5 Kun Yan1,2 Xingyuan He1 Wei Chen1 Tao Lu1 Sheng Xu1 Hongbo Shao2,3 Guoyou Zhang1,4 1
Department of Urban Forest, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, P. R. China 2 Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, P. R. China 3 Institute of Life Sciences, Qingdao University of Science and Technology, Qingdao, P. R. China 4 Laboratory of International Plant Resources, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan
Research Article Variation of Antioxidant System in Pinus armandii under Elevated O3 in an Entire Growth Season Impact of elevated O3 (80 nmol mol1) was studied on antioxidant system in the previous-year needles (P-needles) of Pinus armandii under the background of global climate change. CAT (catalase) did not play an important role in defensing O3 pollution, as no significant change was observed in its activity in P-needles exposed to elevated O3 during the entire growth season. Ascorbate (AsA) was more sensitive to elevated O3, as the O3-induced decrease in its content was observed at Day 45. It suggests that AsA consumption was a crucial way for protecting against O3 pollution. After 75 days of O3 exposure, increase in superoxide dismutase and ascorbate peroxidase activities occurred in P-needles (p < 0.05), whereas glutathione reductase activity was significantly increased after 105 days of O3 treatment (p < 0.05). Dehydroascorbate reductase and monoascorbate reductase activities also increased after 75 days of elevated O3 exposure. However, the elevated antioxidant capability did not suppress the generation of reactive oxygen species, and as a result, increase in malondialdehyde content was noted (p < 0.05). Noticeably, no change was observed in electrolyte leakage during the entire growth season under elevated O3 fumigation. It indicates that O3-induced oxidative stress was not severe in the needles of P. armandii. In other words, P. armandii has certain tolerance to O3 pollution. Keywords: Biomarker; East Asia; Global climate change; Lipid peroxidation Received: April 11, 2012; revised: May 28, 2012; accepted: June 7, 2012 DOI: 10.1002/clen.201200161
1 Introduction Ground-level background O3 concentration has been increasing to current level of 20–45 nmol mol1 in the middle latitude northern hemisphere since pre-industrial times, and this rising trend is expected to persist throughout the century to levels in the range of 42–84 nmol mol1 by 2100 due to the increased emission of O3 precursors [1]. Especially in China, anthropogenic emission of O3 precursors have increased significantly due to rapid urbanization and industrialization, compared with a great reduction in Europe and mild variation in North America [2–4]. Ozone is a phytotoxic air pollutant which can increase the generation of reactive oxygen species (ROS) in plant cell [5–7]. Antioxidant system regulating ROS concentration in plants plays a crucial role in response to high environmental O3 [8]. Antioxidant system consists of antioxidant enzymes and antioxidants. The most abundant antioxidant is ascorbate (AsA). Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) are ROS-scavenging enzymes,
Correspondence: Prof. H. Shao, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China E-mail:
[email protected] Abbreviations: AA, ambient air; AsA, ascorbate; APX, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascorbate reductase; DW, dry weight; EL, electrolyte leakage; EO, elevated O3; GR, glutathione reductase; MDA, malondialdehyde; MDAR, monoascorbate reductase; OTC, open top chamber; P-needles, previous-year needles; ROS, reactive oxygen species; SOD, superoxide dismutase
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while glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monoascorbate reductase (MDAR) act as antioxidantregenerating enzymes. AsA works as substrate for APX to scavenge hydrogen peroxide and also can directly clear ROS [5, 7, 9]. The positive role of AsA is demonstrated by the higher O3-sensitivity in the mutants without AsA synthesis capacity [10] and by plant overexpressing AsA oxidase [11]. SOD cooperates with APX and CAT to scavenge superoxide anion radical and hydrogen peroxide and then limits the production of hydroxyl radical, which can avoid dangerous oxidative damage in plant cell [5, 7, 12]. DHAR, MDAR, and GR cooperate to regenerate glutathione and AsA from their oxidized forms at the expense of reducing power [12]. In previous studies, increase in antioxidants content or antioxidant enzymes activities was widely reported in broadleaf trees as well as conifers under O3 exposure (e.g., [13, 14]), as O3 stimulated the antioxidant defense capability in plants. In contrast, it has been found that O3 decreased antioxidant capacity in Laurus nobilis leaves and Picea abies needles [14, 15]. Besides, high O3 did not affect antioxidant content in Pinus canariensis needles [16]. Much work has been done to explore how elevated O3 affect trees distributed in North America and Mediterranean area, but the endemic tree species in East Asia has not been well concerned. So far, studies on the effects of elevated O3 have mainly focused on important crops such as wheat and rice in Asia [17–19], since it is still a crucial issue to provide enough food for the countries with great
