Comparative Analyses of Physiological Responses of <i>Cynodon dactylon</i> Accessions from Southwest China to Sulfur Dioxide Toxicity

Li, Xi; Wang, Ling; Li, Yiqiao; Sun, Lingxia; Cai, Shizhen; Huang, Zhuo

doi:https://doi.org/10.1155/2014/916595

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Research Article | Open Access

Volume 2014 | Article ID 916595 | https://doi.org/10.1155/2014/916595

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

Xi Li,¹Ling Wang,¹Yiqiao Li,²Lingxia Sun,¹Shizhen Cai,¹and Zhuo Huang¹

Academic Editor: Mehmet Yakup Arica

Received24 Feb 2014

Accepted26 May 2014

Published06 Jul 2014

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.

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 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 ³⁵SO₃ and ³⁵SO₂ 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 SO₂ 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 SO₂ on leaves of 38 wild C. dactylon accessions from Southwest China. Based on injury rate of SO₂ to leaves, nine C. 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 of C. dactylon to SO₂ and provided guideline for selection and development of C. dactylon variations for planting in SO₂ 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°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 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-sensitive C. dactylon accessions were selected out for physiological studies. Leaves 2 cm above the soil from C. dactylon plants treated by SO₂ after 7 days were collected and brought back to laboratory for analysis. Leaves from C. 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 at (Student’s -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 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 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 of C. 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).

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3.2. Changes of Sugar and Proline under SO₂ 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 SO₂ tolerability (Figure 3). Moreover, the soluble sugar and proline contents from all of the high SO₂-tolerant C. dactylon accessions and intermediate SO₂-tolerant C. dactylon accessions were significantly higher than those from any of the three SO₂-sensitive C. 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 any C. dactylon accession, which indicates that both soluble sugar and proline are not induced or inhibited in C. dactylon under SO₂ stress condition (Figure 3).

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3.3. Changes of Photosynthetic Pigments under SO₂ Stress Condition

Contents of photosynthetic pigments in leaves from all of the nine C. dactylon accessions decreased under SO₂ stress condition but showed different patterns with different pigment (Figure 4). Chlorophyll a contents from two intermediate SO₂-tolerant C. dactylon accessions (YN1110 and XZ1206) and from all of the three high SO₂-tolerant C. dactylon accessions were significantly higher than those from any of SO₂-sensitive C. 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-tolerant C. dactylon accessions reduced significantly less than those from SO₂-sensitive C. dactylon accessions. No significant differences of chlorophyll b contents were observed among the high SO₂-tolerant, intermediate SO₂-tolerant, and SO₂-sensitive C. 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 nine C. dactylon accessions. The contents of chlorophyll b showed no significant differences between intermediate SO₂-tolerant and SO₂-sensitive C. dactylon accessions, but significantly less reduction of chlorophyll b content was observed in high SO₂-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 SO₂-tolerant, intermediate SO₂-tolerant, and SO₂-sensitive C. dactylon accessions at 0-day time-point but significantly less reduced along with the increase of SO₂ tolerability of C. dactylon accessions after 7-day SO₂ stress treatment (Figure 4(d)).

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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-sensitive C. 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 nine C. dactylon accessions. When compared within all of the nine C. dactylon accessions, levels of both H₂O₂ and MDA in leaves from high SO₂-tolerant C. dactylon accessions and intermediate SO₂-tolerant C. dactylon accessions were significantly lower than those from SO₂-sensitive C. dactylon accessions (Figures 5(a) and 5(b)).

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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-tolerant C. dactylon accessions, intermediate SO₂-tolerant C. dactylon accessions, and SO₂-sensitive C. 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 nine C. dactylon accessions. However, the increase degree was in a decreasing trend along with SO₂ tolerability of the C. dactylon accessions, displaying highest activities in SO₂-sensitive C. dactylon accessions (more than 120 U/g protein FW) and lowest activities in high SO₂-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 SO₂ stress treatment, but no significant differences were observed within high SO₂-tolerant C. dactylon accessions, intermediate SO₂-tolerant C. dactylon accessions, and SO₂-sensitive C. dactylon accessions (Figure 6(b)). However, we found that POD activities in leaves from SO₂-tolerant C. dactylon accessions were significantly higher than those from SO₂-sensitive C. 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-tolerant C. dactylon accessions, respectively (Figure 6(b)). CAT activities increased in leaves from all of the nine C. dactylon accessions after 7-day SO₂ stress treatment, but no significant differences were observed in leaves from high SO₂-tolerant C. dactylon accessions, intermediate SO₂-tolerant C. dactylon accessions, and SO₂-sensitive C. dactylon accessions either after 7-day SO₂ stress treatment or at 0-day without SO₂ treatment (Figure 6(c)).

