Tree Physiology Advance Access originally published online on December 3, 2008
Tree Physiology 2009 29(1):53-66; doi:10.1093/treephys/tpn005
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Differences in leaf characteristics between ozone-sensitive and ozone-tolerant hybrid aspen (Populus tremula x Populus tremuloides) clones
1 Department of Environmental Science, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland
2 Corresponding author (elina.haikio{at}uku.fi)
3 Faculty of Biosciences, University of Joensuu, P.O. Box 111, FI-80101 Joensuu, Finland
4 Finnish Forest Research Institute, Punkaharju Research Unit, Finlandiantie 18, FI-58450 Punkaharju, Finland
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Abstract |
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The authors analyzed a suite of leaf characteristics that might help to explain the difference between ozone-sensitive and ozone-tolerant hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones. An open-field experiment comprising ambient ozone and 1.5x ambient ozone concentration (about 35 ppb) and two soil nitrogen regimes (60 and 140 kg N ha–1 year–1) was conducted over two growing seasons on potted plants of eight hybrid aspen clones. Four of the clones had previously been determined to be ozone sensitive based on impaired growth in response to elevated ozone concentration. Photosynthetic rate, chlorophyll fluorescence, and concentrations of chlorophyll, protein and carbohydrates were analyzed three times during the second growing season, and foliar phenolic concentrations were measured at the end of the second growing season. Nitrogen amendment counteracted the effects of ozone, but had no effect on growth-related ozone sensitivity of the clones. Ozone-sensitive clones had higher photosynthetic capacity and higher concentrations of Rubisco and phenolics than ozone-tolerant clones, but the effects of ozone were similar in the sensitive and tolerant groups. Nitrogen addition had no effect on phenolic concentration, but elevated ozone concentration increased the concentrations of chlorogenic acid and (+)-catechin. This study suggests that condensed tannins and catechin, but not salicylates or flavonol glycosides, play a role in the ozone tolerance of hybrid aspen.
Keywords: chlorophyll, flavonoids, nitrogen, phenolics, photosynthesis, Rubisco, salicylates, senescence, sensitivity
Received April 22, 2008; Accepted August 24, 2008
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Introduction |
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Tropospheric ozone concentration ([O3]) in the Northern Hemisphere has more than doubled since preindustrial times, from about 10 ppb to the present background concentrations of 25–40 ppb (Vingarzan 2004), and is predicted to increase by 40–70% by the year 2100 (Grenfell et al. 2003, Zeng et al. 2008). In addition to acting as a greenhouse gas, tropospheric O3 affects the land carbon sink by decreasing photosynthesis and thus plant productivity, leading to increased amounts of carbon dioxide in the atmosphere, and doubling the effective radiative forcing due to increased tropospheric ozone alone (Sitch et al. 2007). According to Wittig et al. (2007), an increase in [O3] to 60 ppb would result in a 17% decrease in the photosynthetic capacity of forests. This would negatively affect carbon assimilation and water vapor transfer to the atmosphere affecting terrestrial carbon sinks and regional hydrology. A meta-analysis of ozone effects on the photosynthesis of trees showed that Populus species that are a major component of northern boreal forests and a good candidate for bioenergy are among the species that are most sensitive to elevated tropospheric ozone concentrations (Wittig et al. 2007).
Ozone accelerates foliar senescence in many species (Reich and Lassoie 1985, Pääkkönen et al. 1997, Pell et al. 1999, Ribas et al. 2005) possibly by increasing proteolysis of Rubisco (Brendley and Pell 1998), resulting in declines in net photosynthetic rate and whole-plant carbon gain (Greitner et al. 1994). Degradation of chlorophyll and membranes at the early stage of senescence leads to the generation of reactive oxygen species (ROS) that are normally removed by the constitutive antioxidative system in the apoplast and symplast of leaves (Foyer and Noctor 2005). In senescing leaves, because of protein breakdown, the loss of antioxidative enzymes adds to the increase in ROS (Zimmermann and Zentgraf 2005). Ozone exposure also results directly in the generation of ROS, and the antioxidative defense capacity of the leaves may play a major role in the ozone tolerance of a plant (Pell et al. 1997, Tausz et al. 2007). Apoplastic antioxidants, especially ascorbic acid, make up the first line of defense in the plant cell (Conklin and Barth 2004). In addition to enzymatic and nonenzymatic ROS scavengers, some plant secondary compounds, such as flavonoids, have important antioxidant properties (Cao et al. 1997, Heim et al. 2002). In aspen, secondary compounds comprise 10–35% of leaf dry mass, and considerable intraspecific variation in the amounts of foliar salicylates (glucosides of salicylic acid) and condensed tannins has been found (Hwang and Lindroth 1997, Holton et al. 2003). Elevated [O3] increased the amounts of condensed tannins (Agrell et al. 2005) and decreased the amounts of salicylates in trembling aspen (Holton et al. 2003).
Accelerated senescence of older leaves may also be an adaptive trait allowing nutrient remobilization to younger leaves and permitting compensatory growth in indeterminately growing trees like Populus (Brendley and Pell 1998). Nitrogen deficiency has been found to stimulate ozone-induced senescence (Bielenberg et al. 2001). Correspondingly, enhanced nitrogen supply counteracts O3-induced senescence (Pääkkönen and Holopainen 1995), because the photosynthetic enzymes and pigments are a major sink of nitrogen in leaves. The amounts of secondary compounds also vary with soil fertility: the concentrations of condensed tannins and salicylates are higher in soils with lower nitrogen availability (e.g., Kleiner et al. 1998).
In a previous study, we clustered eight hybrid aspen clones into two equal groups, one group showing O3 sensitivity and the other group showing O3 tolerance, based on their growth responses to elevated [O3] (Häikiö et al. 2007). We now report the effects of elevated [O3] and nitrogen fertilization on senescence-associated parameters and foliar phenolics in 3-year-old potted saplings of these hybrid aspen clones during their second growing season in an open-field ozone fumigation system. By comparing the responses of the two sensitivity groups to elevated [O3], we aimed to identify leaf characteristics that differed between the ozone-sensitive and ozone-tolerant clones. We also studied the role of phenolics, particularly the role of condensed tannins, in defense against oxidative stress; although tannins do not have a broad-spectrum antiherbivore activity (Ayres et al. 1997), they possess good radical-scavenging properties (Hagerman et al. 1998). As increased nutrient availability can result in photosynthate allocation to growth instead of carbon-based allelochemicals (Kleiner et al. 1998), we predicted that nitrogen fertilization would lead to decreased ozone tolerance in our clones. Slow-growing trees, such as beech, are less sensitive to ozone than fast-growing trees such as most of the Populus species (Bortier et al. 2000). Accordingly, we expected to find a positive correlation between high growth rate and ozone sensitivity in our hybrid aspen clones.