Additional correspondence: X. He, e-mail:
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population such as China and India as well as Japan and Korea possessing high population density and less farmland. Under global climate change, sustainable development and ecological environmental planning have become vital concerns. Compared with farmland, forestry ecosystems play more important roles in air pollution clearance. Therefore, it is necessary to ascertain how the endemic trees in Asia response to the continuous increase of surface O3. Pinus armandii, as an endemic conifer species in East Asia, is used for landscape construction and taken as important timber source in China. It has been proved that biomass accumulation was greatly reduced in P. armandii exposed to elevated O3 [20]. However, it did not elucidate the response of antioxidant system to elevated O3. In our study, changes in lipid peroxidation and antioxidant system were detected in needles of P. armandii under elevated O3 exposure, which could help thoroughly evaluate the effects of climatic conditions in the future on this tree species. Ginkgo biloba and Quercus monogolica which are deciduous tree species grow in East Asia as well, and how antioxidant defense system responses to elevated O3 in these trees has been illustrated in our previous studies [21–23]. In this paper, we compared not only the response of antioxidant system to elevated O3 in P. armandii with those in G. biloba and Q. monogolica, but also compared the responses of tree species in East Asia with those in other continents. Consequently, it can be characterized how endemic tree species in East Asia defense the sustaining rise of surface O3 in the context of global climate change.
2 Materials and method
2.3 Measurement of ascorbate content AsA content was assayed according to Davey et al. [25] with some modification, and the detailed protocol has been illuminated previously [23].
2.4 Enzyme extraction and assay The extraction and measuring protocols which were modified from others’ researches have been illuminated in our previous study [23, 26–28].
2.5 Measurement of lipid peroxidation and cell membrane stability The protocols for measuring lipid peroxidation and cell membrane stability have been described in our study [23], and these protocols were modified according to others’ reports [28, 29].
2.6 Statistical analysis One-way ANOVA was done by using SPSS (SPSS, 1999) for all data. The values in this paper are the means of all measurements, and the means were compared through least significant difference test.
3 Results 3.1 Ozone data
The experimental site was established in Shenyang Arboretum, Chinese Academy of Sciences, Liaoning province, China (418460 N, 1238260 E and 41 m above sea level). This area has been described in detail previously [23].
Ozone exposure higher than 40 nmol mol1 can lead to harmful effects on plants [8]. AOT40 indicates aggregate ozone exposure over 40 nmol mol1 in daytime (nmol mol1 h) [30]. Mean value of AOT40 was 89.6, 364.6, 620.4, and 885.4 in ambient air (AA) treatment, whereas it was 5832.0, 16667.1, 29951.1, and 44814.6 in elevated O3 (EO) treatment, respectively on June 12, June 28, July 28, August 28, and September 28 in 2008.
2.2 Open top chambers and plant materials
3.2 MDA content, EL and antioxidant system
We constructed the open top chambers (OTCs) by virtue of previous method [24]. These chambers and ozone treatment method have been described in detail in our previous study [23]. P. armandii trees (5-year-old) were planted into the OTCs in April 15, 2007. In each OTC, there were twenty seedlings which were randomly distributed. In each treatment, three replicate OTCs were utilized. The O3 concentration reached about 80 nmol mol1 in OTCs with O3 treatment while O3 concentration was about 40 nmol mol1 in OTCs of ambient air. The O3 treatment and sampling days are the same with our previous study [23]. The O3 treatment was performed from June 13 to September 28, 2008. Previous-year needles (P-needles, 1-year-old) were collected on June 12, June 28, July 28, August 28, and September 28. In each sampling day, the needles (five bundles of needles per tree) were collected in seven trees which were randomly chosen in each OTC. The collected needles were frozen immediately in liquid nitrogen and stored for measuring of antioxidant enzyme activities and ascorbate (AsA) and malondialdehyde (MDA) contents. Electrolyte leakage (EL) measurement was carried out using other needles. Parallel sample was collected to determine DW by drying needles at 608C to constant weight.
