ZHAO Li-juan, XlE Jing-fang ZHANG Hong, WANG Zhen-tao JlANG Hong-jin GAO Shao-long

1 College of Environment and Resource, Shanxi University, Taiyuan 030006, P.R.China

2 Department of Biology, Xinzhou Teachers University, Xinzhou 034000. P.R.China

3 Shanxi Academy of Analytical Science, Taiyuan 030006, P.R.China

1. lntroduction

Sulfonylurea herbicides are used in several countries to control weeds in cereals and other crops and are highly effective against a wide range of broad-leaved weeds.However, low amounts of herbicide residue in the soil are a serious problem that threatens the growth, development, and productivity of aftercrops. Chlorsulfuron is a sulfonylurea herbicide that is applied pre-emergence or early postemergence to kill broadleaf weeds and some annual grass weeds. Chlorsulfuron is applied at rates of 15–35 g active ingredient per hectare (Brown and Kearney 1991).Chlorsulfuron inhibit acetolactate synthase (ALS) which is a important enzyme in the biosynthesis of valine, leucine,and isoleucine. A previous study reported that a high level of resistance in plants is due to substitutions at Pro197 to sulfonylurea herbicides (Rey-Caballeroet al. 2017).

Chlorsulfuron shows high phytotoxicity even at low application doses. Many studies have shown that chlorsulfuron degrades very slowly in neutral to alkaline soils (Stork 1995; Sarmahet al. 1998; Hollaway 2006).A previous study of wheat seedlings under chlorsufuron stress reported that γ-aminobutyric acid (GABA) might be a protective molecule in carbohydrate metabolism (AL-Quraanet al. 2015).

Cadmium (Cd) is one of the most toxic metals in soil. Its sources include industrial pollution, fertilizer contamination, and wastewater irrigation. It is difficult to mitigate these sources of contamination, particularly wastewater irrigation. Wastewater irrigation is a main irrigating method to alleviate water shortages in agriculture in the northwest, northeast, and central parts of China.These areas are also the main grain-producing areas in China, and the combined areas for crop production and mineral coal production cover approximately 40% of the total cultivable land (Ma 2015). Because of the overlap of land used in the grain and mineral industries, it is inevitable that low levels of contamination with herbicide residue and metals co-occur in agricultural fields. However, the effects of these compounds on growth, development, and production of aftercrops are not clear.

Photosynthesis, one of the main metabolic processes that influence crop production (Li 2015), may be affected by some pollutants. Approximately 40% of all light energy absorption of a healthy plant is released as fluorescence or heat (Martinez-Pe?alveret al. 2011). Light can induce several forms of non-photochemical quenching (NPQ)(Martinez-Pe?alveret al. 2011). NPQ is an indicator that reflects the change in excess heat emissions in plants. It is related to light intensity and the leaf physiological state(Schreiber and Klughammer 2008). Chlorophyll (Chl)fluorescence emission is a common phenomenon in healthly plants, but it only accounts for 2–5% of the energy that is not used in photosynthesis. Changes in Chl fluorescence indicate a decline in the ability of plants to cope with excess light intensity. These changes also indicate declines in protective regulatory mechanisms and in the regulation of photosystem II (Maxwell and Johnson 2000; Papageorgiou and Govindjee 2004). Therefore, any slight change of Chl fluorescence in plants can be used to identify the response of plants to an environmental stress (Demmig-Adamset al.1995).

Previous studies have reported that Chl fluorescence techniques are critical for detecting and evaluating stress in plants (Lichtenthaler and Mieh? 1997; Rohá?eket al. 2008;Boreket al. 2013). Chl fluorescence techniques permit nondestructive sampling on leaves, while retaining accuracy.Chl fluorescence techniques are especially useful because they generate large amounts of data. Some of these data include information about the state of photosynthesis in the leaf, and evidence of unevenness or gradients in Chl fluorescence across the entire leaf (Lichtenthaleret al. 2000;Boreket al. 2013).

Previous studies also have reported that Chl fluorescence can be used to detect changes in photosynthesis in many plant species under herbicide stress (Frankartet al. 2003;Juneauet al. 2007; Ralphet al. 2007; Nestleret al. 2012;Basiet al. 2013; Moustaka and Moustakas 2014). Frankartet al. (2003) proposed that NPQ can be used as a biomarker for herbicide bioassays in the laboratory and in the field.Wilsonet al. (2000) showed that simazine induced the decrease ofΦm(the maximal PSII quantum yield) andΦ′m(the operational PSII quantum yield) inPontederia cordata,andΦ′mwas more sensitive thanΦmfor reflecting the stress of simazine (Barbagalloet al. 2003).

