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Effect of ozone exposure on polyamines in Scots pine trees
Environmental and Experimental Botany 72 (2011) 448–454
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Effect of ozone exposure on polyamines in Scots pine trees Anne Jokela a,∗ , Tytti Sarjala b , Sirkku Manninen c , Satu Huttunen a a
Department of Biology, University of Oulu, P.O. Box 3000, 90014 Oulu, Finland Finnish Forest Research Institute, Parkano, Kaironiementie 15, 39700 Parkano, Finland c Department of Environmental Sciences, P.O. Box 56, 00014 University of Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 13 September 2010 Received in revised form 14 December 2010 Accepted 20 December 2010 Keywords: Ozone Pinus sylvestris Free polyamines Conjugated polyamines Carry-over effect
a b s t r a c t Effects of ozone exposure on polyamines in Pinus sylvestris L. were studied in a long-term experiment. Ten- to 15-year-old Scots pines were exposed to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland. The free, soluble conjugated and insoluble conjugated polyamines were analyzed during the experiment and shortly after termination of exposure as well as at the beginning of the following growing season. A carry-over effect was observed as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Conversely, after termination of the experiment, levels of free polyamines increased in the following growing season, and soluble conjugated polyamines decreased in the developing needles. The post-treatment changes in polyamine concentrations are hypothesized to be caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating the ozone exposure. This suggests that stress-induced injuries to older needles affected metabolism of new developing needles. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ozone (O3 ) enters leaves through stomata and induces various changes in the mesophyll, such as membrane damage, generation of toxic compounds and inhibition of CO2 assimilation, which may decrease photosynthesis and trigger senescence (Leonardi and Langebartels, 1991; Lippert et al., 1996; Mikkelsen and HeideJørgensen, 1996; Lütz et al., 2000). Ozone reacts with components of the cell wall and plasma membranes and produces various reactive oxygen species (ROS) such as H2 O2 in the apoplastic space (Pellinen et al., 2002). Especially high short term peak concentrations of ozone induce active production of ROS by affected cells (Kangasjärvi et al., 2005). Responses that follow ROS burst include changes in gene expression and synthesis, and accumulation of plant stress hormones ethylene, salicylic acid and jasmonic acid (Overmyer et al., 2000; Overmyer et al., 2005). Finally, production of ROS can lead to cell death resembling programmed cell
Abbreviations: ADC, arginine decarboxylase; AOT40, accumulated ozone exposure over a threshold of 40 ppb; FW, fresh weight; ODC, ornithine decarboxylase; OTC, open top chamber; PAO, polyamine oxidase; ROS, reactive oxygen species; SAMdc, S-adenosylmethionine decarboxylase. ∗ Corresponding author. Tel.: +358 8 5531517; fax: +358 8 5531061. E-mail address: anne.jokela@oulu.fi (A. Jokela). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.12.021
death (PCD) which takes place in the hypersensitive response (HR)like lesions (Kangasjärvi et al., 2005; Overmyer et al., 2005). The contribution made by polyamine molecules to plant protection against ozone damage has been documented (Rowland-Bamford et al., 1989; Langebartels et al., 1991; Tuomainen et al., 1996). Drolet et al. (1986) suggested that free polyamines are scavengers of oxygen radicals, while Bors et al. (1989) reported that polyamine conjugates with hydroxycinnamic acids are the main protection mechanism from ozone-triggered ROS accumulation. Addition of exogenous polyamines was found to reduce damage caused by ozone (Scalet et al., 1995) and exposure to elevated ozone level increased intracellular level of polyamines (Peters et al., 1989; Rowland-Bamford et al., 1989; Langebartels et al., 1991; Scalet et al., 1995). It has been proposed that polyamines counteract oxidative damage in plants and protect against ozone stress (Van Buuren et al., 2002; Navakoudis et al., 2003; Groppa and Benavides, 2008). Polyamines are low molecular weight compounds of polycationic nature, ubiquituos in both procaryotes and eucaryotes (Tabor and Tabor, 1984). Diamine putrescine, triamine spermidine and tetramine spermine are the most common polyamines in plants. The polyamine biosynthetic pathway is well characterized: ornithine decarboxylase (ODC) produces putrescine from ornithine with spermidine and spermine being produced by the addition of an aminopropyl group, provided by S-adenosylmethionine
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decarboxylase (SAMdc) action, to putrescine and spermidine, respectively. In bacteria, plants, and (apparently to a lesser extent) in mammals, this pathway is supplemented by arginine decarboxylase (ADC), producing putrescine through a series of intermediates from arginine (Tabor and Tabor, 1984). Polyamine metabolism is altered in response to several environmental stresses, including potassium deficiency, salinity, drought, chilling, heat, hypoxia, ozone, UV-B and UV-C, heavy metal toxicity, mechanical wounding and herbicide treatment as reviewed by Bouchereau et al. (1999) and Groppa and Benavides (2008). More recent studies using molecular approaches support a protective role of polyamines in plant response to abiotic stress (reviewed by Alcázar et al., 2010a). Navakoudis et al. (2007) reported that polyamines play a crucial role in the regulation of photoadaptation in the photosynthetic apparatus and especially putrescine plays a protective role against impact of environmental stresses on the photosynthetic apparatus. A low spermidine/putrescine ratio enhances the tolerance of plants against environmental stressors such as ozone and UV-B (Navakoudis et al., 2003; Sfichi et al., 2004). Takahashi and Kakehi (2010) suggested that the accumulation of putrescine is related to stress tolerance. Spermidine has also been shown to enhance tolerance to environmental stress but spermine plays versatile roles in stress responses. Abiotic stress such as potassium deficiency is known to cause high accumulation of putrescine in Pinus sylvestris L. (Sarjala and Kaunisto, 1993; Sarjala, 1996; Jokela et al., 1997), in which the main pathway of putrescine formation is decarboxylation of arginine by ADC (Vuosku et al., 2006; Jokela et al., unpublished). Putrescine is important as a precursor for the biosynthesis of higher polyamines, but the putrescine level must exceed a certain threshold (Capell et al., 2004) to enhance synthesis of