# Comparative Analyses of Physiological Responses ofCynodon dactylon Accessions from Southwest China to Sulfur Dioxide Toxicity

Authors: Xi Li; Ling Wang; Yiqiao Li; Lingxia Sun; Shizhen Cai; Zhuo Huang
Journal: The Scientific World Journal
 (2014)

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## Abstract

Sulfur dioxide (SO2), a major air pollutant in developing countries, is highly toxic to plants. To achieve better air quality and landscape, planting appropriate grass species in severe SO2 polluted areas is very critical. Cynodon dactylon, a widely used warm season turfgrass species, has good SO2-tolerant ability. In this study, we selected 9 out of 38 C. dactylon accessions from Southwest China as representatives of high, intermediate SO2-tolerant and SO2-sensitive accessions to comparatively analyze their physiological differences in leaves under SO2 untreated and treated conditions. Our results revealed that SO2-tolerant C. dactylon accessions showed higher soluble sugar, proline, and chlorophyll a contents under both SO2 treated and untreated conditions; higher chlorophyll b and carotenoid under SO2 treated condition; lower reactive oxygen species (ROS) level, oxidative damages, and superoxide dismutase (SOD) activities under SO2 treated condition; and higher peroxidase (POD) activities under SO2 untreated condition. Further results indicated that SO2-tolerant C. dactylon accessions had higher sulfur contents under both SO2 treated and untreated conditions, consistent with higher SO activities under both SO2 treated and untreated conditions, and higher SiR activities under SO2 treated condition. Taken together, our results indicated that SO2 tolerance of C. dactylon might be largely related to soluble sugar, proline and chlorophyll a contents, and SO enzyme activity.


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## Body

## 1. Introduction

Sulfur dioxide (SO2), a gaseous pollutant with bad odor in the atmosphere, is mainly emitted from anthropogenic sources. It is estimated that more than 70% of global SO2 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 SO2 into the atmosphere has been increasing quickly. As the biggest developing country in the world, China is leading the world as the biggest SO2 emitter, contributing to about one-fourth of the global emission and more than 90% of East Asia emission since the 1990s [2]. Total SO2 emission in China increased by 53%, from 21.7 Tg (1 Tg = 1012 g) in 2000 to 33.2 Tg in 2006, at an annual growth rate of 7.3%. The SO2 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 SO2 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 SO2 in the atmosphere will be a major concern in developing countries in a long period of forthcoming time.In the atmosphere, when gaseous SO2 meets with water, considerable amounts of SO2 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. SO2 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 SO2 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 SO2, 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 SO2 should be selected out for use. Tree species tolerant to SO2 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 SO2 than woody plants, because the former have a higher S : C ratio than the latter and therefore can take up more SO2 from the atmosphere [8]. Turfgrasses can be generally classified as cool season, warm season, or evergreen types. A few studies on tolerance to SO2 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 ofDactylis glomerata, Festuca rubra, Holcus lanatus, Lolium perenne, andPhleum bertolonii [9]; comparison of stomatal morphology and resistance, membrane permeability, and the uptake and metabolism of 35SO3 and 35SO2 in cool-season species of D.glomerata,F. rubra,H. lanatus, andL. perenne [10]; investigation on the rate of development of tolerance in cool-season species ofF. rubra,L. multiflorum,L. perenne,P. pratense, andPoa pratensis [11]; and genetic nature of tolerance in cool-season species ofL. 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 thatC. dactylon displayed the highest resistance to SO2 among four warm season turfgrasses includingC. dactylon,Eremochloa ophiuroides,Paspalum notatum, andZoysia japonica [13]. In the present study, we firstly compared influences of SO2 on leaves of 38 wildC. dactylon accessions from Southwest China. Based on injury rate of SO2 to leaves, nineC. dactylon accessions representing high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitive to SO2 accessions were selected to comparatively study relationships between SO2 tolerance and several physiological parameters. This study gained some insights into understanding the genetic and molecular mechanisms ofC. dactylon to SO2 and provided guideline for selection and development ofC. dactylon variations for planting in SO2 polluted urban or industrial areas.