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

Sulfur contents in leaves from two intermediate SO₂-tolerant C. dactylon accessions (YN1110 and XZ1206) and all of the three high SO₂-tolerant C. dactylon accessions were significantly higher than those from any of the SO₂-sensitive C. 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 nine C. dactylon accessions in an increasing trend along with increase of SO₂ tolerability of the C. dactylon accessions. Moreover, sulfur contents in leaves from all of the high and intermediate SO₂-tolerant C. dactylon accessions showed significantly higher levels than those from any of the SO₂-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 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-sensitive C. dactylon accessions, 2.4-fold (approximate 12 U/mg protein FW) in intermediate SO₂-tolerant C. dactylon accessions, and 3.4-fold (approximate 17 U/mg protein FW) in high SO₂-tolerant C. dactylon accessions, respectively. More importantly, SiR activities showed significantly higher levels in leaves from high and intermediate SO₂-tolerant C. dactylon accessions than those from SO₂-sensitive C. dactylon accessions, displaying an apparent increasing trend along with SO₂ tolerability of the C. dactylon accessions (Figure 7(b)). SO activities in leaves from any of C. 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-tolerant C. dactylon accessions than those from SO₂-sensitive C. dactylon accessions, displaying an apparent increasing trend along with SO₂ tolerability of the C. dactylon accessions (Figure 7(c)).

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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 of C. 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-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 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 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 SO₂-tolerant mechanisms of C. dactylon and provided guideline for selection and development of C. 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-tolerant C. 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-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 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 of C. dactylon was observed under SO₂ stress condition (Table 2). As a consequence, chlorophyll a, chlorophyll b, and carotenoid contents decreased in C. dactylon under SO₂ stress condition, consistent with previous reports on grass and tree plants [13, 38, 39]. However, SO₂-tolerant C. dactylon accessions showed significantly higher contents of chlorophyll a, chlorophyll b, and carotenoid under SO₂ treated condition, consistent with their less leaf injury SO₂-tolerant C. dactylon accessions observed in this study. Moreover, SO₂-tolerant C. 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 in C. dactylon accessions under both SO₂ treated and untreated conditions indicate that SO₂ tolerance of C. 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 () and bisulfite (H) once it is dissolved in cellular cytoplasm [40]. Furthermore, detoxification reaction of H and to sulfate () 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 on C. dactylon, we measured the ROS level and antioxidant enzyme activities in C. 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 nine C. dactylon accessions under SO₂ stress condition, the SO₂-tolerant C. dactylon accessions showed significantly lower ROS levels and SOD activities, indicating that the SO₂-tolerant C. 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-tolerant C. 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 nine C. dactylon accessions after 7-day SO₂ stress treatment, activities of this antioxidant enzyme from SO₂-tolerant C. dactylon accessions were significantly higher than those from SO₂-sensitive C. 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-tolerant C. 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 in Arabidopsis 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 nine C. dactylon accessions, consistent with previous reports on other plant species [45, 46]. Under SO₂ stress condition, the SO₂-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 SO₂-tolerant C. dactylon accessions showed significantly higher SO activities prior to SO₂ treatment, but no significant differences were observed among the nine C. dactylon accessions. Increased SO activity in SO₂-tolerant C. dactylon accession could convert sulfite to nontoxic sulfate more efficiently than SO₂-sensitive C. dactylon accession for storage, once highly toxic SO₂ gas enters into the C. dactylon cells, which indicates that SO antioxidant enzyme plays an important role in SO₂ 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 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 of C. dactylon in SO₂ polluted areas. To achieve better air quality and landscape effect in SO₂ polluted areas, selection or development of high SO₂-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 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 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 SO₂-tolerant mechanisms of C. dactylon and provided guideline for selection or development of C. dactylon variations for planting in SO₂ 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.

Acknowledgments

This work was supported in part by the Science and Technology Department of Sichuan Province (Grant no. 05JY009-007-4) and the Scientific Research Fund of Sichuan Provincial Education Department (Grant no. 12ZA116).

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Copyright © 2014 Xi Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

Abstract

1. Introduction

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

2.2. Stress Treatment and Experimental Design

2.3. Measurement of Total Soluble Sugars

2.4. Measurement of Proline Content

2.5. Estimation of Chlorophyll and Carotenoid

2.6. Determination of H2O2 Level

2.7. SOD, POD, CAT, SiR, and SO Enzyme Assays

2.8. Estimation of MDA Content

2.9. Estimation of Sulfur Content

2.10. Statistical Analysis

3. Results

3.1. Leaf Injury under SO2 Stress Condition

3.2. Changes of Sugar and Proline under SO2 Stress Condition

3.3. Changes of Photosynthetic Pigments under SO2 Stress Condition

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

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

4. Discussion

5. Conclusion

Conflict of Interests

Authors’ Contribution

Acknowledgments

References

Copyright

2.6. Determination of H₂O₂ Level

3.1. Leaf Injury under SO₂ Stress Condition

3.2. Changes of Sugar and Proline under SO₂ Stress Condition

3.3. Changes of Photosynthetic Pigments under SO₂ Stress Condition

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

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