 µg sheared salmon sperm or herring testis DNA at 65°C. Hybridized membranes were washed twice in 2 × SSC, 0.5% (w/v) SDS and twice in 0.5 × SSC, 0.5% (w/v) SDS at 65°C for 15 min each. Membranes were subsequently exposed to KODAK X-OMAT film at −70°C for 3–5 days as well as to phosphor imager screens (Molecular Dynamics, Sunnyvale, CA, USA). Quantitative analysis of hybridizing bands was performed using Quantity One software (Bio-Rad, Hercules, CA, USA) and the value obtained for each band was corrected for loading differences by values obtained by rehybridizing blots with a 18S ribosomal gene probe from Medicago truncatula.
Ozone-sensitive Bel W3 tobacco plants displayed visible symptoms of injury typical of acute ozone exposure at 10–24 h after 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 to 50% with necrotic lesions on the following day, while younger leaves showed less injury (0–10% of leaf area). Bel B plants did 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 the ozone-sensitive Bel W3 cultivar, while no visible damage could be observed in Bel B plants.
Chlorophyll a fluorescence in O3-treated leaves
Before the ozone treatment the two tobacco cultivars exhibited different patterns of photosynthetic parameters. The strongest difference between the two cultivars maintained in filtered air was the almost two times higher value of NPQ measured in Bel B plants (Table 1). The values of Fv : Fm ratio were 0.777 and 0.781 for Bel W3 and Bel B cultivars, respectively, which were considerably lower than those reported by Bjorkman & Demmig (1987) for dicotyledonous species measured at 77K (0.843).
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
1 – qP
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).
O3 time 0
O3 time 24 h
O3 time 0
O3 time 24 h
In Bel W3 plants, ground fluorescence (F0), maximal fluorescence (Fm) and Fv : Fm ratio did not change when measured at the end of fumigation (time 0). The parameters derived from the quenching analysis also remained unchanged with the exception of the NPQ, which strongly increased compared with untreated plants. When the measurements were carried out 24 h after fumigation (time 24 h) significant differences in some parameters of fluorescence were detected. Indeed, the Fm value and the Fv : Fm ratio strongly decreased in ozone-treated plants. In addition, the state of reduction of the primary acceptor of PSII, QA, as indicated by (1 – qP), as well as the nonphotochemical quenching coefficient increased significantly. A strong decrease in the quantum yield of PSII (ΦPSII) and the efficiency of excitation capture (Φexc) was measured.
In the Bel B cultivar a decrease in Fv : Fm ratio was detected in ozone-treated leaves at the end fumigation (time 0). This decrease was caused by the increased values of F0. However, at 24 h after fumigation (time 24 h) the Fv : Fm ratio has recovered to about control levels (Table 1). The reduction state of the QA and the ΦPSII did not change in ozone-treated Bel B plants during the entire period. An increase in NPQ was observed in this cultivar at time 0 but at time 24 h NPQ was lower compared with control plants. A similar pattern was observed also for the Φexc.
Accumulation of polyamines
Ozone-treated Bel W3 plants showed a 4.5 fold increase in free putrescine content compared with control plants immediately after the end of ozone exposure (Fig. 1b) while the level of conjugated putrescine in both the soluble and insoluble fraction was lower with respect to controls. At this time point the levels of free and conjugated putrescine were not significantly altered in ozone-treated Bel B plants (Fig. 1a). At 24 h after the end of ozone treatment, Bel B and Bel W3 cultivars contained 2.5 and 3.3 fold, respectively, higher levels of free putrescine in plant exposed to ozone compared with control plants (Fig. 1c,d). Ozone-treated BelW3 plants also contained higher levels of soluble and insoluble conjugated putrescine compared with controls whereas no differences were found between control and ozone-treated Bel B plants. The levels of the other polyamines, spermidine, spermine and 1,3-diamino-propane, a product of polyamine oxidation as well as spermidine breakdown 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 significant changes were found (data not shown).
To determine the distribution of polyamines within ozone-damaged leaves, different leaf areas were collected from 2.5 months Bel W3 plants that had been exposed for 1 month at urban tropospheric ozone and the results compared with Bel B plants exposed to the same conditions. During this period (September–October, 1996) the mean ozone concentration was 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).