## 2. Materials and Methods

### 2.1. Plant Materials and Growth Conditions

Thirty-eight wildC. dactylonaccessions 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 wildC. 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 beC. dactylon based on morphological characteristics as described by Harlan [14].Table 1
Geographical origin of 38 wildC. dactylon accessions used in this study.
Order
Accession number
Origin
Habitat
Altitude (m)
Mean annual temperature (°C)
1
SC1102
Pixian, Sichuan
Roadside
560
18.5
2
SC1105
Longquan, Sichuan
Hillside
750
16.5
3
SC1106
Shuangliu, Sichuan
Riverside
510
16.3
4
SC1109
Guangyuan, Sichuan
Roadside
490
16.1
5
SC1115
Zitong, Sichuan
Roadside
610
16.5
6
SC1119
Guanghan, Sichuan
Riverside
420
16.3
7
SC1201
Leshan, Sichuan
Floodland
420
17.4
8
SC1203
Wuhou, Sichuan
City park
540
16.7
9
SC1208
Luxian, Sichuan
Riverside
380
17.8
10
SC1209
Nanchong, Sichuan
Hillside
520
17.4
11
SC1211
Guang’an, Sichuan
Roadside
450
17.1
12
SC1213
Panzhihua, Sichuan
Roadside
1150
20.3
13
SC1217
Meishan, Sichuan
Fieldridge
460
17.1
14
CQ1101
Jiangjin, Chongqing
Roadside
590
18.4
15
CQ1102
Yongchuan, Chongqing
Floodland
340
18.2
16
CQ1107
Bishan, Chongqing
Roadside
530
18.3
17
CQ1108
Jiangbei, Chongqing
Roadside
440
17.5
18
CQ1109
Dianjiang, Chongqing
Fieldridge
420
17.0
19
CQ1112
Liangping, Chongqing
Roadside
520
16.6
20
CQ1116
Wanzhou, Chongqing
Riverside
180
17.7
21
YN1102
Kunming, Yunnan
City park
1910
15.0
22
YN1105
Chuxiong, Yunnan
Roadside
1790
15.8
23
YN1106
Dali, Yunnan
Roadside
1980
15.1
24
YN1107
Lijiang, Yunnan
Riverside
2360
15.8
25
YN1110
Baoshan, Yunnan
Wasteland
2410
16.0
26
YN1201
Yuxi, Yunnan
Roadside
1890
18.2
27
YN1205
Pu’er, Yunnan
Roadside
1750
17.7
28
YN1208
Xishuangbanna, Yunnan
Roadside
890
21.0
29
GZ1103
Guiyang, Guizhou
Riverside
970
15.3
30
GZ1104
Anshun, Guizhou
Roadside
860
14.0
31
GZ1106
Liupanshui, Guizhou
Fieldridge
1810
13.5
32
GZ1109
Zunyi, Guizhou
Roadside
960
15.1
33
GZ1110
Qianlan, Guizhou
Hillside
1050
16.1
34
XZ1205
Lhasa, Tibet
Roadside
3120
7.5
35
XZ1206
Nyingchi, Tibet
Roadside
3430
8.7
36
XZ1208
Bomi, Tibet
Riverside
2680
8.7
37
XZ1209
Baxoi, Tibet
Roadside
3250
10.4
38
XZ1213
Chamdo, Tibet
Floodland
3170
7.6Figure 1
The collection sites of the 38 samples of wildC. dactylonin Southwest China. Provinces where samples were collected are illustrated with number ofC. dactylonaccessions in parentheses underneath in map.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°C, and 79%, respectively. All theC. 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. EachC. dactylon accession was replicated six times. AllC. 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 SO2 stress treatment. All of the selected pots of grass plants were fumigated with SO2 at a concentration of 3.75 mg/L in a custom-made fumigation chamber (85 cm × 85 cm × 40 cm) for 3 h per day over 7 days as described in our previous study [13]. The day when SO2 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 SO2. A SO2 gas detector (Z-1300, Environmental Sensors Co., Boca Raton, FL, USA) was used to measure the concentration of SO2 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 SO2 treatment from each accession served as control. Based on injury rate of SO2 to leaves after 7-day treatment of SO2, three high SO2-tolerant, three intermediate SO2-tolerant, and three SO2-sensitiveC. dactylon accessions were selected out for physiological studies. Leaves 2 cm above the soil fromC. dactylon plants treated by SO2 after 7 days were collected and brought back to laboratory for analysis. Leaves fromC. dactylon plants without SO2 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°C for 40 min and centrifuged at 15,000 ×g for 10 min. The pellets were further extracted twice with another 5 mL of 80% (v/v) ethanol. The supernatants were combined together and depigmented by activated charcoal at 80°C for 30 min. For the determination of soluble total sugars, 0.2 mL of the filtrate was mixed with 3 mL 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°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 μm 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°C for 1 h. The reaction 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 (~0.2 g) were ground in 2 mL of 80% acetone and ethyl alcohol (1 : 1), using a mortar and pestle, and then filtered through 0.45 μm 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 H2O2 Level

For grass protein extraction, about 0.2 g 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 ×g for 15 min at 4°C, the supernatant was used for determination of H2O2 levels as described by Hu et al. [18]. Briefly, 1 mL of the supernatant was mixed with 1 mL of 0.1% titanium sulphate in 20% H2SO4 (v/v) thoroughly for 10 min. After being centrifuged at 15,000 ×g 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 ×g for 15 min at 4°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 H2O2 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 OH- mediated 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 ×g for 20 min, at 4°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°C) for 30 min and cooled immediately on ice to stop the reaction and centrifuged at 15,000 ×g 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-Díaz et al. [29] was applied. Biomass of whole plant dried for 48 h was treated with 95% magnesium nitrate and ashed at 500°C for 8 h. Then the ashed samples were digested in 10 mL of 2 M HCl at 150°C for 60 min. After addition of barium chloride (BaCl2) 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 ± 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 atP
<
0.05 (Student’s t-test).