spermidine and spermine under stress. Takahashi and Kakehi (2010) reviewed that spermine has double-edged roles in cell survival: as a free radical scavenger in the nucleus and as a source of free radicals in the apoplast. Spermine protects DNA from free radical attack and subsequent mutation (Ha et al., 1998). On the other hand, spermine plays a role in defence signaling against pathogens (Yamakawa et al., 1998; Takahashi et al., 2003) which takes place due to accumulation of spermine in the apoplast and subsequent H2 O2 production by the action of polyamine oxidase (PAO) localized in the apoplast (Cona et al., 2006; Kusano et al., 2008; Moschou et al., 2008). Elevated ozone concentrations (about 1.5 × ambient) have decreased seed germination and reduced growth in height, shoot and root biomass production (Prozherina et al., 2009), and manganese content of current year needles as well as increased yellowing and chlorotic mottling of previous year needles in experiment over two growing seasons with young Scots pine seedlings (Utriainen et al., 2000). We exposed 10- to 15-year-old Scots pines to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons and could not measure significant growth effects or changes in needle nutrient concentrations at the end of the experiment (Manninen et al., 2003, 2009). Neither did the trees show any visible injury during the experiment, although the results suggested a slightly decreased chlorophyll a + b/carotenoid ratio in the current year needles of ozone fumigated trees during the second fall (Manninen et al., 2003, 2009). Ozone exposure also lowered concentrations of free polyamines – especially putrescine – after the first exposure year of the experiment (Suorsa et al., 2002) which was considered as a carry-over effect of ozone stress at the beginning of the following growing season. The aim of this study was to measure fluctuations of polyamine levels in Scots pine trees in a long-term experiment with elevated ozone. The polyamine concentration after the second and the third exposure season were compared to those observed after the first exposure season reported by Suorsa et al. (2002). Post-
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treatment concentrations of both free and conjugated polyamines shortly after termination of exposure, as well as at the beginning of the following growing season were studied. We hypothesized that ozone-induced changes in free and conjugated polyamine concentrations are caused by a carry-over effect. 2. Materials and methods Ten- to 15-year-old Scots pines (Pinus sylvestris L.) were planted on the experimental field of the University of Oulu in September 1997. The trees were divided into three treatments: 6 trees in open top chambers (OTCs) with non-filtered ambient air (NF), 6 trees in OTCs exposed to non-filtered ambient air with supplemental ozone (NF + O3 ), and 6 trees on open-field plots (AA). In May, the amount of ozone applied was ambient + 40 ppb, in June ambient + 30 ppb, in July ambient + 20 ppb and in August ambient + 10 ppb. In September, only ambient air was given. The experiment was carried out in the years 1998–2000. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland, where ozone episodes occur in spring and early summer (Laurila and Tuovinen, 1996). The cumulative O3 exposure index AOT40 of the experiment was calculated as a sum of the exceedance of the hourly O3 concentrations above the cut-off of 40 ppb (results presented in Manninen et al., 2003). The needles for polyamine analysis were collected on the 10th of May (needle year classes 98 and 99; n = 3 in NF + O3 and NF treatments and n = 2 in AA treatment) and the 6th of September, 2000 (needle year classes 98, 99 and 00) and the 30th of May (needle year classes 98, 99 and 00) and the 11th of June, 2001 (needle year class 01) when developing stems were also sampled (n = 6 in NF + O3 and NF treatments and n = 2 in AA treatment in September 2000, May 2001 and June 2001). The within-canopy variation effects were kept to a minimum by sampling needles from the same whorl and the same orientation. The needles and stems were immediately frozen in liquid nitrogen and stored at −70 ◦ C until analyzed. The free, perchloric acid soluble conjugated and insoluble conjugated polyamines were analyzed with HPLC (Merck-Hitachi) as described in detail by Sarjala and Kaunisto (1993) and Fornalé et al. (1999). The data was subjected to a non-parametric pairwise comparison with the Mann–Whitney U to test difference between NF + O3 and NF treatments, NF and AA treatments and in addition NF + O3 and AA treatments (SPSS 16.0 Software). 3. Results Ozone concentrations and climatic conditions during the experiment are presented in Table 1 and temperature conditions in OTCs and open field plots for years 1998–2001 (June) in Fig. 1. 3.1. Free polyamines Ozone treatment (NF + O3 ) lowered free spermidine concentration in the needle year class 98 immediately after terminating the ozone exposure in September 2000 (Fig. 2C). In the following spring (May 2001) free spermine concentrations in the 1-year-old needles (needle year class 00, Fig. 2D) were lower under the ozone treatment, whereas ozone increased free spermine concentration in the current year needles, measured in June 2001 (Fig. 2G). Free polyamine concentrations were highest in AA treatment and lowest in NF + O3 treatment in May 2000, although not statistically significantly (data not shown). Immediately after terminating the ozone exposure in September 2000 no differences between NF and AA treatments were observed. However, in the following spring in May 2001, a chamber effect was seen as a higher spermine concentration in the 1-year-old needles under NF than AA (needle year class 00, Fig. 2D). Lower
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Table 1 Ozone concentrations and climatic conditions during the fumigation months. Exposure period
O3 (ppb)
Radiation
Temperature
Relative humidity
May–August 1998–2000a
1998: NF 20 NF + O3 54 AA 22 1999: NF 19 NF + O3 35 AA 22 2000: NF 15 NF + O3 38 AA 17
OTC PAR −28% vs. AA
OTC 13.8 ◦ C vs. AA 12.8 ◦ C
OTC 61% vs. AA 77% (June–August)
OTC PAR −15% vs. AA
OTC 12.1 ◦ C vs. AA 10.5 ◦ C
OTC 61% vs. AA 73%
OTC 16.7 ◦ C vs. AA 15.0 ◦ C
a Average O3 concentrations during the fumigations (8 h per day, 5 days a week) on 4 May–31 August 1998, 23 June–23 August 1999, 7 June–21 August 2000. The walls of the OTCs were replaced by new polycarbonate ones in June 1999.
20
temperature °C
significantly between NF + O3 and AA treatments with respect to spermidine in September 2000 (needle year class 98, Fig. 2C) and June 2001 (needle year class 01, Fig. 2G) and spermine in September 2000 and May 2001 (needle year class 00, Fig. 2A and D).