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Materials and methods |
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Plant material
The material consisted of eight hybrid aspen clones, six of which represent crosses between Finnish origin Populus tremula L. as female parents and Canadian origin Populus tremuloides Michx. as male parents (Clones 1, 14, 55, 110, 193 and 218), one is a cross between P. tremuloides from Canada and P. tremula from Finland (Clone 200), and one is a cross between P. tremuloides from southern Sweden and P. tremula from Finland (Clone 280). The clones were selected for commercial production during the mid-1990s from experimental forests established in southern Finland in the 1950s and 1960s as part of the Finnish hybrid aspen breeding program (Haapanen and Mikola 2008). Hybrid aspen Clones 110, 200, 218 and 280 were previously classified as ozone sensitive, based on impaired growth in the presence of elevated [O3], whereas elevated [O3] did not reduce growth in the ozone-tolerant group, i.e., Clones 1, 14, 55 and 193 (see Figure 2) (cf. Häikiö et al. 2007).
One-year-old micropropagated plants were placed in 7.5-l pots in a peat:sand (2:1, v/v) mixture and transferred to the Ruohoniemi open-air exposure field at Kuopio University Garden (62°53' N and 27°37' E, 80 m a.s.l.) at the end of May 2002. Twenty plants per clone were randomly assigned to each of the eight plots: four plots received an elevated [O3] and four received ambient [O3]. Half of the plants in each plot received high nitrogen fertilization (high-N treatment) and the remainder received low nitrogen fertilization (low-N treatment). The plants were grown in a natural microclimate and were watered daily. The pots were rotated within the plots twice during the growing season. For overwintering, the pots were covered with spruce twigs and natural snowfall.
Ozone fumigation
The realized O3 enhancement based on 24-h mean ozone concentrations was 1.4 and 1.5 times the ambient [O3] in 2002 and 2003, respectively. The mean O3 exposures for 24 h day–1 were 24 and 35 ppb for the ambient ozone and the elevated ozone plots, respectively. In 2002, O3 fumigation was started in July after all the plants had been planted, and continued until the middle of October. In 2003, O3 fumigation was supplied from bud break on May 14 until leaf fall on October 6. Details of the O3 fumigation, the cumulative ozone exposures AOT00 and AOT40, and the mean monthly temperatures for the growing seasons of 2002 and 2003 have been described by Häikiö et al. (2007).
Nitrogen treatment
The plants were subjected to a high or low nitrogen fertilization regime. In 2002, 10 plants per clone in each plot were fertilized either once (Week 27) or twice (Weeks 27 and 29) with a base fertilizer (0.2% solution of Kekkilä Superex-9; 19:5:20 N:P:K) to achieve final N doses of 39 (low-N treatment) and 78 (high-N treatment) kg N ha–1 year–1. In 2003, because of the larger size of the plants, the N doses were increased. All the plants were first fertilized twice (Weeks 22 and 23) with 0.2% solution of Kekkilä Superex-9. In addition to the base fertilization, the plants in the high-N treatment received 100 ml of 0.02 M NH4NO3 solution every 10 days during Weeks 22–34, nine times altogether. The final N doses were 60 and 140 kg N ha–1 year–1 in the low- and high-N treatments, respectively.
Biomass, photosynthesis and chlorophyll fluorescence measurements
At the end of the second growing season (September 2003), a portion of the plants was harvested for biomass analysis as described by Häikiö et al. (2007).
Leaf net photosynthetic rate in saturating light (Asat) was measured three times during the second growing season in 2003 (June 25–26, July 18–24 and August 18–25) on the same sun leaf in the middle of the canopy per clone per treatment on each plot (altogether 160 leaves) with a CI-510 Portable Photosynthesis System (CID, Vancouver, WA). Maximum photochemical efficiency of photosystem II (Fv/Fm) was measured three times in 2003 (July 22, August 12 and August 25) with a portable pulse-modulated FMS2 fluorometer (Hansatech Instruments, Norfolk, UK) on the same leaves that were used for the photosynthetic measurements. Measurements were made on well-watered plants in dry sunny weather between 1000 and 1600 h. For details, see Häikiö et al. (2007).
Pigment, Rubisco, total soluble proteins and total leaf organic nitrogen analyses
Samples for biochemical analyses were taken three times during the second growing season from one tree per clone per treatment per plot from first flush leaves of consecutive branches of the same tree at every sampling: July 16, August 13 and September 11, 2003. A leaf disk (2.05 cm2, about 30 mg in mass) from a sun leaf in the middle of the canopy was punched and frozen in liquid nitrogen and stored at –80 °C. The rest of the leaf was dried and used for the determination of leaf nitrogen. The leaf disk was ground in liquid nitrogen, and 2 ml of extraction buffer (50 mM MES, 20 mM MgCl2, 50 mM 2-mercaptoethanol and 1% Tween-20) was added. Aliquots of the crude extract were taken for the determination of total chlorophylls (Chl a + b), total carotenoids, total soluble proteins (TSPs) and Rubisco. Total chlorophyll and total carotenoid concentrations were determined spectrophotometrically in 80% acetone extracts by the methods by Arnon (1949) and Lichtenthaler and Wellburn (1983), with the corrections of Porra (2002). Soluble proteins were precipitated in 67% (v/v) ethanol, collected by centrifugation and resuspended in 0.1 M NaOH. The protein concentration of the solution was determined by the method by Bradford (1976), with bovine serum albumin as a standard. For the determination of Rubisco, samples of the crude extract were run on native 6% polyacrylamide gels according to Rintamäki et al. (1988). The gels were stained with Coomassie Brilliant Blue R250, destained and scanned, and the intensities of the Rubisco bands were analyzed using Quantity One® 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA) with a known concentration of purified spinach Rubisco (Sigma) as a standard. Total leaf organic nitrogen concentration was determined by the standard Kjeldahl method (Allen 1989).
