1 Introduction

With respect to air pollutants of a regional distribution, ozone (O3) is considered to impose the greatest threat to vegetation (Degl’Innocenti et al. 2002) including trees and forests (Matyssek et al. 1995; Matyssek and Innes 1999; Matyssek and Sandermann 2003). The tropospheric O3 concentration, driven by anthropogenic pollution at local and trans-regional scales, has been increasing for several decades (Ashmore 2005). China has undergone rapid industrialization and urbanization, and the increase in energy consumption from fossil fuels used in motor vehicles will increase local levels of tropospheric O3 (Chen et al. 2010). Negative effects of O3 on vegetation have been known for nearly 50 years, but it is only during the last two decades that O3 has become of serious concern for vegetation in China, especially forest trees. O3 levels in the Beijing area were sufficiently high to induce foliar symptoms in 28 different species including crops (beans, watermelon, and grape vine), Ailanthus, and several pines (Feng et al. 2014). These symptoms were more frequent in rural areas and mountains from northern Beijing, downwind from the city, and less frequent in city gardens (Feng et al. 2014). According to the monitoring ambient O3 concentration during March and May 2006 in Jiaxing County, Zhejiang Province, the maximum instantaneous O3 concentration was 108 ppb, maximum hourly average O3 concentration was 94 ppb, maximum 8-h average O3 concentration was 68 ppb, and 8-h average O3 concentration was 45 ppb (Yao et al. 2008). In subtropical China, we observed maximum 8-h mean and peak O3 concentrations of 72.3 (8 October) and 97 ppb (14:00, 16 October) in Taihe County, Jiangxi Province, which were potentially damaging levels. Elevated O3 can induce visible injury, inhibit photosynthesis, reduce biomass, reduce crop yields, alter carbon (C) allocation to belowground, and impact the soil microbial community (Chen et al. 2009, 2014; Feng et al. 2014; Wang et al. 2007; Zhang et al. 2012). Some studies found that elevated O3 caused visible injuries, decreased photosynthesis, and inhibited growth of subtropical Chinese trees (Liriodendron chinense: Zhang et al. 2011; Metasequoia glyptostroboides: Feng et al. 2008; Zhang et al. 2014; Cinnamomum camphora: Feng et al. 2011; Cyclobalanopsis glauca: 2013). O3 stress also elicits changes in the balance of structural and nonstructural carbohydrates, in one of the most intriguing biochemical responses of plants to O3 (Darrall 1989). However, there is limited information about effects of O3 nonstructural carbohydrates on subtropical Chinese tree species. In this study, two subtropical tree species Phoebe bournei and Pinus massoniana seedlings were used to study the effect of ambient O3 on nonstructural carbohydrates in a rural village of China (Table 1).

Table 1 Nonstructural carbohydrate contents of leaves in Phoebe bournei seedlings under charcoal-filtered air and ambient ozone
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Ph. bournei is a species of tree up to 20 m (66 ft) tall in the Lauraceae family. It is endemic to China where it occurs in Jiangxi, Fujian, Guangdong, Guangxi, Guizhou, Hainan, and Hubei provinces. It is threatened by habitat loss. Pi. massoniana is a species of pine, native to a wide area of central and southern China. The species is a common tree in plantation forestry for replacing or compensating of the loss of the natural forest in southern China. Chinese rosin is obtained mainly from the turpentine of this pine (Pi. massoniana) and slash pine (Pinus elliottii). In this study, seedlings of both species were exposed to filtered air and ambient O3 in open-top chambers (OTCs) to investigate effects of ambient O3 on nonstructural carbohydrates. The hypotheses were that (1) ambient O3 would affect carbohydrate contents; (2) Ph. bournei and Pi. massoniana would respond differently to O3; and (3) carbohydrate contents in fine roots would be more sensitive to O3 than in leaves.