Before exposure to urban tropospheric ozone the leaf polyamine content displayed a different pattern in the two cultivars. The total polyamine content was higher in the ozone-sensitive Bel W3 cultivar compared with the Bel B cultivar (Fig. 2a,b). After 1 month exposure to urban tropospheric ozone, there was an increase in the concentration of diamino-propane (data not shown), putrescine and spermidine in both cultivars due to growth increment (Figs 3 and 4), whereas the spermine content did not show significant changes (data not shown). In Bel B plants (Fig. 3), very few conjugated polyamines were present in the insoluble fraction (8–10 nmol g−1 f. wt for putrescine and spermidine and about 1 nmol g−1 f. wt for spermine). On the other hand, free polyamine content was of the same order of magnitude as in Bel W3 plants. In Bel W3 plants, conjugated putrescine had increased about four times after exposure to urban tropospheric 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 increased moving towards healthy, undamaged tissue.
Activity of polyamine biosynthetic enzymes
ADC and ODC activities were determined in both soluble and particulate leaf fractions of control and ozone-treated plants (Fig. 5). The results are expressed as pmol g−1 f. wt but similar results were obtained when data were expressed on a mg protein basis. Arginase and ornithine transcarbamoylase activities in the soluble fraction were 36% and 29%, respectively. In the particulate fraction ornithine transcarbamoylase activity accounted for 23% of the ODC activity measured while no arginase activity could be recovered. The values in Fig. 5 were corrected accordingly. ODC and ADC were determined using the substrates in trace amount in order to evaluate the physiological activities. ODC was the major enzyme for putrescine biosynthesis in both cultivars and its activity was approx. five- and 8–10-fold higher with respect to ADC in Bel B and Bel W3 plants, respectively. Most activity of each enzyme was concentrated in the soluble fraction except in ozone-treated Bel W3 plants at 24 h (Fig. 5c,d).
Immediately after the end of ozone treatment (time 0), soluble ADC activity was approx. 50% higher in ozone-exposed plants compared with controls in both Bel B and Bel W3 cultivars (Fig. 5a). At 24 h, however, the levels in ozone treated plants had dropped to values similar or inferior to those of control plants (Fig. 5c). A very similar trend was observed for ODC activity following 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 ADC was measured by Northern blot analysis (Fig. 6). In both Bel B and Bel W3 cultivars the ADC transcript level was lower in ozone-exposed plants compared with control plants at time 0 while at time 24 h a small increase could be observed. An almost threefold reduction in ADC transcript level was found in Bel B and Bel W3 control plants at time 24 h compared with time 0. Analysis of PR1 gene expression showed a four–fivefold induction right after ozone treatment in both Bel B and Bel W3 plants although the mRNA level was approx. threefold higher in Bel W3 plants. At 24 h after ozone treatment PR1 mRNA levels had further increased and showed a more than 10-fold induction in both cultivars. Expression of the ODC gene was too low to be detected by Northern blot analysis (data not shown).
Effect of ozone on polyamine metabolism in Bel B and Bel W3 tobacco cultivars
Induction of polyamine biosynthesis has been described as a major physiological switch in the development of ozone tolerance in the Bel B tobacco cultivar (Langebartels et al., 1991). To study the effect of ozone on putrescine accumulation, we analyzed the enzyme activity and mRNA accumulation of its biosynthetic enzymes ADC and ODC.
Putrescine was the main polyamine accumulating in leaves of ozone-treated Bel B and Bel W3 tobacco cultivars. While in Bel W3 plants free putrescine levels increased immediately after ozone treatment (time 0), no changes occurred in Bel B. Both ADC and ODC activities increased in ozone-treated Bel B plants at this timepoint, anticipating the putrescine accumulation observed at 24 h. In ozone-treated Bel W3 plants the increase in free putrescine coincides with a decrease in both soluble and insoluble putrescine conjugates, suggesting a redirection of the conjugation processes. At 24 h after ozone treatment, Bel B and Bel W3 showed a similar increase in free putrescine in comparison to control plants while ADC and ODC enzyme activities had dropped to below control levels indicating that de novo synthesis of putrescine occurred within the 24 h following the end of ozone fumigation.