Leaf carbohydrate analysis
Leaf samples were collected from the ozone-tolerant Clone 55 and the ozone-sensitive Clone 110 (Oksanen et al. 2001, Häikiö et al. 2007) for carbohydrate (starch and soluble sugars) analyses along with the samples for chlorophyll, Rubisco and TSP analyses. Two or three leaves from the same branch were collected, vacuum-dried and ground to a powder with an analysis mill (Janke & Kunkel, Staufen, Germany). For the extraction of soluble sugars, 30–40 mg subsample of ground leaf powder was mixed with 3 ml of 80% ethanol. The sample was incubated at 60 °C for 5 min and centrifuged (2000g, 4 min). The supernatant was collected and the extraction was repeated four times. The combined pellets were saved for starch determination. An aliquot (400 µl) of the combined supernatants was added to 2 ml of anthrone reagent (2 mg anthrone in 1 ml 72% sulfuric acid). Each sample was boiled for 11 min and cooled, and the absorbance at 630 nm was determined (Hansen and Møller 1975). The starch pellet was resuspended in 4 ml of acetate buffer (pH 4.5) and gelatinized in a boiling water bath for 15 min. The starch was hydrolyzed by incubating the sample with 1 ml of amyloglucosidase solution (Sigma, 15 U ml–1) at 50 °C for 24 h. After cooling to room temperature, the samples were centrifuged (5000g, 4 min). An aliquot (400 µl) of the supernatant was mixed with anthrone reagent and the absorbance at 630 nm was determined.
Foliar phenolic composition
Leaf samples were collected on August 26, 2003. Five first flush leaves were collected and pooled (one sample per clone per treatment per plot, altogether 160 samples). The leaves were dried at room temperature at 20% RH according to Julkunen-Tiitto and Sorsa (2001), ground without the midvein, and stored at –20 °C. For extraction, a subsample of 8 mg of ground leaf powder was mixed with 600 µl of ice-cold methanol. The sample was vortexed, left to stand on ice for 15 min, and centrifuged (3 min, 15,000g). The supernatant was collected and the residue was extracted four more times. The supernatants were combined, the methanol was evaporated in a stream of nitrogen, and the dry sample was stored at –20 °C until analyzed for phenolics. The extraction residues were dried and stored at –20 °C for cell wall-bound condensed tannin analysis.
The dry sample was redissolved in 600 µl of methanol:H2O (1:1, v/v). Phenolics were analyzed using a HPLC system (Hewlett–Packard, Avondale, PA) with a binary pump (HP 1050), an autosampler (HP 1050) and a photo diode array detector (HP 1040A), combined with ChemStation for LC 3D (Rev. A.09.01; Agilent Technologies). A 3 µm HP Hypersil ODS column (60 x 4.6 mm2 ID) was used. The eluents were aqueous 1.5% tetrahydrofuran + 0.25% o-phosphoric acid (A) and 100% methanol (B), and the following gradient was used: 0–5 min 100% A, 5–10 min 85% A, 10–20 min 70% A, 20–40 min 50% A and 40–50 min 100% B. The flow rate was 2 ml min–1 and the injection volume was 15 µl. The HPLC runs were monitored at 220, 270, 280, 320 and 360 nm. Identification of the compounds was based on comparisons of retention times and spectral characteristics. The flavonol glycosides were tentatively identified as derivatives. Commercial standards were used for the quantification of the identified compounds: (+)-catechin (Aldrich Chemicals Co., Milwaukee, WI) and chlorogenic acid (Aldrich) for neochlorogenic acid and chlorogenic acid, trans-p-OH-cinnamic acid (Aldrich) for p-OH-cinnamic acid derivatives, kaempferol-3-glucoside (Extrasynthèse, Genay, France) for kaempferol derivatives, quercetin-3-galactoside (Apin Chemicals Ltd., Abingdon, UK) for quercetin derivatives, myricetin-3-rhamnoside (Apin Chemicals Ltd.) for myricetin derivatives, isorhamnetin-3-glucoside (Apin Chemicals Ltd.) for the isorhamnetin derivative, salicin (Sigma, Steinheim, Germany) and tremulacin (Roth, Karlsruhe, Germany). Salicortin and tremuloidin were based on purified standards obtained from Prof. Beat Meier, ETH, Zurich, Switzerland (Julkunen-Tiitto and Sorsa 2001, Heiska et al. 2007). Chromatograms from the HPLC analysis showed the identified peaks at 220 nm (salicylates) and 320 nm (flavonoids and phenolic acids; Figure 1). Soluble condensed tannins were determined from an aliquot of the HPLC extract, and cell wall-bound condensed tannins were analyzed from the dried extraction residue using the acid–butanol test (Porter et al. 1986). Cell wall-bound tannins comprised on average 6% of the whole tannin pool of the leaves. Tannin purified from pooled hybrid aspen leaves was used as a standard.
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Statistical analyses
To determine which foliar characteristics differ between the ozone-tolerant and ozone-sensitive hybrid aspen clones, the clones were divided into two sensitivity groups and the mean value of the four clones in each group (for each plot and each nitrogen treatment, altogether 32 observations) was used. The main effects and interactions of the two concentrations of ozone, two soil nitrogen regimes, two ozone sensitivity groups, and in the case of repeated samplings or measurement, three measuring dates were tested. The plot was used as a replicate. To test for the main effects and interactions of ozone, nitrogen and sensitivity group on total dry mass and concentrations of phenolic compounds of leaves, linear mixed model analysis of variance (ANOVA) using SPSS for Windows Version 14.0 (SPSS, Chicago, IL) was performed, with ozone, nitrogen and sensitivity as fixed factors, and plot as a random factor. Classification of the clones as ozone sensitive or ozone tolerant by the clustering method by Häikiö et al. (2007) was also verified. In the repeated measurements analysis (pigments, Rubisco, TSP, leaf nitrogen, carbohydrates, photosynthesis and fluorescence), ozone, nitrogen, sensitivity and measuring date were used as fixed factors, and plot and replicate as random factors. In case of statistically significant interactions, pairwise comparisons were used for interpretation of the effects of different factors (data not shown). For all statistical tests, the significance level was set at P < 0.05.
The correlation between hybrid aspen clones, foliar phenolic compounds and growth was studied by principal component analysis (PCA) using SIMCA-P 11.5 software (Umetrics AB, Umeå, Sweden). After unit variance scaling of the variables, a model with eight principal components, explaining over 90% of the variation was extracted. The mean values of the replicates for each treatment (control, ozone, high nitrogen and high nitrogen + ozone) per clone were used, so that the input data matrix consisted of 32 samples (eight clones with four treatments) and 26 variables (20 phenolic compounds, total dry mass, height growth, radial growth, Asat, Fv/Fm and Rubisco concentration). These variables were selected for the PCA as they showed differences between the sensitivity groups in the mixed model analysis. Biomass data are from the final harvest in September, whereas photosynthetic rates, chlorophyll fluorescence, Rubisco and phenolic concentrations were assessed at the end of August.