NF+O3
15
NF
10
AA
5 0
3.2. Soluble conjugated polyamines
-5
Ozone treatment increased spermine concentration in the 1year-old needles (needle year class 00) in May 2001 (Fig. 3D) and lowered putrescine concentration in the current year needles in June 2001 (Fig. 3G). Conversely, in stem putrescine concentration was increased by ozone in June 2001 (Fig. 3H). There were no significant differences between treatments in soluble conjugated polyamines in May 2000 (data not shown). A chamber effect was observed as a higher spermidine concentration in the 1-year-old needles (needle year class 99) under NF compared to AA treatment in September 2000 (Fig. 3B). However, in May 2001, the spermidine level was lower under NF in the 2
-10 -15
Fig. 1. Temperature during the experiment. Temperature during January 1998–June 2001 in OTCs (NF + O3 and NF) and open field plot (AA). Monthly mean temperature ◦ C.
putrescine levels in current year needles and stem, and lower spermidine in stem samples in NF than in AA were observed in June 2001 (Fig. 2G and H). Free polyamine concentrations differed B
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Fig. 2. Free polyamines. Concentration of free putrescine, spermidine and spermine (nmol/g FW) in samples collected in September 2000 (A–C), May 2001 (D–F) and June 2001 (G, H). Needle year classes 00 (A, D), 99 (B, E), 98 (C, F) and 01 (G). Stem (H). The different letters indicate statistically significant differences (P < 0.05) between the treatments obtained by pairwise comparison with Mann–Whitney U test.
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C 120
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Fig. 3. Soluble conjugated polyamines. Concentration of soluble conjugated putrescine, spermidine and spermine (nmol/g FW) in samples collected in September 2000 (A–C), May 2001 (D–F) and June 2001 (G, H). Needle year classes 00 (A, D), 99 (B, E), 98 (C, F) and 01 (G). Stem (H). The different letters indicate statistically significant differences (P < 0.05) between the treatments obtained by pairwise comparison with Mann–Whitney U test.
and 3 years old needles (needle year class 99 and 98, Fig. 3E and F). In June 2001 a chamber effect was observed in stem samples containing lower putrescine and spermine concentrations in NF than in AA (Fig. 3H). Soluble conjugated polyamines differed significantly between NF + O3 and AA treatments for putrescine and spermidine in May 2001 (needle year classes 98 and 99, Fig. 3E and F) as well as for putrescine and spermine in stem samples in June 2001, AA treatment having the highest levels of polyamines (Fig. 3H).
No significant differences between treatments were observed in insoluble conjugated polyamines in May 2000 (data not shown), or September 2000 and May 2001 for needle year classes 98 (Fig. 4C and F) and 99 (Fig. 4B and E). A chamber effect was observed as differences between NF and AA treatments in insoluble conjugated putrescine in the current year needles in September 2000 (Fig. 4A) and in spermidine in needle year class 00 in May 2001 (Fig. 4D),
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Fig. 4. Insoluble conjugated polyamines. Concentration of insoluble conjugated putrescine, spermidine and spermine (nmol/g FW) in samples collected in September 2000 (A–C) and May 2001 (D–F). Needle year classes 00 (A, D), 99 (B, E) and 98 (C, F). The different letters indicate statistically significant differences (P < 0.05) between the treatments obtained by pairwise comparison with Mann–Whitney U test.
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AA trees having higher polyamine levels than NF trees. A statistically significant difference between NF + O3 and AA treatments was found for putrescine in May 2001 in the 1-year-old needles, with the highest level detected under AA treatment (needle year class 00, Fig. 4D).
4. Discussion Exposure to elevated ozone in open-top chambers over three growing seasons caused a carry-over effect in Scots pine trees. In our earlier study (Suorsa et al., 2002) ozone induced a reduction in concentration of free polyamines – especially putrescine – in the needles. It was hypothesized that this was caused by a carry-over effect of ozone stress at the beginning of the growing season following the first season of ozone exposure. In the present study, ozone decreased the level of free spermidine in the oldest needle year class in September 2000, immediately after the termination of the third ozone exposure season. Reduction of free spermidine was observed in ozone-exposed trees also – although not significantly – in May 2000, which was the spring following the second exposure season. The oldest needle year class in which the reduction in polyamine levels was observed, developed during the first exposure season of the experiment in 1998. Ozone concentration during the first exposure season was 54 ppb (see Table 1) and decreased the level of free polyamines the following spring (Suorsa et al., 2002), and in addition after the second and third ozone exposure seasons. Therefore reduction of free polyamines in Scots pine trees could be considered as a carry-over effect of ozone stress. Polyamines are known to be closely connected to growth and cell division in Scots pine (Vuosku et al., 2006). So the decrease of net photosynthesis and reduction of shoot growth in the ozoneexposed trees reported by Manninen et al. (2003, 2009) may be related to decreased polyamine levels. In addition, ozone fumigation decreased chlorophyll a + b/carotenoid ratio and increased the amount of epicuticular waxes in needles in this experiment (Manninen et al., 2009). Langebartels et al. (1998) reported that in Pinus sylvestris and Picea abies visible symptoms and needle loss developed in a dose-dependent manner even months after terminating the ozone exposure and concluded that these conifers have some kind of ‘memory’ that may be imprinted by early stress reactions. Stress responses of Picea abies to spring-time frost (Polle et al., 1996) or drought stress (Karlsson et al., 2000) and Vaccinium myrtillus to wintertime desiccation (Tahkokorpi et al., 2007) indicate stress memory or after-effect stress as responses observed long after the stress had ceased. Polyamine levels in needles of Scots pine have seasonal variation – free spermidine and spermine levels being high in spring and showing a clear decrease after May, spermine usually shows very low levels in December (Sarjala and Savonen, 1994). After peaking in May polyamine levels decrease dramatically in June. The differences between individual trees are rather high especially in winter (Sarjala and Savonen, 1994). The polyamine concentrations found in this study are comparable with earlier observations from Scots pine (Sarjala and Savonen, 1994; Sarjala and Kaunisto, 1996; Jokela et al., 1997). The temperature in the chambers was 1.0–1.7 ◦ C higher on average than in open field plots (Table 1) and consequently effective temperature sum increased more rapidly in spring in the chambers (Fig. 1). Spring-time variation in temperature affects phenological events of plants in boreal regions (e.g. Heikinheimo and Lappalainen, 1997). The chamber effect seen in this study as differences in polyamine concentrations between NF and AA trees may be related to different timing of the beginning or ending of growth. Schenone et al. (1994) concluded that the chamber effect is unavoidable in NF OTCs due to micro-climatic alterations caused by the physical structure of the chambers.