## 2.1. Plant Materials and Growth Conditions

Thirty-eight wildC. dactylonaccessions 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 wildC. 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 beC. dactylon based on morphological characteristics as described by Harlan [14].Table 1
Geographical origin of 38 wildC. dactylon accessions used in this study.
Order
Accession number
Origin
Habitat
Altitude (m)
Mean annual temperature (°C)
1
SC1102
Pixian, Sichuan
Roadside
560
18.5
2
SC1105
Longquan, Sichuan
Hillside
750
16.5
3
SC1106
Shuangliu, Sichuan
Riverside
510
16.3
4
SC1109
Guangyuan, Sichuan
Roadside
490
16.1
5
SC1115
Zitong, Sichuan
Roadside
610
16.5
6
SC1119
Guanghan, Sichuan
Riverside
420
16.3
7
SC1201
Leshan, Sichuan
Floodland
420
17.4
8
SC1203
Wuhou, Sichuan
City park
540
16.7
9
SC1208
Luxian, Sichuan
Riverside
380
17.8
10
SC1209
Nanchong, Sichuan
Hillside
520
17.4
11
SC1211
Guang’an, Sichuan
Roadside
450
17.1
12
SC1213
Panzhihua, Sichuan
Roadside
1150
20.3
13
SC1217
Meishan, Sichuan
Fieldridge
460
17.1
14
CQ1101
Jiangjin, Chongqing
Roadside
590
18.4
15
CQ1102
Yongchuan, Chongqing
Floodland
340
18.2
16
CQ1107
Bishan, Chongqing
Roadside
530
18.3
17
CQ1108
Jiangbei, Chongqing
Roadside
440
17.5
18
CQ1109
Dianjiang, Chongqing
Fieldridge
420
17.0
19
CQ1112
Liangping, Chongqing
Roadside
520
16.6
20
CQ1116
Wanzhou, Chongqing
Riverside
180
17.7
21
YN1102
Kunming, Yunnan
City park
1910
15.0
22
YN1105
Chuxiong, Yunnan
Roadside
1790
15.8
23
YN1106
Dali, Yunnan
Roadside
1980
15.1
24
YN1107
Lijiang, Yunnan
Riverside
2360
15.8
25
YN1110
Baoshan, Yunnan
Wasteland
2410
16.0
26
YN1201
Yuxi, Yunnan
Roadside
1890
18.2
27
YN1205
Pu’er, Yunnan
Roadside
1750
17.7
28
YN1208
Xishuangbanna, Yunnan
Roadside
890
21.0
29
GZ1103
Guiyang, Guizhou
Riverside
970
15.3
30
GZ1104
Anshun, Guizhou
Roadside
860
14.0
31
GZ1106
Liupanshui, Guizhou
Fieldridge
1810
13.5
32
GZ1109
Zunyi, Guizhou
Roadside
960
15.1
33
GZ1110
Qianlan, Guizhou
Hillside
1050
16.1
34
XZ1205
Lhasa, Tibet
Roadside
3120
7.5
35
XZ1206
Nyingchi, Tibet
Roadside
3430
8.7
36
XZ1208
Bomi, Tibet
Riverside
2680
8.7
37
XZ1209
Baxoi, Tibet
Roadside
3250
10.4
38
XZ1213
Chamdo, Tibet
Floodland
3170
7.6Figure 1
The collection sites of the 38 samples of wildC. dactylonin Southwest China. Provinces where samples were collected are illustrated with number ofC. dactylonaccessions in parentheses underneath in map.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°C, and 79%, respectively. All theC. 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. EachC. dactylon accession was replicated six times. AllC. 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 SO2 stress treatment. All of the selected pots of grass plants were fumigated with SO2 at a concentration of 3.75 mg/L in a custom-made fumigation chamber (85 cm × 85 cm × 40 cm) for 3 h per day over 7 days as described in our previous study [13]. The day when SO2 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 SO2. A SO2 gas detector (Z-1300, Environmental Sensors Co., Boca Raton, FL, USA) was used to measure the concentration of SO2 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 SO2 treatment from each accession served as control. Based on injury rate of SO2 to leaves after 7-day treatment of SO2, three high SO2-tolerant, three intermediate SO2-tolerant, and three SO2-sensitiveC. dactylon accessions were selected out for physiological studies. Leaves 2 cm above the soil fromC. dactylon plants treated by SO2 after 7 days were collected and brought back to laboratory for analysis. Leaves fromC. dactylon plants without SO2 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°C for 40 min and centrifuged at 15,000 ×g for 10 min. The pellets were further extracted twice with another 5 mL of 80% (v/v) ethanol. The supernatants were combined together and depigmented by activated charcoal at 80°C for 30 min. For the determination of soluble total sugars, 0.2 mL of the filtrate was mixed with 3 mL 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°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 μm 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°C for 1 h. The reaction 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 (~0.2 g) were ground in 2 mL of 80% acetone and ethyl alcohol (1 : 1), using a mortar and pestle, and then filtered through 0.45 μm 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 H2O2 Level

For grass protein extraction, about 0.2 g 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 ×g for 15 min at 4°C, the supernatant was used for determination of H2O2 levels as described by Hu et al. [18]. Briefly, 1 mL of the supernatant was mixed with 1 mL of 0.1% titanium sulphate in 20% H2SO4 (v/v) thoroughly for 10 min. After being centrifuged at 15,000 ×g 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 ×g for 15 min at 4°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 H2O2 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 OH- mediated 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 ×g for 20 min, at 4°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°C) for 30 min and cooled immediately on ice to stop the reaction and centrifuged at 15,000 ×g 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-Díaz et al. [29] was applied. Biomass of whole plant dried for 48 h was treated with 95% magnesium nitrate and ashed at 500°C for 8 h. Then the ashed samples were digested in 10 mL of 2 M HCl at 150°C for 60 min. After addition of barium chloride (BaCl2) 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 ± 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 atP
<
0.05 (Student’s t-test).