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Results |
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Biomass
By the end of the second growing seasons, the high-N treatment had increased the dry mass of hybrid aspen on average by 17% compared with the low-N treatment (N: P = 0.001; Figure 2). Elevated [O3] alone decreased the final dry mass of the ozone-sensitive group by 15%, but increased the dry mass of the ozone-tolerant group by 11% (O3 x G: P = 0.002; Figure 2).
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Photosynthesis and fluorescence
Elevated [O3] decreased and the high-N treatment increased net photosynthetic rates (Figure 3A; Table 1). Photosynthetic rates were reduced by elevated [O3], especially early in the growing season, as indicated by the date x ozone interaction in Table 1. In general, net photosynthetic rates tended to be higher in the ozone-sensitive group than in the ozone-tolerant group (Figure 3A; Table 1).
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The high-N treatment significantly increased the maximum quantum efficiency of photosystem II (Fv/Fm). The ozone-sensitive group had a higher Fv/Fm than the ozone-tolerant group (Figure 3B; Table 1). Elevated [O3] significantly decreased Fv/Fm in ozone-sensitive clones (O3 x G interaction), but the effect of O3 was more obvious at the August measurements than at other measurements times (Figure 3B; Table 1, D x O3 interaction).
Pigments and proteins
Elevated [O3] significantly reduced the concentrations of total chlorophylls and Rubisco (Figure 4A and C; Table 1), but had no effect on total carotenoid, TSP or nitrogen concentrations of the leaves (Figure 4B, D and E; Table 1). The high-N treatment significantly increased the concentrations of chlorophylls, carotenoids, Rubisco, TSP and leaf organic nitrogen (Figure 4A–E; Table 1). The O3-induced reduction in chlorophyll concentration was significant in July and August, but not in September (D x O3 interaction, Table 1). The nitrogen-induced increases in chlorophyll and carotenoid concentrations were especially evident at the last sampling date (D x N interaction, Table 1).
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Ozone-sensitive clones had more Rubisco than ozone-tolerant clones (Figure 4C). The difference in the amount of Rubisco between the sensitivity groups was greatest at the first sampling date, decreasing toward the end of the growing season (D x G interaction, Table 1).
Carbohydrates
Carbohydrates were analyzed from only two clones, the ozone-tolerant Clone 55 and the ozone-sensitive Clone 110. The concentration of soluble sugars increased toward the end of the growing season (Figure 5A). The ozone-sensitive clone had a higher concentration of soluble sugars than the ozone-tolerant clone, especially in the August sampling (D x C interaction, Table 2). Elevated [O3] decreased the concentrations of soluble sugars in June and August, but not in July (D x O3 interaction, Table 2). The O3-induced decrease in the concentrations of soluble sugars was greater in the ozone-sensitive clone in the June sampling and greater in the ozone-tolerant clone in the August sampling (D x O3 x C interaction, Table 2).
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Starch concentrations decreased toward the end of the growing season. The high-N treatment decreased the starch concentration in both clones (Figure 5B; Table 2). The ozone-sensitive clone had twice as much starch as the ozone-tolerant clone. In the ozone-tolerant clone, starch concentration decreased steadily throughout the growing season, whereas in the ozone-sensitive clone, starch degradation started after mid-August (D x C interaction, Figure 5B; Table 2). Elevated [O3] reduced the concentration of starch later in the growing season (July and August), but not in June (D x O3 interaction, Table 2).
Phenolics
The major phenolic compounds of hybrid aspen leaves, condensed tannins and salicylates, accounted for 50% and 25% of all foliar phenolics, respectively. The remaining phenolics comprised flavonoids, i.e. (+)-catechin (a flavan-3-ol), phenolic acids (hydroxycinnamic acid derivatives and the caffeoylquinic acids, chlorogenic acid and neochlorogenic acid) and flavonol glycosides (myricetin, quercetin, kaempferol and isorhamnetin glycosides). There were significantly more condensed tannins and (+)-catechin (a monomer in the synthesis of condensed tannins) in the ozone-tolerant group, but more salicylates in the ozone-sensitive group (Table 3). No differences between the groups in the amounts of total phenolic acids or flavonol glycosides were found, even though there were significant differences between the groups in the concentrations of single compounds (Table 3).
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Significant differences between the sensitivity groups were found in most individual phenolics, except for chlorogenic acid, quercetin derivative 2 and two p-OH-cinnamic acid derivatives (Table 3). In most cases, ozone-sensitive clones had higher phenolic concentrations than ozone-tolerant clones, except for condensed tannins, (+)-catechin and a kaempferol derivative, which were present in higher concentrations in the ozone-tolerant group (Table 3). Elevated [O3] significantly increased the concentrations of (+)-catechin and chlorogenic acid, and decreased the concentrations of p-OH-cinnamic acid derivative 5 and salicin, but the O3-induced decrease in salicin concentration was only observed in the ozone-sensitive group (O3 x G interaction, Table 3).
Principal component analysis was used to study the relationship between the clones and different phenolic compounds. As for the results of the ANOVA presented in Table 3, the clones were separated by the ozone sensitivity groups rather than by ozone or nitrogen treatments along the first principal component (PC) explaining 29% of the variation (Figure 6). The second PC explained 19% of the total variation in the data, and was characterized by high scores in biomass attributes and neochlorogenic acid, which were negatively correlated with condensed tannins and (+)-catechin (Figure 6). The ozone-sensitive Clone 110 was grouped among the ozone-tolerant Clones 1 and 14, probably because of moderately good growth and also because of the negative correlation with (and thus small concentration of) quercetin derivatives 1, 2 and 4, p-OH-cinnamic acid derivative 3 and chlorogenic acid. Fast-growing ozone-tolerant Clones 1 and 14 were characterized by kaempferol, whereas the slow-growing tolerant Clones 55 and 193 contained high concentrations of condensed tannins and (+)-catechin. Salicylates, p-OH cinnamic acid derivatives 4 and 5, and quercetin derivatives 1 and 3 were present in high concentrations in the ozone-sensitive clones (Figure 6).
