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Comparative analyses of physiological responses of Cynodon dactylon accessions from Southwest China to sulfur dioxide toxicity.

1. Introduction

Sulfur dioxide (S[O.sub.2]), a gaseous pollutant with bad odor in the atmosphere, is mainly emitted from anthropogenic sources. It is estimated that more than 70% of global S[O.sub.2] is emitted from anthropogenic sources, half of which is from combustion of fossil fuel [1]. With rapid development of economy in developing countries, emission of S[O.sub.2] into the atmosphere has been increasing quickly. As the biggest developing country in the world, China is leading the world as the biggest S[O.sub.2] emitter, contributing to about one-fourth of the global emission and more than 90% of East Asia emission since the 1990s [2]. Total S[O.sub.2] emission in China increased by 53%, from 21.7 Tg (1 Tg = [10.sup.12] g) in 2000 to 33.2 Tg in 2006, at an annual growth rate of 7.3%. The S[O.sub.2] emission began to decrease after 2006 mainly due to the widespread application of flue-gas desulfurization (FGD) devices at all newly built thermal power units in order to implement a comprehensive national policy strategy of energy conservation and emission reduction since 2005. However, the total S[O.sub.2] emissions are still very high (27.7 Tg) in 2010 due to the dramatic growth of industrial production and energy consumption [3]. Thereafter, high level of S[O.sub.2] in the atmosphere will be a major concern in developing countries in a long period of forthcoming time.

In the atmosphere, when gaseous S[O.sub.2] meets with water, considerable amounts of S[O.sub.2] are converted to sulphurous acid, which is the important component of acid rain. Sulfur is well known to be a basic constituent of sulfur-containing amino acids, iron-sulfur clusters, cofactors, polysaccharides, and lipids for all living organisms. S[O.sub.2] can enter plants via their stomata by the process of photosynthesis and respiration [4]. Plant has the ability to incorporate this kind of inorganic sulfur into sulfur-containing amino acids, proteins, and glutathione (GSH) and sulfur can also serve as the sulfur precursor of sulfur-containing secondary products in plant. However, above a certain threshold, both S[O.sub.2] and acid rain are highly toxic to plants, causing many visible symptoms in the plant like yellowing, chlorosis, bleaching, and even killing foliage depending on the dosages [4]. Because of the harmful effects of S[O.sub.2], some plants cannot grow robustly and even die in severe polluted urban or industrial districts, creating "dead zones" without greenery. To achieve better air quality and landscape effect in such polluted areas, the plants with high resistance to S[O.sub.2] should be selected out for use. Tree species tolerant to S[O.sub.2] were selected out or developed for planting in air polluted areas [5-7].

Turfgrasses were extensively used in a sole manner or in combination with trees for environmental greening. Importantly, grass plants are more resistant to S[O.sub.2] than woody plants, because the former have a higher S : C ratio than the latter and therefore can take up more S[O.sub.2] from the atmosphere [8]. Turfgrasses can be generally classified as cool season, warm season, or evergreen types. A few studies on tolerance to S[O.sub.2] of cool-season grass populations in polluted areas have been carried out in the past decades. These studies mainly focused on identification of tolerant populations from cool-season species of Dactylis glomerata, Festuca rubra, Holcus lanatus, Lolium perenne, and Phleum bertolonii [9]; comparison of stomatal morphology and resistance, membrane permeability, and the uptake and metabolism of [sup.35]S[O.sub.3] and [sup.35]S[O.sub.2] in cool-season species of D. glomerata, F. rubra, H. lanatus, and L. perenne [10]; investigation on the rate of development of tolerance in cool-season species of F. rubra, L. multiflorum, L. perenne, P pratense, and Poa pratensis [11]; and genetic nature of tolerance in cool-season species of L. perenne [12]. In such studies, Cynodon dactylon, a warm season perennial grass species, is not included, which is widely used as turfgrass on sports fields, golf courses, roadsides, and lawns in city or industry districts in warm season. Recently, our comparative study on physiological and growth performances found that C. dactylon displayed the highest resistance to S[O.sub.2] among four warm season turfgrasses including C. dactylon, Eremochloa ophiuroides, Paspalum notatum, and Zoysia japonica [13]. In the present study, we firstly compared influences of S[O.sub.2] on leaves of 38 wild C. dactylon accessions from Southwest China. Based on injury rate of S[O.sub.2] to leaves, nine C. dactylon accessions representing high S[O.sub.2]-tolerant, intermediate S[O.sub.2]-tolerant, and S[O.sub.2]-sensitive to S[O.sub.2] accessions were selected to comparatively study relationships between S[O.sub.2] tolerance and several physiological parameters. This study gained some insights into understanding the genetic and molecular mechanisms of C. dactylon to S[O.sub.2] and provided guideline for selection and development of C. dactylon variations for planting in S[O.sub.2] polluted urban or industrial areas.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions. Thirty-eight wild C. dactylon accessions used in this study were sampled from Sichuan Province, Chongqing municipality, Yunnan Province, Guizhou Province, and Tibet Autonomous Region in Southwestern China between years 2011 and 2012. A complete list of accession descriptions and geographical origins was provided in Table 1 and Figure 1. The wild C. dactylon accessions were collected originally from roadside, riverside, floodland, fieldridge, wasteland, hillside, or city park. All wild accessions used in this study were determined to be C. dactylon based on morphological characteristics as described by Harlan [14].