## 3. Results

### 3.1. Leaf Injury under SO2 Stress Condition

After 7-day fumigation treatment by SO2, injury symptoms appeared on leaves of all the 38C. 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 inC. dactylon accessions from 38.3% in accession YN1205 to 13.3% in accession SC1203 (Table 2). It seemed that accessions originated from city park and hillside had higher SO2 tolerance than other habitat origins (Tables 1 and 2). To further study the physiological response ofC. dactylon to SO2, we selected three accessions of SC1203, SC1209, and GZ1110 as high SO2-tolerant representatives, three accessions of SC1217, YN1110, and XZ1206 as intermediate SO2-tolerant representatives, and three accessions of YN1205, CQ1116, and SC1208 as SO2-sensitive representatives based on the injury rate of leaves and the geographic distribution (Tables 1 and 2).Table 2
Leaf injury rate of 38 wildC. dactylon accessions under SO2 stress condition.
Order
Accession number
Injury rate (%)
1
YN1205
38.3 ± 0.6
2
CQ1116
37.7 ± 1.2
3
SC1208
36.7 ± 0.6
4
SC1106
35.3 ± 1.5
5
GZ1109
34.3 ± 1.5
6
YN1107
32.0 ± 1.7
7
YN1208
31.7 ± 0.6
8
SC1213
30.3 ± 1.5
9
CQ1112
30.3 ± 1.2
10
XZ1209
28.7 ± 1.2
11
XZ1213
28.3 ± 0.6
12
GZ1104
27.3 ± 1.5
13
SC1119
26.3 ± 1.5
14
SC1201
26.3 ± 2.1
15
YN1106
25.7 ± 1.5
16
XZ1205
25.7 ± 2.5
17
CQ1102
25.3 ± 0.6
18
SC1217
25.3 ± 1.5
19
YN1110
25.0 ± 1.0
20
XZ1206
24.7 ± 0.6
21
SC1211
24.7 ± 2.5
22
YN1105
23.7 ± 2.1
23
XZ1208
23.0 ± 1.7
24
GZ1106
22.0 ± 1.0
25
SC1102
21.7 ± 2.1
26
SC1115
20.0 ± 2.6
27
CQ1108
20.0 ± 2.6
28
CQ1109
19.3 ± 1.5
29
YN1201
19.3 ± 0.6
30
SC1109
19.3 ± 1.5
31
GZ1103
19.0 ± 2.0
32
CQ1107
18.7 ± 1.5
33
CQ1101
17.7 ± 1.5
34
YN1102
17.3 ± 1.5
35
SC1105
16.3 ± 1.5
36
GZ1110
15.0 ± 2.0
37
SC1209
13.7 ± 0.6
38
SC1203
13.3 ± 1.5
Note: data are presented by means ± SE (n
=
3).Symptoms ofC. dactylonaccessions in response to SO2. (a) SO2-sensitive representativeC. dactylonaccession CQ1116, (b) intermediate SO2-tolerant representativeC. dactylonaccession SC1217, (c) high SO2-tolerant representativeC. dactylonaccession SC1203, and (d)C. dactylonaccession CQ1116 without SO2 treatment as a control.
(a)
(b)
(c)
(d)


### 3.2. Changes of Sugar and Proline under SO2 Stress Condition

The soluble sugar and proline contents in leaves from all of the nineC. dactylon accessions increased along with the increase of their SO2 tolerability (Figure 3). Moreover, the soluble sugar and proline contents from all of the high SO2-tolerantC. dactylon accessions and intermediate SO2-tolerantC. dactylon accessions were significantly higher than those from any of the three SO2-sensitiveC. dactylon accessions at both 0-day time-point without SO2 treatment and 7-day time-point after SO2 fumigation treatment. However, the soluble sugar and proline contents from 7-day time-point after SO2 fumigation treatment showed no significant change when they were compared with those from 0-day time-point in anyC. dactylon accession, which indicates that both soluble sugar and proline are not induced or inhibited inC. dactylon under SO2 stress condition (Figure 3).Comparison of soluble sugar (a) and proline (b) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)


### 3.3. Changes of Photosynthetic Pigments under SO2 Stress Condition

Contents of photosynthetic pigments in leaves from all of the nineC. dactylon accessions decreased under SO2 stress condition but showed different patterns with different pigment (Figure 4). Chlorophyll a contents from two intermediate SO2-tolerantC. dactylon accessions (YN1110 and XZ1206) and from all of the three high SO2-tolerantC. dactylon accessions were significantly higher than those from any of SO2-sensitiveC. dactylon accessions in an increasing trend along with the increase of SO2 tolerability at 0-day time-point (Figure 4(a)). Under SO2 stress condition, chlorophyll a contents in leaves from intermediate and high SO2-tolerantC. dactylon accessions reduced significantly less than those from SO2-sensitiveC. dactylon accessions. No significant differences of chlorophyll b contents were observed among the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions at 0-day time-point (Figure 4(b)). After 7-day stress treatment by SO2 fumigation, chlorophyll b contents reduced in leaves from all of the nineC. dactylon accessions. The contents of chlorophyll b showed no significant differences between intermediate SO2-tolerant and SO2-sensitiveC. dactylon accessions, but significantly less reduction of chlorophyll b content was observed in high SO2-tolerantC. dactylon accessions. As for total chlorophyll content, it showed a similar pattern with chlorophyll a in leaves from all of the nineC. dactylon accessions (Figure 4(c)). Carotenoid contents showed no significant differences among the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions at 0-day time-point but significantly less reduced along with the increase of SO2 tolerability ofC. dactylon accessions after 7-day SO2 stress treatment (Figure 4(d)).Comparison of chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), and carotenoid (d) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)
(d)