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Nitrogen amendment counteracted the effects of elevated [O3] on chlorophyll and Rubisco
In aspen, onset of senescence is normally determined by photoperiod (Keskitalo et al. 2005). Chloroplast degradation is one of the first signs of senescence, and abiotic stress leading to loss of photosynthetic capacity or membrane integrity might produce a signal for the initiation of senescence (Quirino et al. 2000). In our experiment, however, elevated [O3] reduced the net photosynthetic rate and the chlorophyll concentration, especially early in the growing season. The lower concentration of chlorophyll in O3-fumigated plants may also be associated with delayed bud opening, resulting in a shift in the leaf developmental stage at the beginning of the growing season (Freiwald et al. 2008). We started measuring photosynthetic rates right after the leaves had reached maturity in mid-June, and the greatest O3-induced reduction at this stage supports the idea that maximum ozone sensitivity occurs immediately after full expansion of the leaves (Coleman 1986). The concentration of Rubisco was lower in saplings exposed to elevated [O3] throughout the growing season, resulting in declines in net photosynthetic rate and whole-plant biomass. Elevated [O3] reduced the maximum quantum yield of photosystem II (Fv/Fm) only in the ozone-sensitive clones, although the values stayed high until the end of August and were generally higher in the ozone-sensitive clones. Keskitalo et al. (2005) had also found that Fv/Fm stayed high until the end of September, when chlorophyll concentrations had already decreased by 80%, suggesting that the remaining photosystem II centers remained photosynthetically active until the late stages of senescence. The concentration of carotenoids decreased toward the end of the growing season, but carotenoid degradation was slower than chlorophyll degradation. Our analysis did not distinguish among the different carotenoids, but Keskitalo et al. (2005) had found that β-carotene concentration decreased in parallel with chlorophyll concentrations, whereas the xanthophyll cycle pigments were more stable. Even though there were no significant O3-induced changes in carotenoid concentrations, the slightly higher carotenoid concentration in O3-fumigated plants at the end of the growing season could indicate activation of the xanthophyll cycle in response to increased oxidative stress (Figure 4B).
Plants exposed to declining nitrogen availability and elevated [O3] are reported to have accelerated leaf senescence (Bielenberg et al. 2001). In our study, high-N plants had higher concentrations of chlorophylls, carotenoids, Rubisco and TSP and a higher photosynthetic capacity compared with low-N plants, as found by Pääkkönen and Holopainen (1995) in birch. Under low-N conditions, high availability of carbon relative to nitrogen may lead to sugar accumulation, which in turn affects the photosynthetic rate by end product feedback inhibition, triggering leaf senescence (Wingler et al. 2006). In our study, accumulation of soluble sugars was observed toward the end of the growing season, along with the breakdown of starch, but we found no effect of soil nitrogen supply on the concentrations of soluble sugars. Elevated [O3] reduced the concentration of starch later in the growing season (July and August), and also the concentrations of soluble sugars in June and August, but not in July. Yonekura et al. (2004) had found decreased concentrations of nonstructural carbohydrates in response to ozone in beech; however, in most studies, increased concentrations of soluble sugars and starch, or no effects of elevated [O3] on nonstructural carbohydrates (e.g., in birch, beech and aspen) have been found (Lavola et al. 1994, Lux et al. 1997, Lindroth et al. 2001, Liu et al. 2005). Ozone-induced breakdown of starch in the absence of an effect on photosynthesis might indicate partitioning of assimilated carbon into increased respiration and injury repair processes (Topa et al. 2001).
Ozone-sensitive clones had high Rubisco concentrations and high photosynthetic capacity
In our study, Clones 1, 14 and 280 were considered fast-growing based on dry mass, height growth and radial growth of nontreated potted plants after the second growing season (Häikiö et al. 2007). Of these, Clones 1 and 14 were placed in the ozone-tolerant group and Clone 280 in the ozone-sensitive group (Häikiö et al. 2007). The ozone-sensitive group had higher concentrations of Rubisco and a higher photosynthetic capacity than the ozone-tolerant group. The higher photosynthetic rate may be associated with higher stomatal conductance leading to increased O3 uptake in the ozone-sensitive clones (Wittig et al. 2007). However, there were no differences in the responses of the parameters measured in this study to elevated [O3] between the two groups, as shown by the lack of ozone x group interaction, except for Fv/Fm, which showed a significant decrease in response to elevated [O3] only in the ozone-sensitive group. The higher proportion of total protein in Rubisco in the ozone-sensitive group might indicate less carbon allocation to enzymes related to redox reactions and regeneration of antioxidants, thereby leading to increased sensitivity to O3.
As leaves are most sensitive to O3 after full expansion early in the growing season (Coleman 1986), when ambient [O3] is also high, a significant reduction in net photosynthetic rate of the first flush leaves at this time may lead to reduced biomass accumulation by the end of the growing season. We cannot determine the reason for the decline in photosynthetic rate in early summer, because we did not take the first samples for analyses of pigments, proteins and carbohydrates until July and the samples for analysis of phenolics were not taken until the end of August. However, the O3-induced decline in the concentration of chlorophyll was greater in the first sampling in July than later in the growing season, perhaps indicating a key role of chlorophyll concentration in photosynthetic efficiency.
Does the phenolic profile of the leaves explain the ozone sensitivity of hybrid aspen clones?
The main phenolic compounds of aspen are salicin-based phenolic glycosides (salicylates), hydroxycinnamate derivatives, flavonoids and flavonoid-derived condensed tannins, which may comprise up to 35% of the dry mass of the leaves (Hwang and Lindroth 1997, Tsai et al. 2006). The concentration of phenolics of aspen leaves decreases during the growing season, with young leaves having the highest concentrations of phenolics before leaf maturation (Coleman 1986). The concentrations also change with tree age. In trembling aspen, the concentration of condensed tannins increased sharply with age, primarily between the first and the second year of growth from about 4% to 14% of leaf dry mass. At the same time, the concentrations of salicylates decreased to < 1% (Donaldson et al. 2006). We collected leaf samples for phenolic analysis at the end of the growing season from 3-year-old plants, and found that condensed tannins and salicylates made up 15% and 5% of leaf dry mass, respectively. Environmental factors, such as light availability or soil nutrients, can markedly alter the concentrations of condensed tannins, but only marginally alter the concentrations of salicylates in trembling aspen and birch (Hwang and Lindroth 1997, Keinänen et al. 1999, Stevens and Lindroth 2005). In our study, soil nitrogen status did not affect the foliar phenolic concentration.