The experiments were carried out between April and August, 2013, at Experimental Station of Grass Science, Sichuan Agricultural University, Ya'an, Sichuan Province, China. The experimental location is 600 m in altitude with a humid subtropical climate. Mean annual precipitation, annual temperature, and relative air humidity in the area are 1800 mm, 16.2[degrees]C, and 79%, respectively. All the C. dactylon accessions were planted in plastic pots (18 cm in top diameter, 14 cm in bottom diameter, and 15 cm in depth) filled with typical sandy loam soil in the local place in April. Each C. dactylon accession was replicated six times. All C. dactylon grasses were grown under natural conditions for 2 months with regular watering every day and fertilizing and cutting every four weeks prior to the experimental treatment.

2.2. Stress Treatment and Experimental Design. After two-month growth, three pots of grass plants with nearly the same crown from each accession were chosen from six replications (as mentioned above) for S[O.sub.2] stress treatment. All of the selected pots of grass plants were fumigated with S[O.sub.2] at a concentration of 3.75 mg/L in a custom-made fumigation chamber (85 cm x 85 cm x 40 cm) for 3h per day over 7 days as described in our previous study [13]. The day when S[O.sub.2] fumigation started was designated as day 0. In order to achieve a uniform environment in the chamber, a fan was attached to the chamber ceiling to mix the S[O.sub.2]. A S[O.sub.2] gas detector (Z-1300, Environmental Sensors Co., Boca Raton, FL, USA) was used to measure the concentration of S[O.sub.2] and to keep the gas concentration constant in the chamber during the experiment. After fumigation treatment, grass plants were taken out and grown under natural conditions with regular watering every day. The remaining three pots of grass plants without S[O.sub.2] treatment from each accession served as control. Based on injury rate of S[O.sub.2] to leaves after 7-day treatment of S[O.sub.2], three high S[O.sub.2]-tolerant, three intermediate S[O.sub.2]tolerant, and three S[O.sub.2]-sensitive C. dactylon accessions were selected out for physiological studies. Leaves 2 cm above the soil from C. dactylon plants treated by S[O.sub.2] after 7 days were collected and brought back to laboratory for analysis. Leaves from C. dactylon plants without S[O.sub.2] treatment at day 0 served as control.

2.3. Measurement of Total Soluble Sugars. The total soluble sugars were determined using the anthrone method as previously described by Lu et al. [15] with some modifications. Briefly, 0.2 mg dried leaf samples were extracted in 5 mL of 80% (v/v) ethanol at 80[degrees] C for 40 min and centrifuged at 15,000 xg for 10 min. The pellets were further extracted twice with another 5mL of 80% (v/v) ethanol. The supernatants were combined together and depigmented by activated charcoal at 80[degrees]C for 30 min. For the determination of soluble total sugars, 0.2 mL of the filtrate was mixed with 3mL of 0.15% (w/v) anthrone reagent (0.3 g anthrone was dissolved in 200 mL of 7.74 M H2SO4) and then heated at 90[degrees]C for 20 min. Finally, soluble total sugar level was determined at 620 nm of absorbance using a UV/VIS spectrophotometer Model 723PC (Jinghua Instruments, Shanghai, China).

2.4. Measurement of Proline Content. Proline content was estimated according to the method based on proline's reaction with ninhydrin described by Bates et al. [16] with modification. Briefly, 0.2 g leaf samples were ground in 5 mL 3% (w/v) sulfosalicylic acid and then filtered through 0.45 [micro]m filter paper. Two microliters of filtrate was mixed with equal volumes of ninhydrin reagent and glacial acetic acid. Well mixed solutions were boiled at 100[degrees]C for 1 h. There action was terminated in an iced bath and the chromophore was extracted with 4 mL toluene and its absorbance at 520 nm was determined using a UV/VIS spectrophotometer Model 723PC (Jinghua Instruments, Shanghai, China).

2.5. Estimation of Chlorophyll and Carotenoid. Photosynthetic pigments from the leaves were extracted as described by Lichtenthaler and Wellburn [17] with modification. Leaf samples (~0.2g) were ground in 2mL of 80% acetone and ethyl alcohol (1: 1), using a mortar and pestle, and then filtered through 0.45 [micro]m filter paper. Absorbance of the resulting extracts was measured at three wavelengths 663, 646, and 470 nm for chlorophyll a, chlorophyll b, and carotenoids, respectively, using a UV/VIS spectrophotometer Model 723PC (Jinghua Instruments, Shanghai, China). The amounts of pigments were calculated according to the equations developed by Lichtenthaler and Wellburn [17]. Total chlorophyll was obtained from the sum of chlorophylls a + b.