Elevated O3 markedly elevated MDA content after 75 days exposure ( p < 0.05), and at Day 105, it increases the MDA content by 60.5% ( p < 0.01; Fig. 1A). No significant change was induced by elevated O3 in EL (Fig. 1B). SOD and APX activities were stimulated as well after 75 days of O3 fumigation ( p < 0.05; Fig. 1C and D), while CAT activity did not change greatly during the entire O3 fumigation period (Fig. 1E and F). DHAR and MDAR activities were higher in P-needles in EO treatment than in AA control after 75 days, but the difference was not significant (Fig. 1G and H). Elevated O3 enhanced GR activity after 75 days of exposure, and the increase reached significant level at Day 105 ( p < 0.05; Fig. 1I). AsA content was decreased by elevated O3 at Day 45 ( p < 0.05), and so was at Day 105 ( p < 0.01; Fig. 1F).
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2.1 Experimental site
4 Discussion ROS can bring about lipid peroxidation [31]. MDA content representing the extent of lipid peroxidation associates with the level of O3 exposure [32]. At Day 75, O3 fumigation markedly increased MDA content in the P-needles (Fig. 1A), indicating oxidative stress occurred. EL was not significantly affected by elevated O3 in the P-needles during the entire experimental period (Fig. 1B), suggesting 2013, 41 (1), 5–10
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Figure 1. Effects of elevated O3 on previous-year needles of Pinus armandii. (A) Malondialdehyde (MDA) content; (B) electrolyte leakage, (C) ascorbate peroxidase (APX) activity; (D) superoxide dismutase (SOD) activity, (E) catalase (CAT) activity; (F) ascorbate (AsA) content; (G) dehydroascorbate reductase (DHAR) activity, (H) monoascorbate reductase (MDAR) activity; (I) glutathione reductase (GR) activity. Data in the figure indicate means of three independent OTCs ( standard deviation). Significant difference induced by O3 is indicated by asterisks: p < 0.05 and p < 0.01.
that O3-induced oxidative stress was not severe enough to cause cell membrane injury. Our previous OTC studies also demonstrated that elevated O3 (80 nmol mol1) caused oxidative stress in the leaves of G. biloba and Q. monogolica, however, the significant increase of MDA content occurred at day 45 due to elevated O3 exposure and cell membrane injury indicated by remarkable increase in EL was found at day 75 in their leaves [17, 23]. It suggests that G. biloba and Q. monogolica were more susceptible to O3 than P. armandii. Consistently, Reich [33] proposed that O3 tolerance was higher in conifers than in broadleaf trees, as a result of less O3 uptake subsequent to lower stomatal conductance in needles at elevated O3.
However, in contrast to some Mediterranean conifers such as Pinus halepensis and Pinus pinea, Mediterranean evergreen broadleaf trees are shown to be more tolerant to O3 pollution, because they have sclerophyllous leaves, low gas exchange rates and high antioxidant ability to tolerate oxidative stress [14, 20–29, 34–40]. In addition, Pinus ponderosa, a widely distributed conifer in North America, has been proved to be sensitive to O3 as well, shown by foliar injury, growth reduction, and decreased photosynthetic capacity [41]. Are all East Asian conifer species as tolerant to O3 as P. armandii? Are all conifer species more resistant to O3 than broadleaf ones in East Asia? As for these questions, we cannot offer definite answers at present,
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[54–61]. Hence, various antioxidant strategies were confirmed in different tree species including the endemic ones in East Asia. To summarize, elevated O3 imposed oxidative stress on the P-needles of P. armandii, indicated by the increase of MDA content. Activities of APX and SOD were enhanced by elevated O3, which probably played an important role in alleviating the oxidative stress and provided further evidence of physiological and biochemical indicators as potential biomarkers responding environmental stress [53–61].