### 3.4. Changes of ROS Level and Antioxidant Enzyme Activities under SO2 Stress Condition

As two major indicators for reactive oxygen species (ROS) level and oxidative damage, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were tested in this study. As shown in Figure 5, the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions displayed nearly the same levels of H2O2 and MDA in leaves at 0-day time-point without SO2 treatment (Figures 5(a) and 5(b)). After 7-day SO2 fumigation treatment, levels of both H2O2 and MDA increased in leaves from all of the nineC. dactylon accessions. When compared within all of the nineC. dactylon accessions, levels of both H2O2 and MDA in leaves from high SO2-tolerantC. dactylon accessions and intermediate SO2-tolerantC. dactylon accessions were significantly lower than those from SO2-sensitiveC. dactylon accessions (Figures 5(a) and 5(b)).Comparison of ROS levels reflected by H2O2 (a) and MDA (b) contents in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(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 SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions at 0-day time-point without SO2 treatment (Figure 6(a)). After 7-day SO2 stress treatment, SOD activities increased greatly in leaves from all of the nineC. dactylon accessions. However, the increase degree was in a decreasing trend along with SO2 tolerability of theC. dactylon accessions, displaying highest activities in SO2-sensitiveC. dactylon accessions (more than 120 U/g protein FW) and lowest activities in high SO2-tolerantC. dactylon accessions (more than 60 U/g protein FW) (Figure 6(a)). POD activities increased in leaves from all of the nineC. dactylon accessions after 7-day SO2 stress treatment, but no significant differences were observed within high SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions (Figure 6(b)). However, we found that POD activities in leaves from SO2-tolerantC. dactylon accessions were significantly higher than those from SO2-sensitiveC. dactylon accessions in an increasing trend along with an increase of SO2 tolerability at 0-day time-point without SO2 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 SO2-tolerant and high SO2-tolerantC. dactylon accessions, respectively (Figure 6(b)). CAT activities increased in leaves from all of the nineC. dactylon accessions after 7-day SO2 stress treatment, but no significant differences were observed in leaves from high SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions either after 7-day SO2 stress treatment or at 0-day without SO2 treatment (Figure 6(c)).Comparison of antioxidant enzyme activities in leaves fromC. dactylonaccessions in response to SO2 stress. Antioxidant enzyme activities of SOD (a), POD (b), and CAT (c) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress were compared. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)


### 3.5. Changes of Sulfur Content, SiR, and SO Enzyme Activities under SO2 Stress Condition

Sulfur contents in leaves from two intermediate SO2-tolerantC. dactylon accessions (YN1110 and XZ1206) and all of the three high SO2-tolerantC. dactylon accessions were significantly higher than those from any of the SO2-sensitiveC. dactylon accessions at 0-day time-point without SO2 stress treatment (Figure 7(a)). After 7-day SO2 fumigation treatment, sulfur contents increased in leaves from all of the nineC. dactylon accessions in an increasing trend along with increase of SO2 tolerability of theC. dactylon accessions. Moreover, sulfur contents in leaves from all of the high and intermediate SO2-tolerantC. dactylon accessions showed significantly higher levels than those from any of the SO2-sensitiveC. 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 nineC. dactylon accessions at 0-day time-point without SO2 treatment (Figure 7(b)). After 7-day SO2 stress treatment, SiR activities increased about 2-fold (approximate 10 U/mg protein FW) in SO2-sensitiveC. dactylon accessions, 2.4-fold (approximate 12 U/mg protein FW) in intermediate SO2-tolerantC. dactylon accessions, and 3.4-fold (approximate 17 U/mg protein FW) in high SO2-tolerantC. dactylon accessions, respectively. More importantly, SiR activities showed significantly higher levels in leaves from high and intermediate SO2-tolerantC. dactylon accessions than those from SO2-sensitiveC. dactylon accessions, displaying an apparent increasing trend along with SO2 tolerability of theC. dactylon accessions (Figure 7(b)). SO activities in leaves from any ofC. dactylon accessions after 7-day SO2 fumigation treatment showed nearly the same level with those from 0-day time-point without SO2 treatment (Figure 7(c)). However, SO activity levels were significantly higher in leaves from high and intermediate SO2-tolerantC. dactylon accessions than those from SO2-sensitiveC. dactylon accessions, displaying an apparent increasing trend along with SO2 tolerability of theC. dactylon accessions (Figure 7(c)).Comparison of sulfur contents (a), SiR activities (b), and SO activities (c) in leaves from nine selectedC. dactylon accessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)