2.6. Determination of [H.sub.2][O.sub.2] Level. For grass protein extraction, about 0.2g fresh leaves were ground with liquid nitrogen and then homogenized in extraction buffer (50 mM sodium phosphate buffer, pH 7.8). After centrifugation at 15,000 xg for 15 min at 4[degrees]C, the supernatant was used for determination of [H.sub.2][O.sub.2] levels as described by Hu et al. [18]. Briefly, 1mL of the supernatant was mixed with 1 mL of 0.1% titanium sulphate in 20% [H.sub.2]S[O.sub.4] (v/v) thoroughly for 10 min. After being centrifuged at 15,000 xg for 10 min at room temperature, the absorbance of the supernatant was measured at 410 nm using a UV/VIS spectrophotometer Model 723PC (Jinghua Instruments, Shanghai, China).

2.7. SOD, POD, CAT, SiR, and SO Enzyme Assays. Fresh leave sample (~0.5 g) was homogenized in 5 mL of 0.1 M phosphate buffer (pH 6.8) containing 1 mM EDTA, 1 mM dithiothreitol, and 2% (w/v) polyvinylpyrrolidone (PVP) using a chilled mortar and pestle on ice. The homogenate was centrifuged at 15,000 x g for 15 min at 4[degrees]C, and the supernatant was used for enzyme activity. Soluble protein content was determined following the Bradford method [19] with BSA as standard. Superoxide dismutase (SOD) activity was determined spectrophotometrically at 560 nm based on the measurement of inhibition in the photochemical reduction of nitroblue tetrazolium (NBT) [20, 21]. Peroxidase (POD) activity was determined by the guaiacol oxidation method [22]. Catalase (CAT) activity was determined by measuring the rate of decomposition of [H.sub.2][O.sub.2] at 240 nm, as described by Aebi [23]. Sulfite reductase (SiR) activity was estimated by the coupled SiR/OASTL assay [24, 25] with the addition of NADPH and tungstic acid [26]. Sulfite oxidase (SO) activity was determined by measuring sulphite disappearance using OHmediated discolouring of fuchsine according to Pachmayr's report [27].

2.8. Estimation of MDA Content. Malondialdehyde (MDA) content was determined using the method described by Fu and Huang [28]. Fresh leaf sample (0.2 g) was homogenized with 2 mL of 0.1% (w/v) trichloroacetic acid (TCA) using a chilled mortar and pestle on ice. The homogenate was centrifuged at 15,000 xg for 20 min, at 4[degrees]C, and the supernatant was used for lipid peroxidation analysis. A total of 4 mL of 0.5% thiobarbituric acid (TBA) in 20% TCA was added to 1 mL of the supernatant. The mixture was incubated in hot water (95[degrees]C) for 30 min and cooled immediately on ice to stop the reaction and centrifuged at 15,000 xg for 20 min. Absorbance was measured at 532 and 600 nm, and MDA concentration was estimated by subtracting the nonspecific absorption at 600 nm from the absorption at 532 nm.

2.9. Estimation of Sulfur Content. For sulfur (S) determination, the turbidimetric method described by Reyes-Dlaz et al. [29] was applied. Biomass of whole plant dried for 48 h was treated with 95% magnesium nitrate and ashed at 500[degrees]C for 8 h. Then the ashed samples were digested in 10 mL of 2 M HCl at 150[degrees]C for 60 min. After addition of barium chloride (Ba[Cl.sub.2]) and Tween-80 into the solution, its absorbance was immediately measured using a UV/VIS spectrophotometer Model 723PC (Jinghua Instruments, Shanghai, China) at 440 nm.

2.10. Statistical Analysis. All experiments in this study were repeated at least three times. Statistical analysis (mean [+ or -] standard error) was performed and chart was created using relative tools of Microsoft Excel 2010. All data were analyzed by ANOVA using SPSS 13.0 software package (SPSS Inc., Chicago, USA), and then LSD method was used to detect possible differences among the accessions. Asterisk symbols above the columns in the figures indicate significant differences at P < 0.05 (Student's 1-test).

3. Results

3.1. Leaf Injury under S[O.sub.2] Stress Condition. After 7-day fumigation treatment by S[O.sub.2], injury symptoms appeared on leaves of all the 38 C. dactylon accessions. The visible symptoms consisted of bifacial, marginal, or interval necrosis and chlorosis on leaves at the full stage of development (Figure 2). The necrotic areas ranged from white to brown in color, and the margins of the necrotic areas are mostly irregular and occasionally dark in color. Injury rate of leaves varied in C. dactylon accessions from 38.3% in accession YN1205to 13.3% in accession SC1203 (Table 2). It seemed that accessions originated from city park and hillside had higher S[O.sub.2] tolerance than other habitat origins (Tables 1 and 2). To further study the physiological response of C. dactylon to S[O.sub.2], we selected three accessions of SC1203, SC1209, and GZ1110 as high S[O.sub.2]-tolerant representatives, three accessions of SC1217, YN1110, and XZ1206 as intermediate S[O.sub.2]-tolerant representatives, and three accessions of YN1205, CQ1116, and SC1208 as S[O.sub.2]-sensitive representatives based on the injury rate of leaves and the geographic distribution (Tables 1 and 2).

