Changes in nutrients and decay rate of Ginkgo biloba leaf litter exposed to elevated O₃ concentration in urban area

Study site and experimental treatments

The study was conducted in the Shenyang Arboretum of the Chinese Academy of Sciences (41°46′N, 123°26′E) located in an urban area (He et al., 2009). The arboretum with a mean elevation of 41 m and an area of 5 ha was founded in 1955, mainly planted with native tree species. There are more than 300 tree species and the forest coverage rate was 53.7% (He et al., 2016). Nowadays, it is a near-natural urban forest (He et al., 2003). Affected by warm temperate-zone semi-humid monsoon climate, this area has an annual average temperature of 6.2 to 9.7 °C. The average temperatures in January and July are −12.6 and 27.5 °C, respectively. The maximum temperature is 38.3 °C and the minimum temperature is −30.5 °C (Xu et al., 2014). The average annual precipitation is 755.4 mm. The frost-free period lasts for 150 d yearly (Xu et al., 2005). The flora of the arboretum located area belongs to the intersection of Changbai Mountain, Northern China, and the Mongolian Floras (Xu et al., 2006).

This experiment was carried out in open-top chambers (OTCs). Chambers are 4 m in diameter and 3 m in height, with a 45° sloping frustum and 4-m distance between neighbouring OTCs (Li et al., 2011; Xu et al., 2015). This experiment included control in ambient air (AA, about 40 ppb) and elevated O₃ concentration (EO, 120 ppb). AA and EO had three independent OTC replicates with three OTCs, respectively. Six OTCs were used in total. In 20 May, 2012, nine healthy and uniform five-year-old saplings of G. biloba (1.5 m in average height) from a local nursery were selected and planted in each OTC. During the growing season, the saplings were irrigated twice a week and fertilized once at the beginning of the experiment. After 60 d (20 July), the saplings were fumigated with O₃ for 8 h a day from 9:00-17:00, except in bad weather such as thunderstorm conditions. By the end of fumigation on 5 November, 2012, all the yellow (senescent) leaves of G. biloba from each chamber were harvested and regarded as leaf litter. These senescent leaves under AA and EO were randomly divided into two parts: one part was dried at 65 °C to constant weight for determining the initial chemical compositions and the other was sufficiently dried, and stored in a big glass container and then kept at a cool place under room temperature for the litter decomposition experiments in OTCs next year (2013).

The leaf litters were decomposed according to the litterbag method (Zhang et al., 2015) by placing them in nylon bags (20 cm × 25 cm) with 1 mm² mesh. Before the experiment, each bag was filled with 8 g samples and numbered. On 19 May, 2013, 15 bags were placed into each OTC to touch the topsoil and separate them at 10-cm intervals each other. A total of 45 bags of AA and EO were prepared, respectively. Before putting the litter bags into OTCs, the topsoil (0–10 cm) of each treatment was gathered after removing weeds and other sundries from the ground. Soil samples were mixed uniformly after the stones, roots and other debris were removed. Homogenized fresh soil was passed through a sieve (pore size was 5 mm) and dried under natural conditions. The basic physical and chemical properties of topsoil (0–10 cm) are shown in Table 1. O₃ fumigation was carried out for 8 h each day (9:00–17:00). The samples were collected once a month (Sampling date is 19 every month from June to October) to be decomposed for 150 days in total. O₃ concentrations were monitored every day throughout the growing period (19 May–20 October 2013), at 0.80 m above the ground, in the OTCs of both AA and EO (Fig. 1). The microclimatic conditions in the OTCs during litter decomposition are summarised in Table 2. After washing and drying, the cleaned litters were placed into Kraft envelope bags and oven-dried at a constant temperature of 65° C. Afterwards, leaf litters were weighed and dry mass were pulverised by a high-speed grinding machine (FW-100; Taisite Instrument Co., Ltd., Tianjing, China) and filtered through a sieve (80 meshes, mesh size is 5 mm). The powdered samples were preserved in self-sealing bags for the laboratory measurements.