## 3.1. Leaf Injury under SO2 Stress Condition

After 7-day fumigation treatment by SO2, injury symptoms appeared on leaves of all the 38C. 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 inC. dactylon accessions from 38.3% in accession YN1205 to 13.3% in accession SC1203 (Table 2). It seemed that accessions originated from city park and hillside had higher SO2 tolerance than other habitat origins (Tables 1 and 2). To further study the physiological response ofC. dactylon to SO2, we selected three accessions of SC1203, SC1209, and GZ1110 as high SO2-tolerant representatives, three accessions of SC1217, YN1110, and XZ1206 as intermediate SO2-tolerant representatives, and three accessions of YN1205, CQ1116, and SC1208 as SO2-sensitive representatives based on the injury rate of leaves and the geographic distribution (Tables 1 and 2).Table 2
Leaf injury rate of 38 wildC. dactylon accessions under SO2 stress condition.
Order
Accession number
Injury rate (%)
1
YN1205
38.3 ± 0.6
2
CQ1116
37.7 ± 1.2
3
SC1208
36.7 ± 0.6
4
SC1106
35.3 ± 1.5
5
GZ1109
34.3 ± 1.5
6
YN1107
32.0 ± 1.7
7
YN1208
31.7 ± 0.6
8
SC1213
30.3 ± 1.5
9
CQ1112
30.3 ± 1.2
10
XZ1209
28.7 ± 1.2
11
XZ1213
28.3 ± 0.6
12
GZ1104
27.3 ± 1.5
13
SC1119
26.3 ± 1.5
14
SC1201
26.3 ± 2.1
15
YN1106
25.7 ± 1.5
16
XZ1205
25.7 ± 2.5
17
CQ1102
25.3 ± 0.6
18
SC1217
25.3 ± 1.5
19
YN1110
25.0 ± 1.0
20
XZ1206
24.7 ± 0.6
21
SC1211
24.7 ± 2.5
22
YN1105
23.7 ± 2.1
23
XZ1208
23.0 ± 1.7
24
GZ1106
22.0 ± 1.0
25
SC1102
21.7 ± 2.1
26
SC1115
20.0 ± 2.6
27
CQ1108
20.0 ± 2.6
28
CQ1109
19.3 ± 1.5
29
YN1201
19.3 ± 0.6
30
SC1109
19.3 ± 1.5
31
GZ1103
19.0 ± 2.0
32
CQ1107
18.7 ± 1.5
33
CQ1101
17.7 ± 1.5
34
YN1102
17.3 ± 1.5
35
SC1105
16.3 ± 1.5
36
GZ1110
15.0 ± 2.0
37
SC1209
13.7 ± 0.6
38
SC1203
13.3 ± 1.5
Note: data are presented by means ± SE (n
=
3).Symptoms ofC. dactylonaccessions in response to SO2. (a) SO2-sensitive representativeC. dactylonaccession CQ1116, (b) intermediate SO2-tolerant representativeC. dactylonaccession SC1217, (c) high SO2-tolerant representativeC. dactylonaccession SC1203, and (d)C. dactylonaccession CQ1116 without SO2 treatment as a control.
(a)
(b)
(c)
(d)


## 3.2. Changes of Sugar and Proline under SO2 Stress Condition

The soluble sugar and proline contents in leaves from all of the nineC. dactylon accessions increased along with the increase of their SO2 tolerability (Figure 3). Moreover, the soluble sugar and proline contents from all of the high SO2-tolerantC. dactylon accessions and intermediate SO2-tolerantC. dactylon accessions were significantly higher than those from any of the three SO2-sensitiveC. dactylon accessions at both 0-day time-point without SO2 treatment and 7-day time-point after SO2 fumigation treatment. However, the soluble sugar and proline contents from 7-day time-point after SO2 fumigation treatment showed no significant change when they were compared with those from 0-day time-point in anyC. dactylon accession, which indicates that both soluble sugar and proline are not induced or inhibited inC. dactylon under SO2 stress condition (Figure 3).Comparison of soluble sugar (a) and proline (b) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)


## 3.3. Changes of Photosynthetic Pigments under SO2 Stress Condition

Contents of photosynthetic pigments in leaves from all of the nineC. dactylon accessions decreased under SO2 stress condition but showed different patterns with different pigment (Figure 4). Chlorophyll a contents from two intermediate SO2-tolerantC. dactylon accessions (YN1110 and XZ1206) and from all of the three high SO2-tolerantC. dactylon accessions were significantly higher than those from any of SO2-sensitiveC. dactylon accessions in an increasing trend along with the increase of SO2 tolerability at 0-day time-point (Figure 4(a)). Under SO2 stress condition, chlorophyll a contents in leaves from intermediate and high SO2-tolerantC. dactylon accessions reduced significantly less than those from SO2-sensitiveC. dactylon accessions. No significant differences of chlorophyll b contents were observed among the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions at 0-day time-point (Figure 4(b)). After 7-day stress treatment by SO2 fumigation, chlorophyll b contents reduced in leaves from all of the nineC. dactylon accessions. The contents of chlorophyll b showed no significant differences between intermediate SO2-tolerant and SO2-sensitiveC. dactylon accessions, but significantly less reduction of chlorophyll b content was observed in high SO2-tolerantC. dactylon accessions. As for total chlorophyll content, it showed a similar pattern with chlorophyll a in leaves from all of the nineC. dactylon accessions (Figure 4(c)). Carotenoid contents showed no significant differences among the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions at 0-day time-point but significantly less reduced along with the increase of SO2 tolerability ofC. dactylon accessions after 7-day SO2 stress treatment (Figure 4(d)).Comparison of chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), and carotenoid (d) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)
(d)