Salicylates are thought to act mainly as deterrents to insect herbivores (Hwang and Lindroth 1997, Osier and Lindroth 2006), but the roles of flavonoids and condensed tannins in plant defense are not well established (Ayres et al. 1997). In our study, elevated [O3] did not affect the concentration of total salicylates, but it reduced the concentration of salicin. The higher substituted salicylates, salicortin and tremulacin, typically comprise > 90% of the phenolic glycoside pool (Lindroth et al. 2002). Salicin is both a precursor and a decomposition product of the higher molecular mass salicylates (Ruuhola and Julkunen-Tiitto 2003), but the lower concentration of salicin in plants fumigated with elevated [O3] did not result in lower concentrations of salicortin or tremulacin. Low-molecular mass flavonoids may act as antioxidants in leaves, but condensed tannins with a high degree of polymerization have even better radical-scavenging properties (Hagerman et al. 1998). We found higher concentrations of condensed tannins and their precursor (+)-catechin in O3-fumigated plants, which is consistent with previous results of Holton et al. (2003) and Liu et al. (2005), for example, whereas no effect of elevated [O3] on condensed tannins was found by Lavola et al. (1994) and Lindroth et al. (2001) in birch and trembling aspen. The differences in the responses between studies may be associated with the different ages of the trees in these experiments (Kolb and Matyssek 2001). Chlorogenic acid is an effective scavenger of superoxide (Grace et al. 1998), and as in our experiment, increased concentrations of chlorogenic acid were also found in O3-fumigated birches (Saleem et al. 2001, Peltonen et al. 2005). We found no effect of elevated [O3] on concentrations of total flavonol glycosides in hybrid aspen in contrast to birch (Saleem et al. 2001), indicating that flavonol glycosides do not have a major role in the antioxidative defense system in hybrid aspen.
There was substantial clonal variation in the foliar phenolic profile, as indicated by the PCA (Figure 6). In general, the ozone-sensitive group contained more phenolics than the ozone-tolerant group, and this difference derived mainly from salicylates (the ozone-sensitive clones had more salicylates than the ozone-tolerant clones). Salicylates comprised about 34% and 13% of all phenolics in the ozone-sensitive and ozone-tolerant groups, respectively. The ozone-tolerant group contained higher concentrations of condensed tannins and (+)-catechin than the ozone-sensitive group (75% and 56% of all phenolics in the ozone-tolerant and ozone-sensitive groups, respectively). This finding suggests that condensed tannins protect the ozone-tolerant clones from oxidative stress, whereas the ozone-sensitive group allocates carbon to salicylates that have no antioxidative capacity, but may reduce the susceptibility of these clones to insect herbivores (Hwang and Lindroth 1997).
When the phenolic compounds of the clones were examined individually (data not shown, see Figure 6), Clone 193, that seemed to grow better in elevated [O3] than in ambient air, had low concentrations of salicylates, but a high concentration of chlorogenic acid. The ozone-tolerant Clones 1 and 14 also had low concentrations of salicylates but contained more of the kaempferol derivatives than the other clones. Among the flavonoids, quercetin has the highest antioxidant capacity, followed by (+)-catechin, myricetin and kaempferol (Rice-Evans et al. 1996), but there were no differences in the concentration of the major quercetin derivative (quercetin der. 2) among clones or between ozone treatments. The high concentrations of kaempferol in Clones 1 and 14 might have conferred some ozone tolerance to these clones, with apparently no cost on growth. On the contrary, condensed tannins are expensive in terms of metabolic costs because of the high concentrations found in hybrid aspen, and they are strongly negatively correlated with growth and associated with the slow-growing clones (Figure 6; Gershenzon 1994). Biosynthetic costs of chlorogenic acid are among the cheapest of plant phenolics (Gershenzon 1994), and at the moderately low concentrations found in hybrid aspen, have less effect on growth. Isorhamnetin (3'-methylquercetin) was negatively correlated with growth and was mainly found in the slow-growing ozone-sensitive clones. The methyl group makes isorhamnetin more expensive than the parent compound (Gershenzon 1994), but also less effective in antioxidative capacity (Heim et al. 2002).
In conclusion, we demonstrated that hybrid aspen is sensitive to O3, and even a moderately elevated [O3] lowered the concentrations of chlorophylls and Rubisco, and reduced net photosynthetic rates early in the growing season. Nitrogen amendment did not affect the ozone sensitivity of the clones, but counteracted the effects of elevated [O3] by enhancing the net photosynthetic rate and increasing the concentrations of chlorophyll and proteins, thus lengthening the growing season. We grouped eight hybrid aspen clones into ozone-sensitive and ozone-tolerant clones based on the effect of elevated [O3] on growth; however, we were unable to determine the cause of the decreased growth of the ozone-sensitive clones in response to elevated [O3]. The ozone-sensitive group had more Rubisco and higher photosynthetic capacity, and also higher concentrations of starch and secondary compounds than the ozone-tolerant group, but as we found almost no differences in responses to elevated [O3] between the sensitivity groups, the clonal differences in the primary and secondary metabolism must mostly reflect intraspecific genetic variability. The finding that both ozone-sensitive and ozone-tolerant clones were grouped among the good growers does not support the notion that fast-growing clones are more sensitive to ozone. Fast-growing hybrid aspen is a good candidate for bioenergy (Rytter 2006), and for this purpose it is important to find ozone-tolerant clones with a high initial growth rate. However, susceptibility to other stresses, e.g., herbivory in the case of clones with low concentrations of salicylates, also has to be assessed before large-scale clonal plantations are established.
The concentrations of most phenolic compounds in hybrid aspen seem to be strictly genetically determined and different clones differ greatly in foliar phenolic profiles. Chlorogenic acid and (+)-catechin, and to some extent also condensed tannins, were the only phenolic compounds that were increased by elevated ozone. Ozone-tolerant clones had high concentrations of condensed tannins, whereas the ozone-sensitive clones allocated carbon to salicylates that have no antioxidative properties. High concentration of condensed tannins is an expensive defense mechanism, and it was usually associated with poor growth. Flavonol glycosides do not seem to play a role in defense against oxidative stress of hybrid aspen, despite their antioxidative capacity.
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This study was funded by the Academy of Finland (Project No. 51758), University of Kuopio Environmental Risk Assessment Center (ERAC), and North Savo Regional Fund of the Finnish Cultural Foundation. The authors thank Timo Oksanen for ozone fumigations and technical assistance, Teodor Homentcovschi for fluorescence measurements, Leena Mettinen for carbohydrate analyses, Dr. Åsmund Rinnan for advice on PCA, and Dr. Johanna Riikonen and Dr. Minna Kivimäenpää for valuable comments on the manuscript.
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Agrell J., Kopper B., McDonald E.P., Lindroth R.L. CO2 and O3 effects on host plant preferences of the forest tent caterpillar (Malacosoma disstria). Global Change Biol. (2005) 11:588–599.
Allen S.E. Chemical analysis of ecological materials (1989) London: Blackwell Scientific Publications. 368.