3.2. Changes of Sugar and Proline under S[O.sub.2] Stress Condition. The soluble sugar and proline contents in leaves from all of the nine C. dactylon accessions increased along with the increase of their S[O.sub.2] tolerability (Figure 3). Moreover, the soluble sugar and proline contents from all of the high S[O.sub.2]tolerant C. dactylon accessions and intermediate S[O.sub.2]-tolerant C. dactylon accessions were significantly higher than those from any of the three S[O.sub.2]-sensitive C. dactylon accessions at both 0-day time-point without S[O.sub.2] treatment and 7-daytimepoint after S[O.sub.2] fumigation treatment. However, the soluble sugar and proline contents from 7-day time-point after S[O.sub.2] fumigation treatment showed no significant change when they were compared with those from 0-day time-point in any C. dactylon accession, which indicates that both soluble sugar and proline are not induced or inhibited in C. dactylon under S[O.sub.2] stress condition (Figure 3).

3.3. Changes of Photosynthetic Pigments under S[O.sub.2] Stress Condition. Contents of photosynthetic pigments in leaves from all of the nine C. dactylon accessions decreased under S[O.sub.2] stress condition but showed different patterns with different pigment (Figure 4). Chlorophyll a contents from two intermediate S[O.sub.2]-tolerant C. dactylon accessions (YN1110 and XZ1206) and from all of the three high S[O.sub.2]-tolerant C. dactylon accessions were significantly higher than those from any of S[O.sub.2]-sensitive C. dactylon accessions in an increasing trend along with the increase of S[O.sub.2] tolerability at 0-day time-point (Figure 4(a)). Under S[O.sub.2] stress condition, chlorophyll a contents in leaves from intermediate and high S[O.sub.2]-tolerant C. dactylon accessions reduced significantly less than those from S[O.sub.2]-sensitive C. dactylon accessions. No significant differences of chlorophyll b contents were observed among the high S[O.sub.2]-tolerant, intermediate S[O.sub.2]tolerant, and S[O.sub.2]-sensitive C. dactylon accessions at 0-day time-point (Figure 4(b)). After 7-day stress treatment by S[O.sub.2] fumigation, chlorophyll b contents reduced in leaves from all of the nine C. dactylon accessions. The contents of chlorophyll b showed no significant differences between intermediate S[O.sub.2]-tolerant and S[O.sub.2]-sensitive C. dactylon accessions, but significantly less reduction of chlorophyll b content was observed in high S[O.sub.2]-tolerant C. dactylon accessions. As for total chlorophyll content, it showed a similar pattern with chlorophyll a in leaves from all of the nine C. dactylon accessions (Figure 4(c)). Carotenoid contents showed no significant differences among the high S[O.sub.2]-tolerant, intermediate S[O.sub.2]-tolerant, and S[O.sub.2]-sensitive C. dactylon accessions at 0-day time-point but significantly less reduced along with the increase of S[O.sub.2] tolerability of C. dactylon accessions after 7-day S[O.sub.2] stress treatment (Figure 4(d)).

3.4. Changes of ROS Level and Antioxidant Enzyme Activities under S[O.sub.2] Stress Condition. As two major indicators for reactive oxygen species (ROS) level and oxidative damage, hydrogen peroxide ([H.sub.2][O.sub.2]) and malondialdehyde (MDA) contents were tested in this study. As shown in Figure 5, the high S[O.sub.2]-tolerant, intermediate S[O.sub.2]-tolerant, and S[O.sub.2]-sensitive C. dactylon accessions displayed nearly the same levels of [H.sub.2][O.sub.2] and MDA in leaves at 0-day time-point without S[O.sub.2] treatment (Figures 5(a) and 5(b)). After 7-day S[O.sub.2] fumigation treatment, levels of both [H.sub.2][O.sub.2] and MDA increased in leaves from all of the nine C. dactylon accessions. When compared within all of the nine C. dactylon accessions, levels of both [H.sub.2][O.sub.2] and MDA in leaves from high S[O.sub.2] tolerant C. dactylon accessions and intermediate S[O.sub.2]-tolerant C. dactylon accessions were significantly lower than those from S[O.sub.2]-sensitive C. dactylon accessions (Figures 5(a) and 5(b)).