2 Material and Methods

2.1 Experimental Site

The experimental site is located in the Qianyanzhou Station (115° 03′ 29.2″ E, 26° 44′ 29.1″ N) of the Chinese Academy of Sciences, situated in the typical red earth hilly region of the mid-subtropical monsoon landscape zone of Taihe County, Jiangxi Province, China. The elevation is 60–150 m, and the relative altitude difference within 20–50 m. Meteorological data showed that the mean annual temperature at this site was 17.8 °C, active accumulated temperature above 0 °C was 6543.8 °C and that above 10 °C was 5948.2 °C, annual precipitation was 1471.2 mm, annual evaporation was 259.9 mm, mean relative humidity was 83 %, clear day duration was 1306 h, and global radiation was 4223 MJ/m2.

2.2 O3 Fumigation

The experiments were carried out with three OTCs of 2 m in diameter and 2.2 m in height. The chambers were connected to filter boxes, either without filters or with an activated charcoal O3 decomposition filter and a fan, and run at two air changes per minute. There were two treatments: charcoal-filtered air (CF) and noncharcoal-filtered air (NF, ambient O3). O3 concentration was monitored with an O3 analyzer (Model 49i, Thermo Fisher Scientific Inc., Waltham, MA, USA). Fumigation was during 15 June to 12 November 2014. The 8-h mean concentration during the fumigation period was 6.86 and 32.8 ppb in CF and NF treatments, respectively. During the exposure period, the maximum 8-h mean and peak O3 concentrations were 72.3 (8 October) and 97 ppb (14:00, 16 October), respectively (Fig. 1).

Fig. 1
figure 1

8-h mean O3 concentration during the exposure periods

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2.3 Plants

The experimental plants consisted of Ph. bournei and Pi. massoniana seedlings. In April 2014, 1-year-old nursery-grown container seedlings of Ph. bournei and Pi. massoniana were transplanted to flower pots (20 cm × 30 cm) containing local red soil under ambient air conditions. At the end of May 2014, 15 seedlings of similar height and basal diameter were selected for each species and were transplanted directly into the field in natural soil, and OTCs were set in the field in early June 2014. During the growth season, the seedlings were watered with tap water as needed.

2.4 Sampling and Carbohydrate Analysis

Plants were harvested on 12 November 2014, when the growth season finished. In each OTC, five plants were collected randomly and leaves and fine roots were sampled. Leaves and fine roots were dried at 70 °C and ground to powder through a 2-mm sieve. Carbohydrates were determined by injection of 10-μL sample volume into a high-performance liquid chromatography (HPLC) system using a Sugar-Pak 1 chromatographic column. Column temperature was 70 °C, and distilled water was used as mobile phase (flow rate 0.6 mL/min). Carbohydrates were represented by fructose, glucose, polysaccharide, starch (the sum of residual starch and maltodextrins), water-soluble carbohydrates (WSC) (the sum of glucose, fructose, and sucrose), and total nonstructural carbohydrates (TNCs) (the sum of starch, polysaccharides, and WSC).

2.5 Statistical Analysis

O3 effects on plant response were studied using a single-factor nested experiment that randomly allocated two O3 treatment levels to six OTCs, with three OTCs per O3 level. Independent sample t test was performed for both species to determinate significant differences between individual treatments.

3 Results

Starch and TNC contents in leaves were significantly lower in ambient O3 (NF) than in filtered air (CF) plants, and ambient O3 had no effects on other carbohydrate contents in leaves of Ph. bournei (Table 1). Although ambient O3 did not affect any single nonstructural carbohydrate, TNC content in Pi. massoniana needles was significantly increased in ambient compared to filtered air (Table 1).

Ambient O3 significantly affected most carbohydrate contents, except fructose and WSC, in fine roots. Ambient O3 markedly decreased polysaccharide content in fine roots of Ph. bournei compared to filtered air (p < 0.05). The amounts of sucrose, glucose, WSC, and TNC in fine roots were significantly higher for ambient than filtered air plants in Ph. bournei seedlings (Table 1). While, ambient O3 had no effects on nonstructural carbohydrates in Pi. massoniana fine roots (Table 1).