Arnon D.I. Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol. (1949) 24:1–15.
Ayres M.P., Clausen T.P., MacLean S.F. Jr., Redman A.M., Reichardt. P.B. Diversity of structure and antiherbivore activity in condensed tannins. Ecology (1997) 78:1696–1712.[Web of Science]
Bielenberg D., Lynch J., Pell E. A decline in nitrogen availability affects plant responses to ozone. New Phytol. (2001) 151:413–425.[CrossRef]
Bortier K., De Temmerman L., Ceulemans R. Effects of ozone exposure in open-top chambers on poplar (Populus nigra) and beech (Fagus sylvatica): a comparison. Environ. Pollut. (2000) 109:509–516.[CrossRef][Medline]
Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Brendley B.W., Pell E.J. Ozone-induced changes in biosynthesis of Rubisco and associated compensation to stress in foliage of hybrid poplar. Tree Physiol. (1998) 1817:81–90.
Cao G., Sofic E., Prior R.L. Antioxidant and prooxidant behavior of flavonoids: structure–activity relationships. Free Radic. Biol. Med. (1997) 22:749–760.[CrossRef][Web of Science][Medline]
Coleman J.S. Leaf development and leaf stress: increased susceptibility associated with sink–source transition. Tree Physiol. (1986) 2:289–299.
Conklin P.L., Barth C. Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ. (2004) 27:959–970.[CrossRef]
Donaldson J., Stevens M., Barnhill H., Lindroth R. Age-related shifts in leaf chemistry of clonal aspen (Populus tremuloides). J. Chem. Ecol. (2006) 32:1415–1429.[CrossRef][Web of Science][Medline]
Foyer C.M., Noctor G. Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ. (2005) 28:1056–1071.[CrossRef]
Freiwald V., Häikiö E., Julkunen-Tiitto R., Holopainen J.K., Oksanen E. Elevated ozone modifies the feeding behaviour of the common leaf weevil on hybrid aspen through shifts in developmental, chemical, and structural properties of leaves. Entomol. Exp. Appl. (2008) 128:66–72.
Gershenzon J. The cost of plant chemical defense against herbivory: a biochemical perspective. In: Insect–Plant Interactions—Bernays E.A., ed. (1994) Boca Raton: CRC Press. 105–173.
Grace S.C., Logan B.A., Adams W.W. Seasonal differences in foliar content of chlorogenic acid, a phenylpropanoid antioxidant, in Mahonia repens. Plant Cell Environ. (1998) 21:513–521.
Greitner C.S., Pell E.J., Winner W.E. Analysis of aspen foliage exposed to multiple stresses: ozone, nitrogen deficiency and drought. New Phytol. (1994) 127:579–589.
Grenfell J.L., Shindell D.T., Grewe V. Sensitivity studies of oxidative changes in the troposphere in 2100 using the GISS GCM. Atmos. Chem. Phys. (2003) 3:1267–1283.
Haapanen M, Mikola J. (2008) Metsänjalostus 2050: pitkän aikavälin metsänjalostusohjelma. Metlan Työraportteja/Working Papers of the Finnish Forest Research Institute 71, 50 pp. Available at http://www.metla.fi/julkaisut/workingpapers/2008/mwp071.htm. (in Finnish).
Hagerman A.E., Riedl K.M., Jones G.A., Sovik K.N., Ritchard N.T., Hartzfeld P.W., Riechel T.L. High molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food Chem. (1998) 46:1887–1892.[CrossRef][Web of Science]
Häikiö E., Freiwald V., Silfver T., Beuker E., Holopainen T., Oksanen E. Impacts of elevated ozone and nitrogen on growth and photosynthesis of European aspen (Populus tremula) and hybrid aspen (P. tremula x Populus tremuloides) clones. Can. J. For. Res. (2007) 37:2326–2336.
Hansen J., Møller I. Percolation of starch and soluble carbohydrates from plant tissue for quantitative determination with anthrone. Anal. Biochem. (1975) 68:87–94.[Medline]
Heim K.E., Tagliaferro A.R., Bobilya D.J. Flavonoid antioxidants: chemistry, metabolism and structure–activity relationships. J. Nutr. Biochem. (2002) 13:572–584.[CrossRef][Web of Science][Medline]
Heiska S., Tikkanen O., Rousi M., Julkunen-Tiitto R. Bark salicylates and condensed tannins reduce vole browsing amongst cultivated dark-leaved willows (Salix myrsinifolia). Chemoecology (2007) 17:245–253.
Holton M.K., Lindroth R., Nordheim E. Foliar quality influences tree–herbivore–parasitoid interactions: effects of elevated CO2, O3, and plant genotype. Oecologia (2003) 137:233–244.[CrossRef][Web of Science][Medline]
Hwang S., Lindroth R.L. Clonal variation in foliar chemistry of aspen: effects on gypsy moths and forest tent caterpillars. Oecologia (1997) 111:99–108.[CrossRef][Web of Science]
Julkunen-Tiitto R., Sorsa S. Testing the effects of drying methods on willow flavonoids, tannins, and salicylates. J. Chem. Ecol. (2001) 27:779–789.[CrossRef][Web of Science][Medline]
Keinänen M., Julkunen-Tiitto R., Mutikainen P., Walls M., Ovaska J., Vapaavuori E. Trade-offs in phenolic metabolism of silver birch: effects of fertilization, defoliation, and genotype. Ecology (1999) 80:1970–1986.[Web of Science]
Keskitalo J., Bergquist G., Gardeström P., Jansson S. A cellular timetable of autumn senescence. Plant Physiol. (2005) 139:1635–1648.
Kleiner K.W., Raffa K.F., Ellis D.D., McCown B.H. Effect of nitrogen availability on the growth and phytochemistry of hybrid poplar and the efficacy of the Bacillus thuringiensis cry1A(a) d-endotoxin on gypsy moth. Can. J. For. Res. (1998) 28:1055–1067.
Kolb T.E., Matyssek R. Limitations and perspectives about scaling ozone impacts in trees. Environ. Pollut. (2001) 115:373–393.[CrossRef][Medline]
Lavola A., Julkunen-Tiitto R., Pääkkönen E. Does ozone stress change the primary or secondary metabolites of birch (Betula pendula Roth.)? New Phytol. (1994) 126:637–642.
Lichtenthaler H.K., Wellburn A.R. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. (1983) 11:591–592.