Chemical analyses and experimental statistics

Carbon (C) and nitrogen (N) contents were determined by an elemental analyser (Vario MACRO Cube, Elementar, Germany). Phosphorus (P) and potassium (K) contents were measured by atomic absorption spectrometry (AA800; Perkin Elmer, San Jose, CA, USA) according to the Mo-Sb colorimetric method and flame photometric method, respectively. Lignin content was determined using the ultraviolet spectrophotometric method (Liyama & Wallis, 1988) and total phenol content was measured according to the method of Julkunen-Tiitto (1985) with a minor modification. The condensed tannins and soluble sugar contents were determined by spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan) according to the butyl alcohol-hydrochloric acid method (Porter, Hrstich & Chan, 1986) and anthrone colorimetric method (Dubois et al., 1956), respectively.

Mass remaining of leaf litter over time, expressed as percentage of the initial value, was calculated according to the assumed simple exponential model formulated by Olson (1963). ${\displaystyle Y=A_{\mathrm{t}}∕A_0={e^{-}}^{kt}.}$ Where, Y, A₀ and A_t indicate the remaining rate of litter mass monthly, the initial litter mass (g), and the remaining mass (g) of leaf litter at time t (months), respectively. In addition, e, and k are the base of natural logarithms, and the decomposition coefficient of the litters, respectively. As for t, it is the decomposition time (months) including the half-life of decomposition (t_0.5 = ln(2)∕k).

The remaining rate of nutrient composition was calculated (Pancotto et al., 2003): ${\displaystyle E=\left[\left(M_{\mathrm{t}}\times C_{\mathrm{t}}\right)∕\left(M_0\times C_0\right)\right]\times 100\text{\%}}$ Where, E, M_t, M₀, C₀, and C_t represent the remaining (% initial) of nutrient elements, dry mass (g) of leaf litter at the designated time of decomposition, initial dry mass (g), initial nutrient content (mg g⁻¹), and nutrient content (mg g⁻¹) of leaf litter at the designated time of decomposition, respectively.

Data analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). General linear model (GLM) was used to evaluate and analyse the dynamics of leaf litter mass remaining, changes in C, N, P, lignin, total phenols, condensed tannin and soluble sugar contents with considering the treatments (two levels), decomposition time (continuous variable), as well as their interaction, as independent factors. All data were presented as mean ±  standard deviation. Significant differences between control (ambient air, AA) and elevated O₃ concentration (EO) were tested by T-test.

Changes in chemical compositions in leaf litters of G. biloba after exposure to EO

In comparison with AA, EO decreased C content (by 26.1%), but increased slightly N content (by 11.3%) in leaves of G. biloba by the end of growing season (Fig. 2). EO decreased C/N ratio (by 10.3%). P content decreased under O₃ fumigation. However, changes in C, N, P and C/N were not significant. EO significantly increased the K content by 184.2% (6.31 ± 0.29 vs 17.93 ± 0.40, P < 0.01) compared with AA (Fig. 2).

In addition, EO decreased the contents of lignin (by 8.6%) and condensed tannins (by 17.5%). By contrast, the difference between EO and AA was not significant (Fig. 2). EO significantly decreased the contents of total phenolics (2.82 ± 0.93 vs 1.60 ± 0.44, P < 0.05) and soluble sugar (86.51 ± 19.57 vs 53.76 ± 2.40, P < 0.05) by 43.3% and 37.9% compared with AA, respectively.

Dynamics of leaf litter decomposition of G. biloba exposed to EO

Based on the Olson’s model, the decay constant and the half-life decomposition time of the leaf litters of G. biloba under EO were lower than those AA, showing no significant difference (Table 3). Compared with AA, the mass (dry weight) remaining of leaf litters under EO maintained a higher level at early stage of decomposition (before 60-day sampling point), but decreased with decomposition time after 90 days, and it was 5.8% lower than that of AA at 150-day sampling point (Fig. 3A). No significant difference between AA and EO was observed in mass remaining of G. biloba leaf litter over time. O₃ fumigation reduced the remaining of C in leaf litter and a significant difference between AA and EO was found after 120 days (EO decreased by 8.3% compared with that of AA) (Fig. 3B). By contrast, N remaining showed a lower level after 90 days under EO than that of AA. However, no significant difference was found in N remaining between treatments during decomposition (Fig. 3C). P remaining in leaf litter showed higher level under EO than AA, and decreased dramatically after 60 days and reached a lowest value at 90-day sampling point (11.2%) (Fig. 3D).