Stress-induced low levels of polyamines are possibly related to an increase in ethylene synthesis which could cause premature senescence (Sandermann, 1996; Pandey et al., 2000; Heath, 2008). Accelerated leaf senescence is one of the reported effects of ozone – examples of which include Fagus sylvatica (Nunn et al., 2005; Gielen et al., 2007), Populus tremuloides (Gupta et al., 2005) and Arabidopsis (Miller et al., 1999). Pasqualini et al. (2003) detected a significant decrease in phenolic compounds in the ozone-sensitive Nicotiana tabacum cultivar Bel W3 during ozone exposure. The depletion of phenolic compounds (chlorogenic and caffeic acids) was connected to a reaction with ROS and formation of conjugated polyamines, which have been reported to be efficient radical scavengers (Bors et al., 1989). In Pinus halepensis, the content of spermidine and putrescine increased following treatment with a combination of ozone exposure and simulated acid rain, and conjugated polyamines were suggested as oxygen radical scavengers (Scalet et al., 1995). In both Bel B and Bel W3 N. tabacum cultivars, free putrescine accumulated in undamaged tissues, whereas conjugated putrescine accumulated only in ozone-treated tissue undergoing programmed cell death in Bel W3. Accumulation was caused by redirection of the conjugation pathway, as well as by increase in ADC and ODC activity (Van Buuren et al., 2002). In this study, ozone caused an increase of conjugated polyamines as spermine concentration increased in May 2001 in the 1-year-old needles and putrescine in stem samples in June 2001 suggesting a defense response to ozone-induced formation of oxygen radicals. The afternoon water content of previous year needles was lower in the NF + O3 trees than in NF trees in summer 2000 (Laakso et al., unpublished). The increased amount of epicuticular waxes in the current year needles in NF + O3 treatment at the end of the experiment was considered to be a consequence of earlier observed water stress that may relate to disturbed stomatal functioning and indicate an acclimation process (Manninen et al., 2009). Water stress has been reported to affect polyamine metabolism in plants and polyamine accumulation has effects on drought tolerance (Prabhavathi and Rajam, 2007; Yamaguchi et al., 2007; Alcázar et al., 2010b). Iriti and Faoro (2009) concluded that free and conjugated polyamines improve ozone tolerance by two different mechanisms: by inhibiting ethylene biosynthesis and by direct ROS scavenging. The metabolic shift to ethylene or polyamine biosynthesis can enhance ozone susceptibility or tolerance, respectively, due to the correlation between stress ethylene production and visible ozone injury (Iriti and Faoro, 2009). The results of this study indicate a lack of ability to enhance free polyamine levels during ozone exposure in the oldest needles (needle year class 98) which may be linked to increased levels of ethylene in the ozone-exposed trees. Conversely, after termination of the experiment, in the following growing season levels of free polyamines had increased and soluble conjugated polyamines decreased in the developing needles possibly indicating a recovery process. A further explanation for the higher free polyamine levels in the developing needles may be a consequence of the damaged older needles with reduced strength acting as a carbon sink, and subsequent changes in carbon allocation and growth, which was suggested as a possible effect of ozone by Manninen et al. (2003). Conclusions drawn from this study are that a carry-over effect was observed in Scots pine trees as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Elevated ozone induced post-treatment fluctuation of polyamine concentrations in the following growing season, which was caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. Polyamines are known to be involved both in cell division as well as stress
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responses, and therefore it remains unclear whether the observed polyamine changes in new needles affect growth or stress tolerance of the tree. Additional data on ethylene evolution, antioxidants or phenolics would have given more support to the hypothesis. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating ozone exposure. This suggests that stress-induced injuries to older needle year classes affected metabolism of the developing new needles. Acknowledgements Part of the results were published in Jokela, A., Sarjala, T., Manninen, S. and Huttunen, S. 2004. Ozone-induced polyamine response in Scots pine. In: Kinnunen, H. and Huttunen S. (Eds.) Proceedings of the Meeting ‘Forests under changing climate, enhanced UV and air pollution’, August 25–30, 2004, Oulu, Finland. IUFRO 7 Division Forest Health Project 7.04.00, Impacts of air pollution on forest ecosystems. The project was financed by University of Oulu (Department of Biology; Thule Institute, Focus Area Northern Issues and Environment; Faculty of Sciences (infrastructure)) (S.H.), the Jenny and Antti Wihuri Foundation (S.M.) and the Finnish Cultural Foundation (A.J.). We are grateful to Ms Eeva Pihlajaviita from the Finnish Forest Research Institute, for her technical assistance in polyamine analysis. The language was revised by Ms Sally Ulich. References Alcázar, R., Altabella, T., Marco, F., Bortolotti, C., Reymond, M., Koncz, C., Carrasco, P., Tiburcio, A.F., 2010a. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231, 1237–1249. Alcázar, R., Planas, J., Saxena, T., Zarza, X., Bortolotti, C., Cuevas, J., Bitrián, M., Tiburcio, A.F., Altabella, T., 2010b. Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants over-expressing the homologous Arginine decarboxylase 2 gene. Plant Physiol. Biochem. 48, 547–552. Bors, W., Langebartels, C., Michel, C., Sandermann, H., 1989. Polyamines as radical scavengers and protectans against ozone damages. Phytochemistry 28, 1589–1595. Bouchereau, A., Aziz, A., Larher, F., Martin-Tanguy, J., 1999. Polyamines and environmental challenges: recent development. Plant Sci. 140, 103–125. Capell, T., Bassic, L., Christou, P., 2004. Modulation of polyamine pathway in transgenic rice confers tolerance to drought stress. Proc. Natl. Acad. Sci. U.S.A. 101, 990–991. Cona, A., Rea, G., Angelini, R., Federico, R., Tavladoradi, P., 2006. Functions of amine oxidases in plant development and defense. Trends Plant Sci. 11, 80–88. Drolet, G., Dumbroff, E.B., Legge, R., Thompson, J.E., 1986. Radical scavenging properties of polyamines. Phytochemistry 25, 367–371. Fornalé, S., Sarjala, T., Bagni, N., 1999. Endogenous polyamine content and their metabolism in ectomycorrhizal fungus Paxillus involutus. New Phytol. 143, 581–587. Gielen, B., Löw, M., Deckmyn, G., Metzger, U., Franck, F., Heerdt, C., Matyssek, R., Valcke, R., Ceulemans, R., 2007. Chronic ozone exposure affects leaf senescence of adult beech trees: a chlorophyll fluorescence approach. J. Exp. Bot. 58, 758–795. Groppa, M.D., Benavides, M.P., 2008. Polyamines and abiotic stress: recent advances. Amino Acids 34, 35–45. Gupta, P., Duplessis, S., White, H., Karnosky, D.F., Martin, F., Podila, G.K., 2005. Gene expression patterns of trembling aspen trees following long-term exposure to interacting elevated CO2 and tropospheric O3 . New Phytol. 167, 129–142. Ha, H.C., Sirisoma, N.S., Kuppusamy, P., Zweier, J.L., Woster, P.M., Casero Jr., R.A., 1998. The natural polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. U.S.A. 95, 11140–11145. Heath, R.L., 2008. Modification of the biochemical pathways of plants induced by ozone: what are the varied routes to change? Environ. Pollut. 155, 453–463. Heikinheimo, M., Lappalainen, H., 1997. Dependence of the flower bud burst of some plant taxa in Finland on effective temperature sum: implications for climate change. Ann. Bot. Fenn. 34, 229–243. Iriti, M., Faoro, F., 2009. Chemical diversity and defence metabolism: how plants cope with pathogens and ozone pollution. Int. J. Mol. Sci. 10, 3371–3399. Jokela, A., Sarjala, T., Kaunisto, S., Huttunen, S., 1997. Effects of foliar potassium concentration on morphology, ultrastructure and polyamine concentrations of Scots pine needles. Tree Physiol. 17, 677–686. Kangasjärvi, J., Jaspers, P., Kollist, H., 2005. Signalling and cell death in ozone-exposed plants. Plant Cell Environ. 28, 1021–1036. Karlsson, P.E., Pleijel, H., Pihl Karlsson, G., Medin, E.L., Skärby, L., 2000. Simulations of stomatal conductance and ozone uptake to Norway spruce saplings in open-top chambers. Environ. Pollut. 109, 443–451. Kusano, T., Berberich, T., Tateda, C., Takahashi, Y., 2008. Polyamines: essential factors for growth and survival. Planta 228, 367–381.