Chl fluorescence analysis has also been used to assess the effects of heavy metals on photosynthesis. Ralph and Burchett (1998) reported that lead (Pb) was only mildly toxic toHalophila ovalis. Dezhbanet al. (2015) reported that a certain concentration (1 000 or 2 000 mg L–1) of Cd increased Chlaanda/b, but had no significant effect on Chl fluorescence (Fv/Fm(the maximal photochemical efficiency),Fo(the initial fluorescence), andFm(the maximum fluorescence)) in one-year-old seedlings ofRobinia pseudoacacia.

Moreover, with the increase of the Cd concentration, the changes of Chl fluorescence inAgeratum conyzoideswere different (Sunet al. 2015). For example,Fv/Fmand potential photochemical efficiency of PSII (Fv/Fo) increased, whileFoandFmdecreased. In addition, the quantum yield (ФPSII),electron transfer reaction (ETR), photochemical quenching coefficient (qP), and nonphotochemical quenching coefficient(qN) first increased and then decreased.

The influence of chlorsulfuron herbicide and Cd on Chl fluorescence in maize seedlings has not previously studied, especially the effect of mixed pollutants on Chl fluorescence in maize seedlings. The goal of this study was to investigate changes in enzymatic activity and Chl fluorescence in maize seedlings after exposure to low levels of chlorsulfuron and cadmium residues. Our results elucidate the relationship between changes in enzymatic activity, Chl fluorescence, and stress. The results also demonstrate that Chl fluorescence images can be used as a predictor of stresses.

2. Materials and methods

2.1. Materials

Maize seeds were obtained from Shanxi Qiangsheng Seed Co., Ltd., China. All experiments were performed in the laboratory under controlled conditions: constant temperature of (25±2)°C, relative humidity of 40–60%, and a 12-h:12-h(day:night) cycle with 10.25 W m–2at the soil surface.

The herbicide chlorsulfuron was in the form of 25%Wettable Powder, a commercial formulation (Jiangsu Institute of Ecomones Co., Ltd., Jiangsu, China: Pesticide Registration Certificate no. PD20081325). The concentration of chlorsulfuron chosen for the present study was based on previous reports (Chenet al. 1994; Shan and Cai 1998), as was that of Cd (Antonkiewiczet al. 2006; Baczek-Kwintaet al. 2011a). In the chlorsulfuron treatments, 0.001 mg chlorsulfuron kg–1DM (dry matter) of soil was applied to the soil before the seedlings were planted. In the Cd treatments,5 mg Cd kg–1DM of soil (in the form of CdCl2) was applied to the soil before the seedlings were planted. The control pots contained the same type of soil but without added chlorsulfuron or Cd.

Seeds were planted after the herbicide and Cd (as CdCl2)were added to the soil. Single-exposure experiments were performed using 5 mg kg–1Cd or 0.001 mg kg–1chlorsulfuron.The combined experiments with Cd and chlorsulfuron used 5 mg kg–1Cd and 0.001 mg kg–1chlorsulfuron. Each experiment was carried out three times. Each treatment included 60 seedlings. No nutritious solution was applied.

2.2. Germination and growth

Seed germination and seedling growth were conducted according to the ISO11269-2 (ISO 2013) guidelines.Detailed guidelines for the specific steps can be found in Zhaoet al. (2016). After 3 weeks, half of samples were harvested, the content of chlorophyll and malondialdehyde,and enzyme activities were analyzed; additional samples were used for the analyses of the major fluorescence parameters of Chl.

2.3. Germination and growth analysis

The length of roots and shoots were measured with a ruler, and the wet weight of shoots was measured with an electronic balance.

2.4. Chlorophyll and lipid peroxide analysis

Chlorophyll analysisChlorophyll analysis was conducted to determine the amount of chlorophyll in leaves. Fresh plant samples (0.1 g) were homogenized in 8 mL 80% acetone(pH 7.8), then centrifuged at 7 000 r min–1for 10 min. The supernatant was measured for absorbance at 663 and 645 nm and total chlorophyll was calculated (Porraet al.1989).