## 3.4. Changes of ROS Level and Antioxidant Enzyme Activities under SO2 Stress Condition

As two major indicators for reactive oxygen species (ROS) level and oxidative damage, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were tested in this study. As shown in Figure 5, the high SO2-tolerant, intermediate SO2-tolerant, and SO2-sensitiveC. dactylon accessions displayed nearly the same levels of H2O2 and MDA in leaves at 0-day time-point without SO2 treatment (Figures 5(a) and 5(b)). After 7-day SO2 fumigation treatment, levels of both H2O2 and MDA increased in leaves from all of the nineC. dactylon accessions. When compared within all of the nineC. dactylon accessions, levels of both H2O2 and MDA in leaves from high SO2-tolerantC. dactylon accessions and intermediate SO2-tolerantC. dactylon accessions were significantly lower than those from SO2-sensitiveC. dactylon accessions (Figures 5(a) and 5(b)).Comparison of ROS levels reflected by H2O2 (a) and MDA (b) contents in leaves from nine selectedC. dactylonaccessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(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 SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions at 0-day time-point without SO2 treatment (Figure 6(a)). After 7-day SO2 stress treatment, SOD activities increased greatly in leaves from all of the nineC. dactylon accessions. However, the increase degree was in a decreasing trend along with SO2 tolerability of theC. dactylon accessions, displaying highest activities in SO2-sensitiveC. dactylon accessions (more than 120 U/g protein FW) and lowest activities in high SO2-tolerantC. dactylon accessions (more than 60 U/g protein FW) (Figure 6(a)). POD activities increased in leaves from all of the nineC. dactylon accessions after 7-day SO2 stress treatment, but no significant differences were observed within high SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions (Figure 6(b)). However, we found that POD activities in leaves from SO2-tolerantC. dactylon accessions were significantly higher than those from SO2-sensitiveC. dactylon accessions in an increasing trend along with an increase of SO2 tolerability at 0-day time-point without SO2 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 SO2-tolerant and high SO2-tolerantC. dactylon accessions, respectively (Figure 6(b)). CAT activities increased in leaves from all of the nineC. dactylon accessions after 7-day SO2 stress treatment, but no significant differences were observed in leaves from high SO2-tolerantC. dactylon accessions, intermediate SO2-tolerantC. dactylon accessions, and SO2-sensitiveC. dactylon accessions either after 7-day SO2 stress treatment or at 0-day without SO2 treatment (Figure 6(c)).Comparison of antioxidant enzyme activities in leaves fromC. dactylonaccessions in response to SO2 stress. Antioxidant enzyme activities of SOD (a), POD (b), and CAT (c) in leaves from nine selectedC. dactylonaccessions in response to SO2 stress were compared. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)


## 3.5. Changes of Sulfur Content, SiR, and SO Enzyme Activities under SO2 Stress Condition

Sulfur contents in leaves from two intermediate SO2-tolerantC. dactylon accessions (YN1110 and XZ1206) and all of the three high SO2-tolerantC. dactylon accessions were significantly higher than those from any of the SO2-sensitiveC. dactylon accessions at 0-day time-point without SO2 stress treatment (Figure 7(a)). After 7-day SO2 fumigation treatment, sulfur contents increased in leaves from all of the nineC. dactylon accessions in an increasing trend along with increase of SO2 tolerability of theC. dactylon accessions. Moreover, sulfur contents in leaves from all of the high and intermediate SO2-tolerantC. dactylon accessions showed significantly higher levels than those from any of the SO2-sensitiveC. 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 nineC. dactylon accessions at 0-day time-point without SO2 treatment (Figure 7(b)). After 7-day SO2 stress treatment, SiR activities increased about 2-fold (approximate 10 U/mg protein FW) in SO2-sensitiveC. dactylon accessions, 2.4-fold (approximate 12 U/mg protein FW) in intermediate SO2-tolerantC. dactylon accessions, and 3.4-fold (approximate 17 U/mg protein FW) in high SO2-tolerantC. dactylon accessions, respectively. More importantly, SiR activities showed significantly higher levels in leaves from high and intermediate SO2-tolerantC. dactylon accessions than those from SO2-sensitiveC. dactylon accessions, displaying an apparent increasing trend along with SO2 tolerability of theC. dactylon accessions (Figure 7(b)). SO activities in leaves from any ofC. dactylon accessions after 7-day SO2 fumigation treatment showed nearly the same level with those from 0-day time-point without SO2 treatment (Figure 7(c)). However, SO activity levels were significantly higher in leaves from high and intermediate SO2-tolerantC. dactylon accessions than those from SO2-sensitiveC. dactylon accessions, displaying an apparent increasing trend along with SO2 tolerability of theC. dactylon accessions (Figure 7(c)).Comparison of sulfur contents (a), SiR activities (b), and SO activities (c) in leaves from nine selectedC. dactylon accessions in response to SO2 stress. Mean values are presented with vertical error bars representing the standard deviations (n
=
3). The asterisk symbols indicate significant differences between SO2-sensitive accessions and SO2-tolerant accessions.
(a)
(b)
(c)