To address the relationship between the changes of ROS level and the antioxidant enzyme activities, three major antioxidant enzymes, including SOD, POD, and CAT, were analyzed for their enzyme activities. SOD activities showed no significant differences (about 30 U/g protein FW) in leaves from high S[O.sub.2]-tolerant C. dactylon accessions, intermediate S[O.sub.2]-tolerant C. dactylon accessions, and S[O.sub.2]-sensitive C. dactylon accessions at 0-day time-point without S[O.sub.2] treatment (Figure 6(a)). After 7-day S[O.sub.2] stress treatment, SOD activities increased greatly in leaves from all of the nine C. dactylon accessions. However, the increase degree was in a decreasing trend along with S[O.sub.2] tolerability of the C. dactylon accessions, displaying highest activities in S[O.sub.2]sensitive C. dactylon accessions (more than 120 U/g protein FW) and lowest activities in high S[O.sub.2]-tolerant C. dactylon accessions (more than 60 U/g protein FW) (Figure 6(a)). POD activities increased in leaves from all of the nine C. dactylon accessions after 7-day S[O.sub.2] stress treatment, but no significant differences were observed within high S[O.sub.2]tolerant C. dactylon accessions, intermediate S[O.sub.2]-tolerant C. dactylon accessions, and S[O.sub.2]-sensitive C. dactylon accessions (Figure 6(b)). However, we found that POD activities in leaves from S[O.sub.2]-tolerant C. dactylon accessions were significantly higher than those from S[O.sub.2]-sensitive C. dactylon accessions in an increasing trend along with an increase of S[O.sub.2] tolerability at 0-day time-point without S[O.sub.2] treatment, displaying nearly 1.4-fold increase (15433/11183 U/g protein FW) and 1.7-fold increase (18866/11183 U/g protein FW) of enzyme activities in intermediate S[O.sub.2]-tolerant and high S[O.sub.2]-tolerant C. dactylon accessions, respectively (Figure 6(b)). CAT activities increased in leaves from all of the nine C. dactylon accessions after 7-day S[O.sub.2] stress treatment, but no significant differences were observed in leaves from high S[O.sub.2]-tolerant C. dactylon accessions, intermediate S[O.sub.2]-tolerant C. dactylon accessions, and S[O.sub.2]sensitive C. dactylon accessions either after 7-day S[O.sub.2] stress treatment or at 0-day without S[O.sub.2] treatment (Figure 6(c)).

3.5. Changes of Sulfur Content, SiR, and SO Enzyme Activities under S[O.sub.2] Stress Condition. Sulfur contents in leaves from two intermediate S[O.sub.2]-tolerant C. dactylon accessions (YN1110 andXZ1206) and all of the three high S[O.sub.2]-tolerant C. dactylon accessions were significantly higher than those from any of the S[O.sub.2]-sensitive C. dactylon accessions at 0-day timepoint without S[O.sub.2] stress treatment (Figure 7(a)). After 7-day S[O.sub.2] fumigation treatment, sulfur contents increased in leaves from all of the nine C. dactylon accessions in an increasing trend along with increase of S[O.sub.2] tolerability of the C. dactylon accessions. Moreover, sulfur contents in leaves from all of the high and intermediate S[O.sub.2]-tolerant C. dactylon accessions showed significantly higher levels than those from any of the S[O.sub.2]-sensitive C. dactylon accessions (Figure 7(a)). SiR activities were nearly in the same levels (about 5 U/mg protein FW) in leaves from all of the nine C. dactylon accessions at 0-day time-point without S[O.sub.2] treatment (Figure 7(b)). After 7-day S[O.sub.2] stress treatment, SiR activities increased about 2fold (approximate 10 U/mg protein FW) in S[O.sub.2]-sensitive C. dactylon accessions, 2.4-fold (approximate 12 U/mg protein FW) in intermediate S[O.sub.2]-tolerant C. dactylon accessions, and

3.4-fold (approximate 17 U/mg protein FW) in high S[O.sub.2] tolerant C. dactylon accessions, respectively. More importantly, SiR activities showed significantly higher levels in leaves from high and intermediate S[O.sub.2]-tolerant C. dactylon accessions than those from S[O.sub.2]-sensitive C. dactylon accessions, displaying an apparent increasing trend along with S[O.sub.2] tolerability of the C. dactylon accessions (Figure 7(b)). SO activities in leaves from any of C. dactylon accessions after 7day S[O.sub.2] fumigation treatment showed nearly the same level with those from 0-day time-point without S[O.sub.2] treatment (Figure 7(c)). However, SO activity levels were significantly higher in leaves from high and intermediate S[O.sub.2]-tolerant C. dactylon accessions than those from S[O.sub.2]-sensitive C. dactylon accessions, displaying an apparent increasing trend along with S[O.sub.2] tolerability of the C. dactylon accessions (Figure 7(c)).