The content of malondialdehyde (MDA)MDA was determined, following Yan and Lu (2013). A 0.5-g sample of fresh tissue was ground in 5 mL 0.1% trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 7 000 r min–1for 30 min, and 2 mL of the supernatant was mixed with 2 mL of 0.5% thiobarbituric acid in 20% TCA.The mixture was heated at 95°C for 30 min, chilled on ice,and centrifuged at 7 000 r min–1for 10 min. The absorbance of the supernatant was measured at 532 nm. The values were corrected for nonspecific absorption by subtracting the absorbance read at 600 nm.

2.5. Assay of enzyme activities

Fresh tissues (0.1 g) were ground in ice-cooled 50 mmol L–1phosphate buffered saline (pH 7.8) containing 1% (w/v)polyvinylpyrrolidone. The homogenate was centrifuged at 4 000 r min–1at 4°C for 20 min. The supernatant was immediately used for assaying enzyme activities.

Superoxide dismutase (SOD) activity was evaluated by measuring the inhibition of the photochemical reduction of nitro-blue tetrazolium (NBT) (Jianget al. 2010). The reactant mixture contained 50 mmol L–1phosphate buffered saline(pH 7.8), 130 mmol L–1L-methionine, 0.075 mmol L–1NBT,0.1 mmol L–1ethylenediaminetetraacetic acid (EDTA)-Na2,and 400 μL enzyme extract. The absorbance was measured at 560 nm.

Guaiacol peroxidase (POD) activity was assayed on the basis of guaiacol oxidation using H2O2(Zhouet al. 2008).The reactant mixture contained 100 mmol L–1phosphate buffered saline (pH 6.0), 28 μL guaiacol, 19 μL 30% H2O2,and 100 μL enzyme extract. The activity was assayed following the change of absorbance at 470 nm because of guaiacol oxidation.

Catalase (CAT) activity was assayed by the consumption of H2O2(Li 2001). The reaction mixture contained 0.15 mol L–1phosphate buffered saline (pH 7.0), 30% H2O2, and enzyme extract. Activity was assayed following the change of absorbance at 240 nm.

Glutathione-S-transferase (GST) activity measurement was measured based on the method of Yan and Lu (2013).The reaction mixture consisted of 50 mmol L–1potassium phosphate (pH 7.4), 1 mmol L–1reduced glutathione (GSH),1 mmol L–11-chloro-2,4-dinitro-benzene (CDNB) (10 mmol L–1CDNB dissolved in ethanol), and 50 μL enzyme extract.The reaction was initiated by adding CDNB. The change of absorbance at 340 nm was recorded for 1 min.

2.6. Analyses of major fluorescence parameters of Chl

According to the ISO11269-2 guidelines, all parameters were determined after 21 days under chlorsulfuron and/or Cd stress (Zhaoet al. 2016). The level of chlorsulfuronand/or Cd-induced stress to plants was estimated by analyzing major fluorescence parameters of Chl (Handy FluorCam FC 1000-H, PSI, Brno, Czech Republic), which revealed PSII activity. We left maize seedlings in darkness for 5 min (Krauseet al. 1982) to minimize this fluorescence during measurements and to open all reaction centers.Continuous irradiation of 0.5 μmol m?2s?1light intensity was used to measureFoof maize seedlings’ leaves. A saturating pulse of intensity (1 200 μmol m?2s?1) was used to measureFm(Hussain and Reigosa 2015; Guisande-Collazoet al. 2016). These values were used to calculateFv=Fm?FoandFv/Fm. The other parametersFv′/Fm′ (PSII quantum yield of light adapted sample at steady-state),NPQ,qP, and Rfd (chlorophyll fluorescence decline ratio in steady-state) were calculated under a saturating pulse of intensity at 1 200 μmol m?2s?1and an actinic light of 205 μmol m?2s?1.

2.7. Statistical analysis

Values in the current study were the means of four replicates.Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (ver. 18).Significant differences between germination and growth,enzymatic activity, the amounts of chlorophyll and lipid peroxide, and the change in fluorescence parameters of Chl were estimated using two-way ANOVA.

3. Results

3.1. Germination of maize

Chlorsulfuron and Cd had no significant effect (P=0.124)on the germination of maize. The emergence rates, which were at least 90% in each treatment group, were not affected by chlorsulfuron, Cd, or the combination of both pollutants.