The mRNA levels of the ADC and ODC genes could not be correlated with their enzyme activities. However, because ADC and ODC assays were determined at non-saturating conditions a change in enzyme activity does not necessarily reflect a change in protein level. In addition, a transient increase in mRNA levels might have occurred before time 0. ADC seems to be mainly regulated at the post-transcriptional level in tobacco (Burtin & Michael, 1997) as well as in several other plant species (Rastogi et al., 1993; Watson & Malmberg, 1996; Primikirios & Roubelakis-Angelakis, 2001). Our mRNA data are consistent with a post-transcriptional regulation of ADC during ozone stress in tobacco. Similarly, the low ODC mRNA accumulation may be explained by the complicated and tight regulation of this enzyme, which occurs at multiple levels (Canellakis et al., 1981).
The accumulation of free and conjugated putrescine in the Bel W3 cultivar is apparently in contrast with the data obtained by Langebartels et al. (1991) even if care was taken to use plants of the same age, to analyze the same leaf numbers and to use similar ozone dosage and exposure time. Part of the discrepancy might be explained by our finding that free putrescine accumulates mainly in undamaged tissues, while conjugated putrescine is predominant in necrotic tissues of Bel W3 plants. Biochemical analysis by Langebartels et al. (1991) was carried out on leaf discs taken with a cork borer which is difficult when performed on fragile and chlorotic tissue. Therefore, the tissue analyzed might have been predominantly taken from undamaged parts of the leaves, thereby underestimating the level of conjugated putrescine. In addition, insoluble conjugated polyamines (bound to the cell wall components), which constitute the majority of ozone-induced polyamines (Bagni et al., 2000), were not included in their analysis.
Several lines of evidence indicate that ozone induces a HR-like programmed cell death in Bel W3 plants, for example, the nduction of a biphasic oxidative burst, the biosynthesis of SA and ethylene, the induction of pathogenesis-related and antioxidant 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 and conjugated putrescine in ozone-damaged Bel W3 leaves is similar to that seen in tobacco mosaic virus (TMV) infected tobacco leaves that display HR cell death (Torrigiani et al., 1997), confirming that ozone-induced lesions are similar, if not identical, to those induced by an incompatible plant–pathogen interaction.
Due to its scavenging capabilities, soluble conjugated putrescine that accumulates in necrotic lesions and surrounding zones might play a role in attenuating ROS production thereby limiting lesion spread. Alternatively, conjugated polyamines could play a role in plant defense mechanisms as demonstrated for example by the specific accumulation of a phenolic conjugate, p-coumaroyl-hydroxyagmatine with antifungal activity in a broad spectrum resistance reaction controlled by the mlo resistance alleles in barley (von Ropenack 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 putrescine plays a role in conferring tolerance in Bel B plants. However, free putrescine might be important in counteracting accelerated senescence during chronic ozone exposure by stabilizing chloroplast 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 regulation of plant defense responses and in ozone-induced cell death and is influenced by several interacting signal transduction pathways that involve ROS, SA, jasmonic acid and ethylene (Rao et al., 2000). A recent study on mastoparan-induced HR in isolated mesophyll cells suggested a complex relationship between photosynthesis and cell death (Allen et al., 1999). To elucidate the role of photosynthesis in ozone-induced cell death 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) no alteration in chlorophyll fluorescence parameters was visible with the exception of an increase in the NPQ value. However, 24 h after the end of fumigation, the Fv : Fm ratio dramatically decreased because of a substantial decrease in the Fm value. The ΦPSII also underwent a significant decrease due essentially to the strong increase in 1 – qP parameter. Because 1 – qP is a measure for the reduction state of the primary quinone acceptor, these data indicate a less effective reoxidation of this electron acceptor suggesting that a fraction of the PSII traps was closed during actinic illumination. These closed traps, which are unable to undergo charge separation and to take part in linear photosynthetic electron transport, should lead to decreased quantum yield. The increase in NPQ value was not sufficient to eliminate the photosynthetic damage in this cultivar. Allen et al. (1999) induced HR in isolated Asparagus sprengeri using a G-protein mastoparan (MP). A large increase in NPQ of chl a fluorescence accompanied the initial stage of the oxidative burst while (1 – qP) did not change and a similar response was found in Bel W3 tobacco plants immediately after the end of the O3 exposure. As the oxidative burst continued, a large increase in (1 – qP) was observed like in Bel W3 tobacco plants 24 h after the start of the fumigation. It should be noted, that these measurements were done on cells undergoing HR, whereas measurements on tobacco Bel W3 plants were performed on parts of the leaf that did not show any visible damage.