4. Discussion

S[O.sub.2], a major air pollutant in developing countries, is highly toxic to plants once they are exposed to high doses of S[O.sub.2] above the threshold. C. dactylon is a widely used warm season turfgrass on sports fields, golf courses, roadsides, and lawns in city or industry districts. Our previous study indicated that growth rate of C. dactylon was affected and visible symptoms appeared on leaves under S[O.sub.2] stress condition; however this species has much better S[O.sub.2]-tolerant ability among warm season turfgrasses [13]. C. dactylon is wildly distributed in South America, Africa, Europe, and South Asia and displays abundant genetic diversities worldwide [3033]. To achieve better air quality and landscape effect in S[O.sub.2] polluted areas, selection or development of high S[O.sub.2]-tolerant C. dactylon variations for planting in such regions is desired. In this study, we selected 9 out of 38 wild C. dactylon accessions from Southwest China as representatives of high, intermediate S[O.sub.2]-tolerant, and S[O.sub.2]-sensitive accessions based on the injury degree of S[O.sub.2] to leaves and the geographic distribution and then comparatively analyzed their physiological differences under S[O.sub.2] untreated and treated conditions. Our results indicated that S[O.sub.2] tolerance of C. dactylon might be largely related to soluble sugar, proline and chlorophyll a contents, and SO enzyme activities. To the best of our knowledge, this is the first comprehensive study of physiological differences in C. dactylon accessions of warm season turfgrasses. This study gained some insights into understanding the genetic and molecular S[O.sub.2]-tolerant mechanisms of C. dactylon and provided guideline for selection and development of C. dactylon variations for planting in S[O.sub.2] polluted urban or industrial areas.

Soluble sugars and proline, as two major compatible solutes in the cytoplasm and organelle, play important roles under multiple stress conditions, such as drought and salinity [34, 35]. In this study, we observed that S[O.sub.2]-tolerant C. dactylon accessions showed significantly higher soluble sugar and proline contents under both S[O.sub.2] treated and untreated conditions (Figure 3), suggesting that both of them might be related to S[O.sub.2] tolerance. C. dactylon accessions originated from habitats of hillside and city park have much higher S[O.sub.2] tolerance than those from other habitats (Tables 1 and 2), suggesting that the increased soluble sugar and proline contents most probably evolved from drought and S[O.sub.2] stress adaptation. However, increased soluble sugar and proline contents in S[O.sub.2]-tolerant C. dactylon accessions are not likely involved in osmotic pressure but more likely involved in maintaining cell membrane stability, synthesis of other compounds, supply of energy, action as regulators of gene expression, and signal molecules based on their multiple functions [36]. Thereafter, soluble sugar and proline contents can be considered as marker for selection of C. dactylon variations with high S[O.sub.2] tolerability.

Chlorophyll (including chlorophylls a and b) and carotenoid are known as the two important pigments in chloroplast of tree and grass plant leaves. The important role of pigments is to absorb certain wavelengths from sunlight and then convert the unusable sunlight energy into usable chemical energy during photosynthesis. Chlorophyll a is the primary pigment for photosynthesis in plants [37]. In this study, leaf injury of C. dactylon was observed under S[O.sub.2] stress condition (Table 2). As a consequence, chlorophyll a, chlorophyll b, and carotenoid contents decreased in C. dactylon under S[O.sub.2] stress condition, consistent with previous reports on grass and tree plants [13, 38, 39]. However, S[O.sub.2]-tolerant C. dactylon accessions showed significantly higher contents of chlorophyll a, chlorophyll b, and carotenoid under S[O.sub.2] treated condition, consistent with their less leaf injury S[O.sub.2]tolerant C. dactylon accessions observed in this study. Moreover, S[O.sub.2]-tolerant C. dactylon accessions had significantly higher content of chlorophyll a under S[O.sub.2] untreated condition. Now that chlorophyll a is the primary pigment for photosynthesis in plants, significantly higher contents of chlorophyll a in C. dactylon accessions under both S[O.sub.2] treated and untreated conditions indicate that S[O.sub.2] tolerance of C. dactylon might be largely related to content of chlorophyll a.

Early study showed that S[O.sub.2] gas after entering leaves of plant is converted into sulfite (S[O.sub.3.sup.2-]]) and bisulfite (HS[O.sub.3.sup.2-]]) once it is dissolved in cellular cytoplasm [40]. Furthermore, detoxification reaction of HS[O.sub.3.sup.2-]] and S[O.sub.3.sup.2-]] to sulfate (S[O.sub.4.sup.2-]]) in plants leads to production of many kinds of ROS, such as superoxide radical ([??]), hydrogen peroxide ([H.sub.2][O.sub.2]), and hydroxyl radical ([??]) 41]. Excessive ROS are highly reactive and toxic to plants, which could cause oxidative damage to membranes, DNA, proteins, photosynthetic pigments, and lipids [42]. To protect plant cells from ROS damage, plant developed antioxidant enzymes to deal with the excessive ROS in plant cells. SOD, POD, and CAT are considered as three major antioxidant enzymes. To analyze the oxidative effect of S[O.sub.2] on C. dactylon, we measured the ROS level and antioxidant enzyme activities in C. dactylon accessions. Although both ROS levels (reflected by [H.sub.2][O.sub.2] and MDA contents) and antioxidant enzyme activities (reflected by SOD, POD, and CAT) increased in all of the nine C. dactylon accessions under S[O.sub.2] stress condition, the S[O.sub.2]-tolerant C. dactylon accessions showed significantly lower ROS levels and SOD activities, indicating that the S[O.sub.2]-tolerant C. dactylon accessions have much stronger antioxidant ability and less damage occurs to them by S[O.sub.2]. Moreover, lower SOD activity was theoretically consistent with lower ROS level in the S[O.sub.2]-tolerant C. dactylon accessions under S[O.sub.2] stress condition, which is in agreement with previous report [43]. Although POD activities were nearly at the same level in leaves from all of the nine C. dactylon accessions after 7day S[O.sub.2] stress treatment, activities of this antioxidant enzyme from S[O.sub.2]-tolerant C. dactylon accessions were significantly higher than those from S[O.sub.2]-sensitive C. dactylon accessions. Taken together, we suggest that significantly higher activity of POD prior to S[O.sub.2] treatment might be devoted to the increased antioxidant ability in S[O.sub.2]-tolerant C. dactylon accessions.