4 Discussion

Our monitoring data showed that ambient O3 concentration in Taihe County was relatively high. Differing from most findings, the high level of ambient O3 concentration appeared in October, not in summer (June–August)—because during June–August, it was always rainy, and in October, it was sunny with high air temperature and little wind. Ambient O3 concentration nearly exceeded the Level II Standard of Chinese National Air Quality (based on US standards) of 50 nL/L (daily maximum 8-h mean) (Fig. 1). Thus, ambient O3 in Taihe County reached damaging levels, with possible effects on tree species.

In leaves of Ph. bournei, ambient O3 decreased starch content compared to filtered air, consistent with reports of Thomas et al. (2005, 2006) who found that starch concentrations in beech leaves decreased in ambient compared with filtered air. Neufeld et al. (2012) also found that starch concentrations decreased in leaves from sensitive compared to tolerant coneflower plants at Purchase Knob in the Great Smoky Mountains National Park, USA. Conversely, Günthardt-Goerg et al. (1993) found starch enrichment birch leaves after exposed to O3. While, ambient O3 had no impact on starch content in needles of Pi. massoniana in the present study, which was accordant with the finding that in an open-air experiment, the starch concentrations in Aleppo pine needles were not significantly affected in one season of exposure (Anttonen et al. 1998). Some elevated O3 exposure experiments showed that elevated O3 significantly decreased starch content in needles of tree species, including ponderosa (Pinus ponderosa; Andersen et al. 1997), spruce (Picea abies; Thomas et al. 2005), and Aleppo pines (Pinus halepensis; Anttonen et al. 1998). But, Wellburn and Wellburn (1994) found starch enrichment in Aleppo pine (Pi. halepensis) needles after fumigation with O3 using light microscopy. When young Norway spruce was grown in plots along a gradient of increasing O3 pollution in Switzerland, starch concentration in the needles increased gradually (Braun et al. 2004). In spruce from Wengernalp in the Swiss Alps, the pool size of starch was significantly higher in the needles of plants in ambient compared with filtered air (Lux et al. 1997).

Ambient O3 decreased TNC content in leaves of Ph. bournei, which was coincident with some previous results especially with crops (spring wheat: Gelang et al. 2001; Sild et al. 2002). Sugar maple (Acer saccharum) seedlings were fumigated with ambient, 3.0 × ambient O3 in OTCs for 3 years, and elevated O3 reduced TNC concentrations in spring cohort leaves, but there was no significant O3 effects on TNC concentrations of leaves produced in summer cohort leaves (Topa et al. 2001). While, TNC content in leaves of 1-year-old beech splings Fagus sylvatica was significantly higher in ambient plants than filtered plants (Lux et al. 1997). Contents of soluble sugars (sucrose, glucose, and fructose) were enhanced in older birch (Betula pendula) leaves of the O3 and low fertilization treatment (Landolt et al. 1997).

Contrary to Ph. bournei, ambient O3 increased TNC content in needles of Pi. massoniana compared to filtered air. In spruce (Pi. abies, 2-year-old seedlings), glucose, fructose, and TNC content in needles were significantly higher in ambient air than in filtered air plants (Lux et al. 1997). When 3-year-old Norway spruce (Pi. abies) were exposed to O3 at 100 or 20 ppb (control), O3 caused a decrease in ethanol-soluble carbohydrate content in needles (Barnes et al. 1990). After 2-year-old red spruce (Picea rubens) seedlings were exposed to various levels of O3 in OTCs in Ithaca, NY, USA, for one growth season, there was a trend toward reduced total sugar content in foliage during later autumn in high O3 treatment (Alscher et al. 1989). After spruce saplings were fumigated with either charcoal-filtered or ambient air for three growing seasons, O3 fumigation decreased starch concentrations in needles (Thomas et al. 2005).