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Effect of ozone exposure on polyamines in Scots pine trees Anne Jokela a,∗ , Tytti Sarjala b , Sirkku Manninen c , Satu Huttunen a a
Department of Biology, University of Oulu, P.O. Box 3000, 90014 Oulu, Finland Finnish Forest Research Institute, Parkano, Kaironiementie 15, 39700 Parkano, Finland c Department of Environmental Sciences, P.O. Box 56, 00014 University of Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 13 September 2010 Received in revised form 14 December 2010 Accepted 20 December 2010 Keywords: Ozone Pinus sylvestris Free polyamines Conjugated polyamines Carry-over effect
a b s t r a c t Effects of ozone exposure on polyamines in Pinus sylvestris L. were studied in a long-term experiment. Ten- to 15-year-old Scots pines were exposed to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland. The free, soluble conjugated and insoluble conjugated polyamines were analyzed during the experiment and shortly after termination of exposure as well as at the beginning of the following growing season. A carry-over effect was observed as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Conversely, after termination of the experiment, levels of free polyamines increased in the following growing season, and soluble conjugated polyamines decreased in the developing needles. The post-treatment changes in polyamine concentrations are hypothesized to be caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating the ozone exposure. This suggests that stress-induced injuries to older needles affected metabolism of new developing needles. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ozone (O3 ) enters leaves through stomata and induces various changes in the mesophyll, such as membrane damage, generation of toxic compounds and inhibition of CO2 assimilation, which may decrease photosynthesis and trigger senescence (Leonardi and Langebartels, 1991; Lippert et al., 1996; Mikkelsen and HeideJørgensen, 1996; Lütz et al., 2000). Ozone reacts with components of the cell wall and plasma membranes and produces various reactive oxygen species (ROS) such as H2 O2 in the apoplastic space (Pellinen et al., 2002). Especially high short term peak concentrations of ozone induce active production of ROS by affected cells (Kangasjärvi et al., 2005). Responses that follow ROS burst include changes in gene expression and synthesis, and accumulation of plant stress hormones ethylene, salicylic acid and jasmonic acid (Overmyer et al., 2000; Overmyer et al., 2005). Finally, production of ROS can lead to cell death resembling programmed cell
Abbreviations: ADC, arginine decarboxylase; AOT40, accumulated ozone exposure over a threshold of 40 ppb; FW, fresh weight; ODC, ornithine decarboxylase; OTC, open top chamber; PAO, polyamine oxidase; ROS, reactive oxygen species; SAMdc, S-adenosylmethionine decarboxylase. ∗ Corresponding author. Tel.: +358 8 5531517; fax: +358 8 5531061. E-mail address: anne.jokela@oulu.fi (A. Jokela). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.12.021
death (PCD) which takes place in the hypersensitive response (HR)like lesions (Kangasjärvi et al., 2005; Overmyer et al., 2005). The contribution made by polyamine molecules to plant protection against ozone damage has been documented (Rowland-Bamford et al., 1989; Langebartels et al., 1991; Tuomainen et al., 1996). Drolet et al. (1986) suggested that free polyamines are scavengers of oxygen radicals, while Bors et al. (1989) reported that polyamine conjugates with hydroxycinnamic acids are the main protection mechanism from ozone-triggered ROS accumulation. Addition of exogenous polyamines was found to reduce damage caused by ozone (Scalet et al., 1995) and exposure to elevated ozone level increased intracellular level of polyamines (Peters et al., 1989; Rowland-Bamford et al., 1989; Langebartels et al., 1991; Scalet et al., 1995). It has been proposed that polyamines counteract oxidative damage in plants and protect against ozone stress (Van Buuren et al., 2002; Navakoudis et al., 2003; Groppa and Benavides, 2008). Polyamines are low molecular weight compounds of polycationic nature, ubiquituos in both procaryotes and eucaryotes (Tabor and Tabor, 1984). Diamine putrescine, triamine spermidine and tetramine spermine are the most common polyamines in plants. The polyamine biosynthetic pathway is well characterized: ornithine decarboxylase (ODC) produces putrescine from ornithine with spermidine and spermine being produced by the addition of an aminopropyl group, provided by S-adenosylmethionine
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decarboxylase (SAMdc) action, to putrescine and spermidine, respectively. In bacteria, plants, and (apparently to a lesser extent) in mammals, this pathway is supplemented by arginine decarboxylase (ADC), producing putrescine through a series of intermediates from arginine (Tabor and Tabor, 1984). Polyamine metabolism is altered in response to several environmental stresses, including potassium deficiency, salinity, drought, chilling, heat, hypoxia, ozone, UV-B and UV-C, heavy metal toxicity, mechanical wounding and herbicide treatment as reviewed by Bouchereau et al. (1999) and Groppa and Benavides (2008). More recent studies using molecular approaches support a protective role of polyamines in plant response to abiotic stress (reviewed by Alcázar et al., 2010a). Navakoudis et al. (2007) reported that polyamines play a crucial role in the regulation of photoadaptation in the photosynthetic apparatus and especially putrescine plays a protective role against impact of environmental stresses on the photosynthetic apparatus. A low spermidine/putrescine ratio enhances the tolerance of plants against environmental stressors such as ozone and UV-B (Navakoudis et al., 2003; Sfichi et al., 2004). Takahashi and Kakehi (2010) suggested that the accumulation of putrescine is related to stress tolerance. Spermidine has also been shown to enhance tolerance to environmental stress but spermine plays versatile roles in stress responses. Abiotic stress such as potassium deficiency is known to cause high accumulation of putrescine in Pinus sylvestris L. (Sarjala and Kaunisto, 1993; Sarjala, 1996; Jokela et al., 1997), in which the main pathway of putrescine formation is decarboxylation of arginine by ADC (Vuosku et al., 2006; Jokela et al., unpublished). Putrescine is important as a precursor for the biosynthesis of higher polyamines, but the putrescine level must exceed a certain threshold (Capell et al., 2004) to enhance synthesis of spermidine and spermine under stress. Takahashi and Kakehi (2010) reviewed that spermine has double-edged roles in cell survival: as a free radical scavenger