## 4. Discussion

SO2, a major air pollutant in developing countries, is highly toxic to plants once they are exposed to high doses of SO2 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 ofC. dactylon was affected and visible symptoms appeared on leaves under SO2 stress condition; however this species has much better SO2-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 [30–33]. To achieve better air quality and landscape effect in SO2 polluted areas, selection or development of high SO2-tolerantC. dactylon variations for planting in such regions is desired. In this study, we selected 9 out of 38 wildC. dactylon accessions from Southwest China as representatives of high, intermediate SO2-tolerant, and SO2-sensitive accessions based on the injury degree of SO2 to leaves and the geographic distribution and then comparatively analyzed their physiological differences under SO2 untreated and treated conditions. Our results indicated that SO2 tolerance ofC. 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 inC. dactylon accessions of warm season turfgrasses. This study gained some insights into understanding the genetic and molecular SO2-tolerant mechanisms ofC. dactylon and provided guideline for selection and development ofC. dactylon variations for planting in SO2 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 SO2-tolerantC. dactylon accessions showed significantly higher soluble sugar and proline contents under both SO2 treated and untreated conditions (Figure 3), suggesting that both of them might be related to SO2 tolerance.C. dactylon accessions originated from habitats of hillside and city park have much higher SO2 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 SO2 stress adaptation. However, increased soluble sugar and proline contents in SO2-tolerantC. 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 ofC. dactylon variations with high SO2 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 ofC. dactylon was observed under SO2 stress condition (Table 2). As a consequence, chlorophyll a, chlorophyll b, and carotenoid contents decreased inC. dactylon under SO2 stress condition, consistent with previous reports on grass and tree plants [13, 38, 39]. However, SO2-tolerantC. dactylon accessions showed significantly higher contents of chlorophyll a, chlorophyll b, and carotenoid under SO2 treated condition, consistent with their less leaf injury SO2-tolerantC. dactylon accessions observed in this study. Moreover, SO2-tolerantC. dactylon accessions had significantly higher content of chlorophyll a under SO2 untreated condition. Now that chlorophyll a is the primary pigment for photosynthesis in plants, significantly higher contents of chlorophyll a inC. dactylon accessions under both SO2 treated and untreated conditions indicate that SO2 tolerance ofC. dactylon might be largely related to content of chlorophyll a.Early study showed that SO2 gas after entering leaves of plant is converted into sulfite (SO
3
2
-) and bisulfite (HSO
3
2
-) once it is dissolved in cellular cytoplasm [40]. Furthermore, detoxification reaction of HSO
3
2
- and SO
3
2
- to sulfate (SO
4
2
-) in plants leads to production of many kinds of ROS, such as superoxide radical (O2−∙), hydrogen peroxide (H2O2), and hydroxyl radical (OH∙) [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 SO2 onC. dactylon, we measured the ROS level and antioxidant enzyme activities inC. dactylon accessions. Although both ROS levels (reflected by H2O2 and MDA contents) and antioxidant enzyme activities (reflected by SOD, POD, and CAT) increased in all of the nineC. dactylon accessions under SO2 stress condition, the SO2-tolerantC. dactylon accessions showed significantly lower ROS levels and SOD activities, indicating that the SO2-tolerantC. dactylon accessions have much stronger antioxidant ability and less damage occurs to them by SO2. Moreover, lower SOD activity was theoretically consistent with lower ROS level in the SO2-tolerantC. dactylon accessions under SO2 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 nineC. dactylon accessions after 7-day SO2 stress treatment, activities of this antioxidant enzyme from SO2-tolerantC. dactylon accessions were significantly higher than those from SO2-sensitiveC. dactylon accessions. Taken together, we suggest that significantly higher activity of POD prior to SO2 treatment might be devoted to the increased antioxidant ability in SO2-tolerantC. dactylon accessions.SO2 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 inArabidopsis thaliana and/or tomato plants [44–47]. Transcriptional analyses indicate that SiR is induced by SO2 but SO is constitutively expressed in natural plant [45, 46]. In this study, we found that SiR activity level was significantly increased under SO2 stress condition but SO activity level had almost no change under SO2 treated and untreated conditions in leaves from all of the nineC. dactylon accessions, consistent with previous reports on other plant species [45, 46]. Under SO2 stress condition, the SO2-tolerantC. 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 SO2-tolerantC. dactylon accessions showed significantly higher SO activities prior to SO2 treatment, but no significant differences were observed among the nineC. dactylon accessions. Increased SO activity in SO2-tolerantC. dactylon accession could convert sulfite to nontoxic sulfate more efficiently than SO2-sensitiveC. dactylon accession for storage, once highly toxic SO2 gas enters into theC. dactylon cells, which indicates that SO antioxidant enzyme plays an important role in SO2 tolerance inC. 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 SO2-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 ofC. dactylon in SO2 polluted areas. To achieve better air quality and landscape effect in SO2 polluted areas, selection or development of high SO2-tolerantC. dactylon variations is desired. In this study, we selected 9 out of 38C. dactylon accessions from Southwest China as representatives of high, intermediate SO2-tolerant, and SO2-sensitive accessions and then comparatively analyzed their physiological differences under SO2 untreated and treated conditions. Our results indicated that SO2 tolerance ofC. 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 SO2-tolerant mechanisms ofC. dactylon and provided guideline for selection or development ofC. dactylon variations for planting in SO2 polluted urban or industrial areas.



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*Source: 916595-2014-07-06.xml*