and future work should involve more endemic tree species, in particular the precious species and dominate species [41–48]. In the first 45 days of O3 exposure, change in antioxidant enzymes activities was not significant (Fig. 1C–I), indicating that the O3 impact was too low to induce the response of enzymatic defense system in P. armandii. In contrast, AsA content responded to elevated O3 more rapidly, as a significant decrease occurred at day 45 (Fig. 1F). Similarly, the higher sensitivity of AsA to elevated O3 was also found in Q. monogolica. However, the O3-induced response pattern of AsA in P. armandii was contrary to that in Q. monogolica as well as G. biloba in the initial 45 days [22, 23]. Decrease in AsA content (Fig. 1F) could be considered as a consequence to resist the oxidative stress induced by elevated O3 in P. armandii needles, and it might reduce the possibility of ROS to attack membrane lipids, so no increase of MDA content occurred at day 45 (Fig. 1A). Similarly, loss of AsA content has been reported in needles of P. abies (3-year-old) under elevated O3 exposure (200 nmol mol1) for 63 days in controlled chamber [42]. More interestingly, contrary result that AsA content was markedly increased by elevated O3 (100 nmol mol1, 120 days controlled chamber treatment) has been demonstrated in needles of P. abies (4-year-old) as well [43], and it was suggested to play an important role in preventing O3-induced decline of photosynthesis. In contrast with observation in controlled chamber, twice-ambient O3 concentration (67 nmol mol1) showed no obvious effect on AsA content in needles of old-growth P. abies (52-year-old) [44] and young P. canariensis (15-month-old) [16] using a free-air fumigation method in Kranzberg Forest, Germany. The reason why trees in field site are less sensitive to O3 may be due to large natural variation of environmental factors which may limit the O3 uptake. From above analysis, the mechanism of AsA response in trees at elevated O3 still remains obscure, although its importance in resisting oxidative stress was unquestionable [42–48]. In the prolonged O3 fumigation period (from August 28 to September 28 in 2008), activities of SOD and APX were induced to a higher level in P. armandii (Fig. 1C and D), which should be attributed to the mild increase in ROS. Compared with APX, change in CAT activity was not significant during the entire O3 fumigation period (Fig. 1E). It suggests that APX was more susceptible to oxidative stress in contrast to CAT in the needles, and similar proposal with probable reason also has been reported in the O3 study on Q. monogolica [23]. Due to prolonged O3 exposure, activities of APX, CAT, and SOD declined significantly, resulting in the aggravation of oxidative damage in leaves of Q. monogolica [23]. In contrast, enhancement of SOD and APX activities strengthened the antioxidant defense capacity in the needles of P. armandii, and alleviated the O3-induced oxidative stress in some extent, since no increase in EL was recorded in this study (Fig. 1B). However, Polle et al. [15] demonstrated that long-term (180 days) O3 fumigation (80 nmol mol1) led to remarkable decrease in SOD and CAT activities in P. abies (5-year-old) needles, indicating that responses of antioxidant enzymes to O3 were different among conifer species. DHAR, MDAR, and GR have been proved to be important in resisting oxidative stress by means of genetic transformation methods [45–53]. However, they did not response positively in P. armandii at elevated O3 except for the significant increase in GR activity just at Day 105. Thus, reduction in AsA content could not be avoided in the needles (Fig. 1F). Compared with P. armandii and Q. monogolica, activities of DHAR, MDAR, and GR increased significantly in O3-exposed leaves of G. biloba [23] and Liriodendron tulipifera [48], which could efficiently promote antioxidant regeneration process and enhance antioxidant defense capacity in consequence
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Acknowledgments This work was jointly funded by the National Natural Science Foundation of China (31170573), the National Natural Science Foundation of China Important Project (90411019), the National Science and Technology Pillar Program of China (2008BAJ10B04), the Opening Foundation of the State Key Lab of Crop Biology, Shandong Agriculture University (2011KF02), One Hundred-Talent Plan of Chinese Academy of Sciences and the CAS/SAFEA International Partnership Program for Creative Research Teams. We express our sincere thanks to Prof. Dali Tao for critical reading of the manuscript. The authors have declared no conflict of interest.
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