S[O.sub.2] gas after entering leaves of plant can be converted into either sulfate by SO to enter into oxidative pathway or sulfide by SiR to enter into reductive pathway [44]. Overexpression of both SO and SiR showed more tolerance to sulfur dioxide toxicity in Arabidopsis thaliana and/or tomato plants [44-47]. Transcriptional analyses indicate that SiR is induced by S[O.sub.2] but SO is constitutively expressed in natural plant [45, 46]. In this study, we found that SiR activity level was significantly increased under S[O.sub.2] stress condition but SO activity level had almost no change under S[O.sub.2] treated and untreated conditions in leaves from all of the nine C. dactylon accessions, consistent with previous reports on other plant species [45, 46]. Under S[O.sub.2] stress condition, the S[O.sub.2]-tolerant C. dactylon accessions showed higher levels of both SiR and SO activities and contained higher sulfur content in leaves as corresponding consequence. More importantly, we found that the S[O.sub.2]-tolerant C. dactylon accessions showed significantly higher SO activities prior to S[O.sub.2] treatment, but no significant differences were observed among the nine C. dactylon accessions. Increased SO activity in S[O.sub.2]-tolerant C. dactylon accession could convert sulfite to nontoxic sulfate more efficiently than S[O.sub.2]-sensitive C. dactylon accession for storage, once highly toxic S[O.sub.2] gas enters into the C. dactylon cells, which indicates that SO antioxidant enzyme plays an important role in S[O.sub.2] tolerance in C. dactylon.

5. Conclusion

C. dactylon, a warm season perennial grass species, is widely used as turfgrass on sports fields, golf courses, roadsides, and lawns in city or industry districts in warm season. Although this species has much better S[O.sub.2]-tolerant ability among warm season turfgrasses, its growth rate will be affected and visible symptoms like yellowing, chlorosis, bleaching, and even killing foliage will appear on leaves of C. dactylon in S[O.sub.2] polluted areas. To achieve better air quality and landscape effect in S[O.sub.2] polluted areas, selection or development of high S[O.sub.2]-tolerant C. dactylon variations is desired. In this study, we selected 9 out of 38 C. dactylon accessions from Southwest China as representatives of high, intermediate S[O.sub.2]-tolerant, and S[O.sub.2]-sensitive accessions and then comparatively analyzed their physiological differences under S[O.sub.2] untreated and treated conditions. Our results indicated that S[O.sub.2] tolerance of C. dactylon might be largely related to soluble sugar, proline and chlorophyll a contents, and SO enzyme activities. This study gained some insights into understanding the genetic and molecular S[O.sub.2]-tolerant mechanisms of C. dactylon and provided guideline for selection or development of C. dactylon variations for planting in S[O.sub.2] polluted urban or industrial areas.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors' Contribution

Xi Li and Ling Wang contributed equally to this paper.

http://dx.doi.org/10.1155/2014/916595

Acknowledgments

This work was supported in part by the Science and Technology Department of Sichuan Province (Grant no. 05JY009007-4) and the Scientific Research Fund of Sichuan Provincial Education Department (Grant no. 12ZA116).

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Xi Li, (1) Ling Wang, (1) Yiqiao Li, (2) Lingxia Sun, (1) Shizhen Cai, (1) and Zhuo Huang (1)

(1) College of Landscape Architecture, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang, Sichuan 611130, China

(2) Business School, Sichuan Agricultural University, Dujiangyan, Sichuan 611830, China

Correspondence should be addressed to Xi Li; 781221015@qq.com

Received 24 February 2014; Accepted 26 May 2014; Published 6 July 2014

Academic Editor: Mehmet Yakup Arica

TABLE 1: Geographical origin of 38 wild C. dactylon accessions used in
this study.