The remaining of lignin in leaf litters of G. biloba showed a significant decreasing trend regardless of O₃ treatment during decomposition (Fig. 3E, Table 4, P < 0.001). Compared with the AA, the remaining of lignin under EO was quite lower after 120 days, indicating an increasing of lignin decomposition rate. However, no significant effect was observed in the remaining of lignin under EO (Table 4, P = 0.249). After 60 days, the remaining of total phenol content showed a significant decrease under EO at each sampling point (Fig. 3F), compared with AA (P < 0.05). A significant interactive effect of O₃ concentration and decomposition time was found for the total phenol remaining (Table 4, P = 0.002). EO showed no significant effect on the decomposition of condensed tannins during the experiment (Fig. 3G, Table 4, P = 0.326). During decomposition, the remaining of soluble sugar showed a significant higher lever at each sampling point except for the 90-day sampling point under EO than AA (P < 0.05) (Fig. 3H).

Effect of elevated O₃ concentration on chemical composition of G. biloba leaf litter

The results of our study showed that elevated O₃ concentration (120 ppb) increased N and K contents in leaf litter of G. biloba, but significantly decreased the total phenol content, as well as the C/N and lignin/N ratios. However, the P and lignin contents, as well as the C/P ratio, did not change significantly. Actually, elevated O₃ concentration usually changes the chemical composition of plants (Calatayud et al., 2011; Shang et al., 2018). Booker et al. (2005) found that the changes in N and lignin contents of soybean leaf litter increase significantly, while the soluble sugar content significantly decreased under elevated O₃ concentration (74 ppb), in agreement with our results in this study. Parsons, Lindroth & Bockheim (2004) tested the impact of high O₃ concentration (55 ppb) on leaf litter of Betula papyrifera for 12 months. The results demonstrated that the contents of N, lignin, and condensed tannins, as well as the C/N ratio of leaf litter, showed no significant change under elevated O₃ concentration compared to tests in ambient air conditions.

In our study, the increasing of N content in G. biloba leaves after high O₃ exposure may be a helpful response to prevent the damage caused by O₃ to some extent (Cao et al., 2016). High N concentration in leaves could make plants adapt to O₃ stress through leaf turnover (Tjoelker & Luxmoore, 1991; Shang et al., 2018). In addition, the C/N ratio was an important indicator for the degree of coordination of C and N metabolism. Reduction of C/N in plants under elevated O₃ concentration showed that the growth of plants was inhibited (Zheng et al., 2011), in agreement with our result that C/N ratio decreased under O₃ fumigation. Zhang et al. (2011) demonstrated that elevated O₃ concentration promoted K absorption during growth of plants. Furthermore, K increased N absorption in order to transfer it into proteins under adverse environment. The increase of K contents in our study was possibly one of the reasons why N content increased under elevated O₃ concentration.

Plant secondary substances play a pivotal role in scavenging the high level of oxygen species caused by ozone at the end of O₃ fumigation (He et al., 2009). Generally, the secondary metabolic enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) are involved in the synthesis of secondary substances (Tiwari, Sangwan & Sangwan, 2016). Luo & Zhang (2010) found that elevated O₃ concentration significantly decreased the lignin contents of plants by inhibiting the activities of PAL, POD and PPO. In our study, the decrease of lignin content in G. biloba leaves could be due to the inhibition of activities of relevant enzyme for synthesising lignin by elevated O₃ concentration.

Phenolic compounds are the main products of secondary metabolism and are important defence substances in plants. Some research demonstrated that elevated O₃ concentrations increased the contents of phenolic compounds in leaves (Yamaji et al., 2003; Peltonen, Vapaavuori & Julkunen-Titto, 2005). In our previous studies, elevated O₃ concentration (80 ppb) significantly increased the total phenolics and condensed tannin contents in leaves of Quercus mongolica, leading to the increasing of the antioxidant capacity of plants to O₃ stress (Zhang et al., 2009). In this study, elevated O₃ concentration decreased the contents of condensed tannins and total phenols of G. biloba leaves. It could be due to the produce of a large number of free radicals under O₃ stress, leading to many antioxidant substances including phenolic compounds were consumed by the end of the growing season (He et al., 2009). Therefore, the decreases of condensed tannins and total phenolic content in G. biloba leaves may increase the sensitivity of the leaf injuries to O₃ (Yamaji et al., 2003). Actually, no effect of elevated O₃ concentrations on the contents of condensed tannins and phenolic compounds was reported by some studies (Lavola, Julkunen-Tiitto & Paakkonen, 1994; Lindroth et al., 2001), not in agreement with our current results due to the differences in tree species and O₃ concentrations to some extent.