In fine roots of Ph. bournei, ambient O3 increased sucrose, glucose, starch, and TNC contents and decreased the amount of polysaccharide, which was consistent with some studies. In beech saplings at Schönenbuch in Switzerland, the amount of soluble carbohydrates increased and starch contents mostly decreased in roots in ambient compared with filtered air (Lux et al. 1997). Using 14C labeling, Topa et al. (2004) found that O3 had no significant effect on partitioning of recently assimilated 14C into starch and the ethanol-soluble fraction in most root tissues in sugar maple. O3 fumigation resulted in a significant increase of the fine-root monosaccharide concentrations in young beech trees, but no other carbohydrate groups were affected (Thomas et al. 2006). Enrichment of carbohydrates in fine roots of Ph. bournei seedlings in ambient compared with filtered air is consistent with O3 increasing belowground C allocation. Pregitzer et al. (2008) found that long-term exposure to elevated O3 increased aspen fine-root production and mortality. This response, which suggested that O3 increased C allocation belowground, may have resulted from the loss of O3-sensitive individuals from the population rather than a physiological response at the individual tree level. Studies with tree seedlings have also shown stimulation of belowground activity (e.g., increased soil CO2 efflux) in response to O3 (Andersen and Scagel 1997). While the findings of the present study on effects of O3 on carbohydrates in Ph. bournei roots contradict the results of some other broadleaf tree species, which have found that O3 decreases carbohydrate contents of roots. At Zugerberg in the lower Alps, the starch concentration was lower in roots of beech seedlings (F. sylvatica) when grown in ambient compared with filtered air (Braun and Fluckiger 1995). When young beech (F. sylvatica) was grown in pots along a gradient of O3 pollution in Switzerland, the monosaccharide (Braun et al. 2004) and starch (Thomas et al. 2002) concentration in fine roots showed a decreasing trend with increasing O3.

In fine roots of Ph. bournei, ambient O3 increased nonstructural carbohydrates. This change in allocation toward root TNC reserves may be survival strategy for Ph. bournei which was under O3 stress. Upon outplanting, root TNC reserves are important for seedlings to survive the stressful environmental conditions, such as high water stress encountered immediately after planting (Grossnickle 2005). Stored carbohydrate reserves are important for the establishment and growth of seedlings, particularly in deciduous species that need to rely on the stored reserves to initiate leaf area and new root growth without current photosynthesis (Kozlowski and Pallardy 2002; Sprugel 2002; Landhäusser 2011). Although photosynthesis is reduced in aspen under stress conditions, some carbon continues to be assimilated and is used to build carbohydrate reserves rather than structural growth (Martens et al. 2007; Galvez et al. 2011; Landhäusser et al. 2012). Under these stress conditions, an asynchrony between carbon supply and the immediate carbon demand for growth develops as a result of the termination of height growth (Chapin et al. 1990), allowing the plant to divert photosynthates to reserves instead of growth (Körner 1991; Galvez et al. 2011). A similar accumulation of TNC reserves has been found in Oxytropis sericea, a perennial herb, where slower growing plants allocated proportionally more C to root reserves than faster growing plants (Wyka 2000). This prioritization of photosynthates to the root system over shoots may be an ecological adaptation for a species which naturally regenerates from its root system after disturbances such as fire, drought, and defoliation kill the aboveground portion of the plant (Frey et al. 2003).