in the nucleus and as a source of free radicals in the apoplast. Spermine protects DNA from free radical attack and subsequent mutation (Ha et al., 1998). On the other hand, spermine plays a role in defence signaling against pathogens (Yamakawa et al., 1998; Takahashi et al., 2003) which takes place due to accumulation of spermine in the apoplast and subsequent H2 O2 production by the action of polyamine oxidase (PAO) localized in the apoplast (Cona et al., 2006; Kusano et al., 2008; Moschou et al., 2008). Elevated ozone concentrations (about 1.5 × ambient) have decreased seed germination and reduced growth in height, shoot and root biomass production (Prozherina et al., 2009), and manganese content of current year needles as well as increased yellowing and chlorotic mottling of previous year needles in experiment over two growing seasons with young Scots pine seedlings (Utriainen et al., 2000). We exposed 10- to 15-year-old Scots pines to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons and could not measure significant growth effects or changes in needle nutrient concentrations at the end of the experiment (Manninen et al., 2003, 2009). Neither did the trees show any visible injury during the experiment, although the results suggested a slightly decreased chlorophyll a + b/carotenoid ratio in the current year needles of ozone fumigated trees during the second fall (Manninen et al., 2003, 2009). Ozone exposure also lowered concentrations of free polyamines – especially putrescine – after the first exposure year of the experiment (Suorsa et al., 2002) which was considered as a carry-over effect of ozone stress at the beginning of the following growing season. The aim of this study was to measure fluctuations of polyamine levels in Scots pine trees in a long-term experiment with elevated ozone. The polyamine concentration after the second and the third exposure season were compared to those observed after the first exposure season reported by Suorsa et al. (2002). Post-
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treatment concentrations of both free and conjugated polyamines shortly after termination of exposure, as well as at the beginning of the following growing season were studied. We hypothesized that ozone-induced changes in free and conjugated polyamine concentrations are caused by a carry-over effect. 2. Materials and methods Ten- to 15-year-old Scots pines (Pinus sylvestris L.) were planted on the experimental field of the University of Oulu in September 1997. The trees were divided into three treatments: 6 trees in open top chambers (OTCs) with non-filtered ambient air (NF), 6 trees in OTCs exposed to non-filtered ambient air with supplemental ozone (NF + O3 ), and 6 trees on open-field plots (AA). In May, the amount of ozone applied was ambient + 40 ppb, in June ambient + 30 ppb, in July ambient + 20 ppb and in August ambient + 10 ppb. In September, only ambient air was given. The experiment was carried out in the years 1998–2000. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland, where ozone episodes occur in spring and early summer (Laurila and Tuovinen, 1996). The cumulative O3 exposure index AOT40 of the experiment was calculated as a sum of the exceedance of the hourly O3 concentrations above the cut-off of 40 ppb (results presented in Manninen et al., 2003). The needles for polyamine analysis were collected on the 10th of May (needle year classes 98 and 99; n = 3 in NF + O3 and NF treatments and n = 2 in AA treatment) and the 6th of September, 2000 (needle year classes 98, 99 and 00) and the 30th of May (needle year classes 98, 99 and 00) and the 11th of June, 2001 (needle year class 01) when developing stems were also sampled (n = 6 in NF + O3 and NF treatments and n = 2 in AA treatment in September 2000, May 2001 and June 2001). The within-canopy variation effects were kept to a minimum by sampling needles from the same whorl and the same orientation. The needles and stems were immediately frozen in liquid nitrogen and stored at −70 ◦ C until analyzed. The free, perchloric acid soluble conjugated and insoluble conjugated polyamines were analyzed with HPLC (Merck-Hitachi) as described in detail by Sarjala and Kaunisto (1993) and Fornalé et al. (1999). The data was subjected to a non-parametric pairwise comparison with the Mann–Whitney U to test difference between NF + O3 and NF treatments, NF and AA treatments and in addition NF + O3 and AA treatments (SPSS 16.0 Software). 3. Results Ozone concentrations and climatic conditions during the experiment are presented in Table 1 and temperature conditions in OTCs and open field plots for years 1998–2001 (June) in Fig. 1. 3.1. Free polyamines Ozone treatment (NF + O3 ) lowered free spermidine concentration in the needle year class 98 immediately after terminating the ozone exposure in September 2000 (Fig. 2C). In the following spring (May 2001) free spermine concentrations in the 1-year-old needles (needle year class 00, Fig. 2D) were lower under the ozone treatment, whereas ozone increased free spermine concentration in the current year needles, measured in June 2001 (Fig. 2G). Free polyamine concentrations were highest in AA treatment and lowest in NF + O3 treatment in May 2000, although not statistically significantly (data not shown). Immediately after terminating the ozone exposure in September 2000 no differences between NF and AA treatments were observed. However, in the following spring in May 2001, a chamber effect was seen as a higher spermine concentration in the 1-year-old needles under NF than AA (needle year class 00, Fig. 2D). Lower
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Table 1 Ozone concentrations and climatic conditions during the fumigation months. Exposure period
O3 (ppb)
Radiation
Temperature
Relative humidity
May–August 1998–2000a
1998: NF 20 NF + O3 54 AA 22 1999: NF 19 NF + O3 35 AA 22 2000: NF 15 NF + O3 38 AA 17
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putrescine levels in current year needles and stem, and lower spermidine in stem samples in NF than in AA were observed in June 2001 (Fig. 2G and H). Free polyamine concentrations differed B
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and 3 years old needles (needle year class 99 and 98, Fig. 3E and F). In June 2001 a chamber effect was observed in stem samples containing lower putrescine and spermine concentrations in NF than in AA (Fig. 3H). Soluble conjugated polyamines differed significantly between NF + O3 and AA treatments for putrescine and spermidine in May 2001 (needle year classes 98 and 99, Fig. 3E and F) as well as for putrescine and spermine in stem samples in June 2001, AA treatment having the highest levels of polyamines (Fig. 3H).