Lindroth R.L., Kopper B.J., Parsons W.F.J., Bockheim J.G., Karnosky D.F., Hendrey G.R., Pregitzer K.S., Isebrands J.G., Sober J. Consequences of elevated carbon dioxide and ozone for foliar chemical composition and dynamics in trembling aspen (Populus tremuloides) and paper birch (Betula papyrifera). Environ. Pollut. (2001) 115:395–404.[Medline]
Lindroth R.L., Osier T.L., Barnhill H.R.H., Wood S.A. Effects of genotype and nutrient availability on phytochemistry of trembling aspen (Populus tremuloides Michx.) during leaf senescence. Biochem. Syst. Ecol. (2002) 30:297–307.[CrossRef][Web of Science]
Liu L., King J.S., Giardina C.P. Effects of elevated concentrations of atmospheric CO2 and tropospheric O3 on leaf litter production and chemistry in trembling aspen and paper birch communities. Tree Physiol. (2005) 25:1511–1522.
Lux D., Leonardi S., Müller J., Wiemken A., Flückiger W. Effects of ambient ozone concentration on contents of non-structural carbohydrates in young Picea abies and Fagus sylvatica. New Phytol. (1997) 137:399–409.
Oksanen E., Amores G., Kokko H., Santamaria J.M., Kärenlampi L. Genotypic variation in growth and physiological responses of Finnish hybrid aspen (Populus tremuloides x P. tremula) to elevated tropospheric ozone concentration. Tree Physiol. (2001) 21:1171–1181.
Osier T., Lindroth R. Genotype and environment determine allocation to and costs of resistance in quaking aspen. Oecologia (2006) 148:293–303.[CrossRef][Web of Science][Medline]
Pääkkönen E., Holopainen T. Influence of nitrogen supply on the response of clones of birch (Betula pendula Roth.) to ozone. New Phytol. (1995) 129:595–603.
Pääkkönen E., Holopainen T., Kärenlampi L. Differences in growth, leaf senescence and injury, and stomatal density in birch (Betula pendula Roth.) in relation to ambient levels of ozone in Finland. Environ. Pollut. (1997) 9630:117–127.
Pell E.J., Schlagnhaufer C.D., Arteca R.N. Ozone-induced oxidative stress: mechanisms of action and reaction. Physiol. Plant. (1997) 100:264–273.[CrossRef]
Pell E.J., Sinn J.P., Brendley B.W., Samuelson L., Vinten-Johansen C., Tien M., Skillman J. Differential response of four tree species to ozone-induced acceleration of foliar senescence. Plant Cell Environ. (1999) 22:779–790.
Peltonen P.A., Vapaavuori E., Julkunen-Tiitto R. Accumulation of phenolic compounds in birch leaves is changed by elevated carbon dioxide and ozone. Global Change Biol. (2005) 11:1305–1324.
Porra R.J. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth. Res. (2002) 73:149–156.[CrossRef][Web of Science][Medline]
Porter L.J., Hrstich L.N., Chan B.G. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry (1986) 25:223–230.[CrossRef][Web of Science]
Quirino B.F., Noh Y., Himelblau E., Amasino R.M. Molecular aspects of leaf senescence. Trends Plant Sci. (2000) 5:278–282.[CrossRef][Web of Science][Medline]
Reich P.B., Lassoie J.P. Influence of low concentrations of ozone on growth, biomass partitioning and leaf senescence in young hybrid poplar plants. Environ. Pollut. (1985) 39:39–51.
Ribas A., Peñuelas J., Elvira S., Gimeno B.S. Ozone exposure induces the activation of leaf senescence-related processes and morphological and growth changes in seedlings of Mediterranean tree species. Environ. Pollut. (2005) 134:291–300.[CrossRef][Medline]
Rice-Evans C., Miller N.J., Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. (1996) 20:933–956.[CrossRef][Web of Science][Medline]
Rintamäki E., Keys A.J., Parry M.A.J. Comparison of the specific activity of ribulose-1,5-bisphosphate carboxylase-oxygenase from some C3 and C4 plants. Physiol. Plant. (1988) 74:326–331.[CrossRef]
Ruuhola T., Julkunen-Tiitto R. Trade-off between synthesis of salicylates and growth of micropropagated Salix pentandra. J. Chem. Ecol. (2003) 29:1565–1588.[CrossRef][Web of Science][Medline]
Rytter L. A management regime for hybrid aspen stands combining conventional forestry techniques with early biomass harvests to exploit their rapid early growth. Forest Ecol. Manag. (2006) 236:422–426.
Saleem A., Loponen J., Pihlaja K., Oksanen E. Effects of long-term open-field ozone exposure on leaf phenolics of European silver birch (Betula pendula Roth.). J. Chem. Ecol. (2001) 27:1049–1062.[Medline]
Sitch S., Cox P.M., Collins W.J., Huntingford C. Indirect radiative forcing of climate change through ozone effects on the land–carbon sink. Nature (2007) 448:791–794.[CrossRef][Medline]
Stevens M., Lindroth R. Induced resistance in the indeterminate growth of aspen (Populus tremuloides). Oecologia (2005) 145:297–305.
Tausz M., Grulke N.E., Wieser G. Defense and avoidance of ozone under global change. Environ. Pollut. (2007) 147:525–531.[Medline]
Topa M.A., Vanderklein D.W., Corbin A. Effects of elevated ozone and low light on diurnal and seasonal carbon gain in sugar maple. Plant Cell Environ. (2001) 24:663–677.
Tsai C., Harding S.A., Tschaplinski T.J., Lindroth R.L., Yuan Y. Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol. (2006) 172:47–62.[CrossRef][Medline]
Vingarzan R. A review of surface ozone background levels and trends. Atmos. Environ. (2004) 38:3431–3442.
Wingler A., Purdy S., MacLean J.A., Pourtau N. The role of sugars in integrating environmental signals during the regulation of leaf senescence. J. Exp. Bot. (2006) 57:391–399.
Wittig V.E., Ainsworth E.A., Long S.P. To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last 3 decades of experiments. Plant Cell Environ. (2007) 30:1150–1162.[Medline]
Yonekura T., Yoshidome M., Watanabe M., Honda Y., Ogiwara I., Izuta T. Carry-over effects of ozone and water stress on leaf phenological characteristics and bud frost hardiness of Fagus crenata seedlings. Trees Struct. Funct. (2004) 18:581–588.
Zeng G., Pyle J.A., Young P.J. Impact of climate change on tropospheric ozone and its global budgets. Atmos. Chem. Phys. (2008) 8:369–387.
Zimmermann P., Zentgraf U. The correlation between oxidative stress and leaf senescence during plant development. Cell. Mol. Biol. Lett. (2005) 10:515–534.[Web of Science][Medline]
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