Different from Ph. bournei, ambient O3 had no effects on nonstructural carbohydrates in fine roots of Pi. massoniana, compared to the filtered treatment. This can be explained like that ambient O3 had not yet affected the growth of Pi. massoniana and Pi. massoniana need not take adaptation measures to build root carbohydrate reserves to survive the stressful environmental conditions like Ph. bournei did. But, most of other studies showed that O3 decreased carbohydrate contents in roots of conifer species. The amounts of starch, glucose, and fructose in fine roots were significantly reduced in ponderosa pine seedlings exposed to the highest concentration of O3 compared to controls (Andersen et al. 1997). When young spruce were grown in pots along an increasing gradient of O3 pollution in Switzerland, starch concentrations decreased (Braun et al. 2004). Lux et al. found that ambient O3 in different elevation in Switzerland had different effects on carbohydrates in spruce roots, starch content in spruce from the lowland was significantly higher in the finest roots of the ambient air trees than in the filtered air trees, while in trees from the highland roots contained significantly less soluble carbohydrates, and also, the starch contents were slightly decreased in ambient air as compared to filtered air (Lux et al. 1997). After spruce saplings were fumigated with charcoal-filtered or ambient air for three growing seasons, sugar alcohol, disaccharide, and trisaccharide concentrations of fine roots showed no reaction to O3 fumigation, and monosaccharides were significantly enhanced by O3 as well as the total soluble carbohydrate concentrations (Thomas et al. 2005). Under the forest site conditions of the San Bernardino Mountains (California, USA) which have been exposed to elevated O3 regimes for several decades, mature trees of ponderosa and Jeffery pines (Pinus jeffreyi) showed depressed monosaccharide concentrations in fine and coarse roots (Grulke et al. 2001). Three-year-old red spruce (Pi. rubens) seedlings were exposed to various levels of O3 in OTCs in Ithaca, NY, USA, for two consecutive growing season, and seasonal means of starch content of roots decreased linearly with increasing doses of O3, but in contrast, seasonal mean sugar content and rate of sugar accumulation of roots increased linearly (Amundson et al. 1991).

The present study showed that carbohydrate contents in Ph. bournei were much more sensitive to O3 than in Pi. massoniana, and nonstructural carbohydrate contents were much more easily affected in roots than in leaves. This is supported by existing findings. For mature trees, a higher sensitivity toward O3 of deciduous compared to coniferous trees may be expected (Samuelson and Kelly 2001). A research showed that in addition to ozone pollution reducing the strength of trees to hold carbon in the northern temperate mid-latitudes by reducing tree growth, it also indicates that broad-leaf trees, such as poplars, are more sensitive to ozone pollution than conifers, such as pines, and that root growth is suppressed more than aboveground growth (The Free Library 2008). Manes et al. (2012) assessed the effects of tree diversity on the removal of tropospheric O3 in Rome, Italy, in 2 years (2003 and 2004), which found that the deciduous broadleaf trees took up the most O3 and conifer the least except under drought conditions. Several studies have observed O3-induced changes to the root system, such as root growth reductions, chromosomal aberrations, and metabolic changes in root tips, prior to noticeable effects on the shoot, possibly because shoots have easy access to C for repair (Andersen 2003; Calvo et al. 2007; Grantz et al. 2003; Miller et al. 1997; Mortensen 1998; Wonisch et al. 1999; Zouzoulas et al. 2009). Vollsnes et al. (2010) also found that O3 exposure induced earlier changes in belowground than aboveground growth in Trifolium subterraneum seedlings—they thought that, although leaves are the primary sites of tropospheric O3 exposure, O3 may cause greater and earlier disruption of belowground growth with long-term consequences for productivity.

5 Conclusion

This study is the first to research the effects of ambient O3 on nonstructural carbohydrate contents in subtropical tree species in China. The ambient O3 in Taihe County, Jiangxi Province, affected the amounts of nonstructural carbohydrate in leaves and roots of both Ph. bournei and Pi. massoniana. However, Pi. massoniana was less sensitive than Ph. bournei to O3, and O3-induced changes in amounts of carbohydrates were much greater in roots than in leaves. This study shows that ambient O3 has harmful effects on tree species in subtropical China. In this study, seedlings were exposed for just one growth season, it needs much longer research, and physiological mechanism needs to be studied that changes carbon metabolites.