No significant differences between treatments were observed in insoluble conjugated polyamines in May 2000 (data not shown), or September 2000 and May 2001 for needle year classes 98 (Fig. 4C and F) and 99 (Fig. 4B and E). A chamber effect was observed as differences between NF and AA treatments in insoluble conjugated putrescine in the current year needles in September 2000 (Fig. 4A) and in spermidine in needle year class 00 in May 2001 (Fig. 4D),
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AA trees having higher polyamine levels than NF trees. A statistically significant difference between NF + O3 and AA treatments was found for putrescine in May 2001 in the 1-year-old needles, with the highest level detected under AA treatment (needle year class 00, Fig. 4D).
4. Discussion Exposure to elevated ozone in open-top chambers over three growing seasons caused a carry-over effect in Scots pine trees. In our earlier study (Suorsa et al., 2002) ozone induced a reduction in concentration of free polyamines – especially putrescine – in the needles. It was hypothesized that this was caused by a carry-over effect of ozone stress at the beginning of the growing season following the first season of ozone exposure. In the present study, ozone decreased the level of free spermidine in the oldest needle year class in September 2000, immediately after the termination of the third ozone exposure season. Reduction of free spermidine was observed in ozone-exposed trees also – although not significantly – in May 2000, which was the spring following the second exposure season. The oldest needle year class in which the reduction in polyamine levels was observed, developed during the first exposure season of the experiment in 1998. Ozone concentration during the first exposure season was 54 ppb (see Table 1) and decreased the level of free polyamines the following spring (Suorsa et al., 2002), and in addition after the second and third ozone exposure seasons. Therefore reduction of free polyamines in Scots pine trees could be considered as a carry-over effect of ozone stress. Polyamines are known to be closely connected to growth and cell division in Scots pine (Vuosku et al., 2006). So the decrease of net photosynthesis and reduction of shoot growth in the ozoneexposed trees reported by Manninen et al. (2003, 2009) may be related to decreased polyamine levels. In addition, ozone fumigation decreased chlorophyll a + b/carotenoid ratio and increased the amount of epicuticular waxes in needles in this experiment (Manninen et al., 2009). Langebartels et al. (1998) reported that in Pinus sylvestris and Picea abies visible symptoms and needle loss developed in a dose-dependent manner even months after terminating the ozone exposure and concluded that these conifers have some kind of ‘memory’ that may be imprinted by early stress reactions. Stress responses of Picea abies to spring-time frost (Polle et al., 1996) or drought stress (Karlsson et al., 2000) and Vaccinium myrtillus to wintertime desiccation (Tahkokorpi et al., 2007) indicate stress memory or after-effect stress as responses observed long after the stress had ceased. Polyamine levels in needles of Scots pine have seasonal variation – free spermidine and spermine levels being high in spring and showing a clear decrease after May, spermine usually shows very low levels in December (Sarjala and Savonen, 1994). After peaking in May polyamine levels decrease dramatically in June. The differences between individual trees are rather high especially in winter (Sarjala and Savonen, 1994). The polyamine concentrations found in this study are comparable with earlier observations from Scots pine (Sarjala and Savonen, 1994; Sarjala and Kaunisto, 1996; Jokela et al., 1997). The temperature in the chambers was 1.0–1.7 ◦ C higher on average than in open field plots (Table 1) and consequently effective temperature sum increased more rapidly in spring in the chambers (Fig. 1). Spring-time variation in temperature affects phenological events of plants in boreal regions (e.g. Heikinheimo and Lappalainen, 1997). The chamber effect seen in this study as differences in polyamine concentrations between NF and AA trees may be related to different timing of the beginning or ending of growth. Schenone et al. (1994) concluded that the chamber effect is unavoidable in NF OTCs due to micro-climatic alterations caused by the physical structure of the chambers.
Stress-induced low levels of polyamines are possibly related to an increase in ethylene synthesis which could cause premature senescence (Sandermann, 1996; Pandey et al., 2000; Heath, 2008). Accelerated leaf senescence is one of the reported effects of ozone – examples of which include Fagus sylvatica (Nunn et al., 2005; Gielen et al., 2007), Populus tremuloides (Gupta et al., 2005) and Arabidopsis (Miller et al., 1999). Pasqualini et al. (2003) detected a significant decrease in phenolic compounds in the ozone-sensitive Nicotiana tabacum cultivar Bel W3 during ozone exposure. The depletion of phenolic compounds (chlorogenic and caffeic acids) was connected to a reaction with ROS and formation of conjugated polyamines, which have been reported to be efficient radical scavengers (Bors et al., 1989). In Pinus halepensis, the content of spermidine and putrescine increased following treatment with a combination of ozone exposure and simulated acid rain, and conjugated polyamines were suggested as oxygen radical scavengers (Scalet et al., 1995). In both Bel B and Bel W3 N. tabacum cultivars, free putrescine accumulated in undamaged tissues, whereas conjugated putrescine accumulated only in ozone-treated tissue undergoing programmed cell death in Bel W3. Accumulation was caused by redirection of the conjugation pathway, as well as by increase in ADC and ODC activity (Van Buuren et al., 2002). In this study, ozone caused an increase of conjugated polyamines as spermine concentration increased in May 2001 in the 1-year-old needles and putrescine in stem samples in June 2001 suggesting a defense response to ozone-induced formation of oxygen radicals. The afternoon water content of previous year needles was lower in the NF + O3 trees than in NF trees in summer 2000 (Laakso et al., unpublished). The increased amount of epicuticular waxes in the current year needles in NF + O3 treatment at the end of the experiment was considered to be a consequence of earlier observed water stress that may relate to disturbed stomatal functioning and indicate an acclimation process (Manninen et al., 2009). Water stress has been reported to affect