The response of Bel B plants that did not show any visible damage was different. Indeed, the decrease in Fv/Fm ratio measured in ozone-treated plants was due essentially to an increase in F0 value at time 0, which is associated with photoinhibitory damage. This may indicate an alteration of the reaction centres of PSII in energy utilization. On the other hand, Φexc was also reduced by ozone and this reduction was correlated with a large increase in NPQ. The PSII reaction centres were apparently not damaged but they were not able to carry out electron transport. The decrease of Φexc indicates also an increased probability that a photon absorbed by the PSII antennae is being dissipated as heat and demonstrates the occurrence of a stress-induced down-regulation of photosynthesis. However, the fast recovery of the Fv : Fm ratio 24 h after the end of the fumigation indicated that chronic photoinhibition was not taking place. This is also shown by the fact that the ΦPSII and 1 – qP are not altered in ozonated plants in comparison to the controls. Together, these data are indicative of an active mechanism of stress tolerance. Down-regulation of PSII reaction centres seems to dissipate excitation energy when active centres are closed, and their slightly lower trapping efficiency is reflected in the increase in dark-adapted F0, which commonly precedes and is then sustained during photoinhibition (Critchley & Russel, 1994). It is important to underline that Bel B plants showed a strong stomatal closure upon ozone exposure (data not shown, Heggestadt, 1991; Sandermann, 1996). Therefore, it is likely that photosynthesis in ozone-exposed plants was not limited by a decline in PSII efficiency, this being rather a regulatory adjustment of PSII efficiency to a decreased carbon availability. This determines a minor demand for reducing equivalent (NADPH and ATP) in the Calvin cycle that, in turn, causes a diminution in the electron transport rate. The complete recovery 24 h after the end of the fumigation is consistent with this hypothesis.
In conclusion, photosynthesis is affected differently by ozone exposure in Bel B and Bel W3 plants. In ozone-tolerant Bel B a regulatory mechanism plays a key role in the ozone-response that includes stomata closure and a reduction in CO2 assimilation rate. This results in a decrease of PSII photochemistry efficiency that might be important in limiting oxidative stress. In addition, defense responses are induced as shown by the induction of putrescine biosynthesis and the expression of PR1. Therefore, Bel B plants are able to perceive ozone stress and to induce a series of defense mechanisms without activating hypersensitive cell death. Bel W3 plants, on the other hand, are apparently impaired in one or more signal transduction pathways that are necessary to control the cellular redox state. In these plants ozone activates localized cell death as well as photosynthetic damage in other parts of the leaf.
This work was partially supported by funds from the University of Bologna for selected research topics, special project ‘Apoptosis’ (N.B.) and funds for Research Training Grant of European Commission (M.L.B., N.B.), and by the financed Project ‘Molecular mechanisms involved in plant responses to ozone’ funds from MURST (Ministry of University and Scientific and Technological Research, G.F.S.). We thank Dr J. Ryals (formerly at Novartis) for providing the tobacco PR-1 clone, Dr A.J. Michael (Institute of Food Research, Norwich, UK) for providing the tobacco ADC and ODC clones and Dr M.J. Harrison (The S.R. Noble Foundation, Ardmore, OK) for providing the 18S ribosomal clone from Medicago truncatula. Seeds of tobacco cultivars Bel B and Bel W3 were kindly provided by Dr G. Lorenzini. We are grateful to Mr G. Bugamelli for precious help in growing plants.