Soluble sugar plays an important role in plant metabolism under adverse environments (Liu et al., 2004). Wang et al. (2011) reported that O₃ fumigation (60 ppb, 50% above ambient air) significantly decreased the soluble sugar content of rice in each growth stage. However, Lu et al. (2012) found that the content of soluble sugar in the leaves of Mangifera indica increased under O₃ concentration (50 ppb), but decreased significantly under a high-O₃ concentration (200 ppb), in agreement with our result that the soluble sugar contents in leaves of G. biloba decreased significantly under elevated O₃ concentration (120 ppb). The reason for this was probably due to the accumulation of glycolytic enzymes in leaves, which accelerated the degradation of sugar components (Tiwari, Sangwan & Sangwan, 2016).

Effect of elevated O₃ concentration on the decomposition rate of G. biloba leaf litter

Elevated O₃ concentration not only changes the chemical composition of leaf litter, but also indirectly affects the litter decomposition (Baldantoni, Fagnano & Alfani, 2011). In this study, the remaining mass of dry weight of G. biloba leaf litter showed significant positive correlation with the C, N, P, and lignin contents as well as the ratios of C/N and lignin/N, regardless of O₃ treatment (Table 5). This indicated that the higher the contents of C, N, P, and lignin, the higher the mass remaining, and the lower the decomposition rate (Bonanomi et al., 2013). In this study, the mass remaining are larger under elevated O₃ concentration than under ambient air at early decomposition stage, which implied that the decomposition of leaf litter slowed down under O₃ fumigation. This is consistent with most of the previous studies showing that elevated O₃ has adverse effects on litter decomposition (Parsons, Bockheim & Lindroth, 2008; Liu et al., 2009; Baldantoni, Fagnano & Alfani, 2011). At the late stage of decomposition (after 90 days), the mass remaining of leaf litter showed higher level under ambient air than elevated O₃ treatment, which indicated that O₃ slightly increased the decay rate of leaf litter by impacting litter quality in this study. Indeed, Litter quality is the most important factor affecting mass loss and decay rates of nutrients (Bonanomi et al., 2010).

In fact, O₃ exposure has not always led to reduction in litter decomposition rate of tree species (Scherzer, Rebbeck & Boerner, 1998; Kainulainen, Holopainen & Holopainen, 2003). In our recent study, we observed that N mineralization and lignin degradation in leaf litters of Q. mongolica under elevated O₃ concentration (120 ppb) were inhibited during early stage of decomposition, but promoted at later stage of decomposition (Su et al., 2016). Indeed, as the major component in leaf litter, lignin has a complex structure and is difficult to decompose (Peng & Liu, 2002). During the decomposition in this experiment, the remaining rates of lignin, condensed tannin and total phenols were lower under elevated O₃ concentration than under ambient air at late decomposition stage, which implied that elevated O₃ concentration promoted the decomposition of leaf litter. The slight promotion of the decomposition rate of leaf litter mainly resulted from the decrease of leaf litter quality induced by O₃ fumigation. Besides, exposure of O₃ at the late decomposition stage of the experiment might accelerate the oxidation and decomposition of secondary metabolic substances (Su et al., 2016).

Unlike the remaining of lignin, condensed tannin and total phenols, the remaining of soluble sugar showed higher values under elevated O₃ concentration than ambient air at any sampling point during litter decomposition. The lower decay rate observed for ozone-exposed leaves could be due to changes in both structural and functional molecules that we did not study here or decreases in the activities of enzymes relating to carbohydrate metabolism under O₃ exposure (Peace, Lea & Darrall, 1995). In addition, the higher value of carbohydrate remaining was maintained under O₃ fumigation during decomposition and becoming much smaller difference between AA and EO by the end of the experiment, which might result from inhibiting effect of O₃ on decay rates of leaf litter with high initial values at early decomposition stage. The similar results were observed in some previous studies (Fioretto et al., 2005; Liu et al., 2009).