polyamine metabolism in plants and polyamine accumulation has effects on drought tolerance (Prabhavathi and Rajam, 2007; Yamaguchi et al., 2007; Alcázar et al., 2010b). Iriti and Faoro (2009) concluded that free and conjugated polyamines improve ozone tolerance by two different mechanisms: by inhibiting ethylene biosynthesis and by direct ROS scavenging. The metabolic shift to ethylene or polyamine biosynthesis can enhance ozone susceptibility or tolerance, respectively, due to the correlation between stress ethylene production and visible ozone injury (Iriti and Faoro, 2009). The results of this study indicate a lack of ability to enhance free polyamine levels during ozone exposure in the oldest needles (needle year class 98) which may be linked to increased levels of ethylene in the ozone-exposed trees. Conversely, after termination of the experiment, in the following growing season levels of free polyamines had increased and soluble conjugated polyamines decreased in the developing needles possibly indicating a recovery process. A further explanation for the higher free polyamine levels in the developing needles may be a consequence of the damaged older needles with reduced strength acting as a carbon sink, and subsequent changes in carbon allocation and growth, which was suggested as a possible effect of ozone by Manninen et al. (2003). Conclusions drawn from this study are that a carry-over effect was observed in Scots pine trees as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Elevated ozone induced post-treatment fluctuation of polyamine concentrations in the following growing season, which was caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. Polyamines are known to be involved both in cell division as well as stress
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responses, and therefore it remains unclear whether the observed polyamine changes in new needles affect growth or stress tolerance of the tree. Additional data on ethylene evolution, antioxidants or phenolics would have given more support to the hypothesis. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating ozone exposure. This suggests that stress-induced injuries to older needle year classes affected metabolism of the developing new needles. Acknowledgements Part of the results were published in Jokela, A., Sarjala, T., Manninen, S. and Huttunen, S. 2004. Ozone-induced polyamine response in Scots pine. In: Kinnunen, H. and Huttunen S. (Eds.) Proceedings of the Meeting ‘Forests under changing climate, enhanced UV and air pollution’, August 25–30, 2004, Oulu, Finland. IUFRO 7 Division Forest Health Project 7.04.00, Impacts of air pollution on forest ecosystems. The project was financed by University of Oulu (Department of Biology; Thule Institute, Focus Area Northern Issues and Environment; Faculty of Sciences (infrastructure)) (S.H.), the Jenny and Antti Wihuri Foundation (S.M.) and the Finnish Cultural Foundation (A.J.). We are grateful to Ms Eeva Pihlajaviita from the Finnish Forest Research Institute, for her technical assistance in polyamine analysis. The language was revised by Ms Sally Ulich. References Alcázar, R., Altabella, T., Marco, F., Bortolotti, C., Reymond, M., Koncz, C., Carrasco, P., Tiburcio, A.F., 2010a. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231, 1237–1249. Alcázar, R., Planas, J., Saxena, T., Zarza, X., Bortolotti, C., Cuevas, J., Bitrián, M., Tiburcio, A.F., Altabella, T., 2010b. Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants over-expressing the homologous Arginine decarboxylase 2 gene. Plant Physiol. Biochem. 48, 547–552. Bors, W., Langebartels, C., Michel, C., Sandermann, H., 1989. Polyamines as radical scavengers and protectans against ozone damages. Phytochemistry 28, 1589–1595. Bouchereau, A., Aziz, A., Larher, F., Martin-Tanguy, J., 1999. Polyamines and environmental challenges: recent development. Plant Sci. 140, 103–125. Capell, T., Bassic, L., Christou, P., 2004. Modulation of polyamine pathway in transgenic rice confers tolerance to drought stress. Proc. Natl. Acad. Sci. U.S.A. 101, 990–991. Cona, A., Rea, G., Angelini, R., Federico, R., Tavladoradi, P., 2006. Functions of amine oxidases in plant development and defense. Trends Plant Sci. 11, 80–88. Drolet, G., Dumbroff, E.B., Legge, R., Thompson, J.E., 1986. Radical scavenging properties of polyamines. Phytochemistry 25, 367–371. Fornalé, S., Sarjala, T., Bagni, N., 1999. Endogenous polyamine content and their metabolism in ectomycorrhizal fungus Paxillus involutus. New Phytol. 143, 581–587. Gielen, B., Löw, M., Deckmyn, G., Metzger, U., Franck, F., Heerdt, C., Matyssek, R., Valcke, R., Ceulemans, R., 2007. Chronic ozone exposure affects leaf senescence of adult beech trees: a chlorophyll fluorescence approach. J. Exp. Bot. 58, 758–795. Groppa, M.D., Benavides, M.P., 2008. Polyamines and abiotic stress: recent advances. Amino Acids 34, 35–45. Gupta, P., Duplessis, S., White, H., Karnosky, D.F., Martin, F., Podila, G.K., 2005. Gene expression patterns of trembling aspen trees following long-term exposure to interacting elevated CO2 and tropospheric O3 . New Phytol. 167, 129–142. Ha, H.C., Sirisoma, N.S., Kuppusamy, P., Zweier, J.L., Woster, P.M., Casero Jr., R.A., 1998. The natural polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. U.S.A. 95, 11140–11145. Heath, R.L., 2008. Modification of the biochemical pathways of plants induced by ozone: what are the varied routes to change? Environ. Pollut. 155, 453–463. Heikinheimo, M., Lappalainen, H., 1997. Dependence of the flower bud burst of some plant taxa in Finland on effective temperature sum: implications for climate change. Ann. Bot. Fenn. 34, 229–243. Iriti, M., Faoro, F., 2009. Chemical diversity and defence metabolism: how plants cope with pathogens and ozone pollution. Int. J. Mol. Sci. 10, 3371–3399. Jokela, A., Sarjala, T., Kaunisto, S., Huttunen, S., 1997. Effects of foliar potassium concentration on morphology, ultrastructure and polyamine concentrations of Scots pine needles. Tree Physiol. 17, 677–686. Kangasjärvi, J., Jaspers, P., Kollist, H., 2005. Signalling and cell death in ozone-exposed plants. Plant Cell Environ. 28, 1021–1036. Karlsson, P.E., Pleijel, H., Pihl Karlsson, G., Medin, E.L., Skärby, L., 2000. Simulations of stomatal conductance and ozone uptake to Norway spruce saplings in open-top chambers. Environ. Pollut. 109, 443–451. Kusano, T., Berberich, T., Tateda, C., Takahashi, Y., 2008. Polyamines: essential factors for growth and survival. Planta 228, 367–381.
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