Order   Accession            Origin              Habitat
        number

1        SC1102         Pixian, Sichuan         Roadside
2        SC1105        Longquan, Sichuan        Hillside
3        SC1106        Shuangliu, Sichuan       Riverside
4        SC1109        Guangyuan, Sichuan       Roadside
5        SC1115         Zitong, Sichuan         Roadside
6        SC1119        Guanghan, Sichuan        Riverside
7        SC1201         Leshan, Sichuan         Floodland
8        SC1203          Wuhou, Sichuan         City park
9        SC1208         Luxian, Sichuan         Riverside
10       SC1209        Nanchong, Sichuan        Hillside
11       SC1211        Guang'an, Sichuan        Roadside
12       SC1213        Panzhihua, Sichuan       Roadside
13       SC1217         Meishan, Sichuan       Fieldridge
14       CQ1101       Jiangjin, Chongqing       Roadside
15       CQ1102       Yongchuan, Chongqing      Floodland
16       CQ1107        Bishan, Chongqing        Roadside
17       CQ1108       Jiangbei, Chongqing       Roadside
18       CQ1109       Dianjiang, Chongqing     Fieldridge
19       CQ1112       Liangping, Chongqing      Roadside
20       CQ1116        Wanzhou, Chongqing       Riverside
21       YN1102         Kunming, Yunnan         City park
22       YN1105         Chuxiong, Yunnan        Roadside
23       YN1106           Dali, Yunnan          Roadside
24       YN1107         Lijiang, Yunnan         Riverside
25       YN1110         Baoshan, Yunnan         Wasteland
26       YN1201           Yuxi, Yunnan          Roadside
27       YN1205           Puer, Yunnan          Roadside
28       YN1208      Xishuangbanna, Yunnan      Roadside
29       GZ1103         Guiyang, Guizhou        Riverside
30       GZ1104         Anshun, Guizhou         Roadside
31       GZ1106       Liupanshui, Guizhou      Fieldridge
32       GZ1109          Zunyi, Guizhou         Roadside
33       GZ1110         Qianlan, Guizhou        Hillside
34       XZ1205           Lhasa, Tibet          Roadside
35       XZ1206         Nyingchi, Tibet         Roadside
36       XZ1208           Bomi, Tibet           Riverside
37       XZ1209           Baxoi, Tibet          Roadside
38       XZ1213          Chamdo, Tibet          Floodland

Order   Altitude     Mean annual
           (m)       temperature
                     ([degrees]C)

1          560           18.5
2          750           16.5
3          510           16.3
4          490           16.1
5          610           16.5
6          420           16.3
7          420           17.4
8          540           16.7
9          380           17.8
10         520           17.4
11         450           17.1
12        1150           20.3
13         460           17.1
14         590           18.4
15         340           18.2
16         530           18.3
17         440           17.5
18         420           17.0
19         520           16.6
20         180           17.7
21        1910           15.0
22        1790           15.8
23        1980           15.1
24        2360           15.8
25        2410           16.0
26        1890           18.2
27        1750           17.7
28         890           21.0
29         970           15.3
30         860           14.0
31        1810           13.5
32         960           15.1
33        1050           16.1
34        3120           7.5
35        3430           8.7
36        2680           8.7
37        3250           10.4
38        3170           7.6

TABLE 2: Leaf injury rate of38 wild C. dactylon accessions
under S[O.sub.2] stress condition.

Order   Accession    Injury rate (%)
        number

1         YN1205     38.3 [+ or -] 0.6
2         CQ1116     377 [+ or -] 1.2
3         SC1208     36.7 [+ or -] 0.6
4         SC1106     35.3 [+ or -] 1.5
5         GZ1109     34.3 [+ or -] 1.5
6         YN1107     32.0 [+ or -] 1.7
7         YN1208     31.7 [+ or -] 0.6
8         SC1213     30.3 [+ or -] 1.5
9         CQ1112     30.3 [+ or -] 1.2
10        XZ1209     28.7 [+ or -] 1.2
11        XZ1213     28.3 [+ or -] 0.6
12        GZ1104     27.3 [+ or -] 1.5
13        SC1119     26.3 [+ or -] 1.5
14        SC1201     26.3 [+ or -] 2.1
15        YN1106     25.7 [+ or -] 1.5
16        XZ1205     25.7 [+ or -] 2.5
17        CQ1102     25.3 [+ or -] 0.6
18        SC1217     25.3 [+ or -] 1.5
19        YN1110     25.0 [+ or -] 1.0
20        XZ1206     24.7 [+ or -] 0.6
21        SC1211     24.7 [+ or -] 2.5
22        YN1105     23.7 [+ or -] 2.1
23        XZ1208     23.0 [+ or -] 1.7
24        GZ1106     22.0 [+ or -] 1.0
25        SC1102     21.7 [+ or -] 2.1
26        SC1115     20.0 [+ or -] 2.6
27        CQ1108     20.0 [+ or -] 2.6
28        CQ1109     19.3 [+ or -] 1.5
29        YN1201     19.3 [+ or -] 0.6
30        SC1109     19.3 [+ or -] 1.5
31        GZ1103     19.0 [+ or -] 2.0
32        CQ1107     18.7 [+ or -] 1.5
33        CQ1101     17.7 [+ or -] 1.5
34        YN1102     17.3 [+ or -] 1.5
35        SC1105     16.3 [+ or -] 1.5
36        GZ1110     15.0 [+ or -] 2.0
37        SC1209     13.7 [+ or -] 0.6
38        SC1203     13.3 [+ or -] 1.5

Note: data are presented by means [+ or -] SE (n = 3).
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