3.2. Analysis of length of shoot and root

The results of two-way ANOVA (Table 1) indicated that chlorsulfuron had a significant influence on the shoot and root growth of maize seedlings (P＜0.001). Cadmium had no significant effect (P=0.601) on shoot growth, but promoted root growth (P=0.013). The results also indicated that there was an interaction between chlorsulfuron and Cd on shoot and root growth (P=0.003, 0.016).

Twenty-one days after exposure to 0.001 mg kg–1chlorsulfuron, the inhibition ratios on shoot and root length were 28 and 50%, respectively (Fig. 1). However, Cd promoted shoot and root growth compared with the control.The inhibition ratios were –14% for shoot and –31% for root (Fig. 1).

After exposure to the chlorsulfuron and cadmium mixture,the inhibition ratios on shoot and root length were 47 and 51%, respectively (Fig. 1). This result indicated that the addition of Cd aggravated the toxicity of chlorsulfuron.

3.3. Wet weight of maize seedlings

The results of two-way ANOVA (Table 1) indicated that:chlorsulfuron had a significant effect on the wet weight of maize seedlings (P＜0.01); Cd did not have a significant effect on the wet weight (P=0.377); and there was no interaction between chlorsulfuron and Cd (P=0.456).

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Table 1 (Continued from preceding page)

Compared with control, chlorsulfuron reduced wet weight by 55.8% (Fig. 1); the combined pollutants led to a significant decrease in wet weight (63%, Fig. 1). This result indicated that chlorsulfuron was the main factor causing the decrease of wet weight.

3.4. Chlorophyll analysis

The results of two-way ANOVA (Table 1) indicated that chlorsulfuron and/or Cd had significant effects on chlorophyll in maize seedlings (P＜0.01). There was also an interaction between chlorsulfuron and Cd (P＜0.01) in terms of chlorophyll content.

Both chlorsulfuron and Cd caused significant decreases in the amount of chlorophyll (49 and 31%, respectively Fig. 2). Compared with chlorsulfuron, the addition of Cd alleviated the downward trend in chlorophyll content.

3.5. Malondialdehyde (MDA) analysis

The results of two-way ANOVA (Table 1) indicated that chlorsulfuron and Cd did not significantly affect chlorophyll of maize seedlings (P=0.41, 0.201). There was no interaction between chlorsulfuron and Cd (P=0.522). The results also indicated that there was no significant accumulation of MDA in all treatments (Fig. 2).

3.6. Activity levels of antioxidant enzymes under stress

The results of two-way ANOVA (Table 1) indicated that chlorsulfuron had a significant effect on SOD, POD, and GST of maize seedlings, and no significant effect on CAT activity.Cd had a significant effect on CAT and GST activities of maize seedlings, but no significant effect on SOD and POD activities. This result also indicated that there was an interaction between chlorsulfuron and Cd on CAT and GST,and no interaction on SOD and POD.

Chlorsulfuron caused a significant increase in SOD(14%, Fig. 3), while POD and GST were significantly decreased (13 and 43%, Fig. 3). Cd caused CAT and GST to decrease significantly (65 and 6.3%, respectively, Fig. 3).Compared with chlorsulfuron, the addition of Cd alleviated the downward trend of GST (17%, Fig. 3). Compared with Cd, the addition of chlorsulfuron alleviated the downward trend of CAT (35%, Fig. 3). This result also indicated an interaction between chlorsulfuron and Cd on CAT and GST.

Fig. 2 Effects of CK (control check), chlorsulfuron, cadmium(Cd), and the combination of chlorsulfuron and Cd on chlorophyll and malondialdehyde (MDA) of 21-day-old maize seedlings in soil (mean net response with standard error bars). All units(nominal values) are in mg AI per kg dry soil. ＊＊, P＜0.01, twotailed t-test. FW, fresh weight.

Fig. 3 Effects of CK (control check), chlorsulfuron, cadmium(Cd), and both chlorsulfuron and Cd on glutathione-S-transferase (GST), superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) of 21-day-old maize seedlings in soil (mean net response with standard error bars).All units (nominal values) are in mg AI per kg dry soil. ＊, P＜0.05;＊＊, P＜0.01, two-tailed t-test. FW, fresh weight.

3.7. Effects on PSll photochemistry

The images ofFv′/Fm′, NPQ,qP, and Rfd are shown in Fig. 4.This figure illustrates the differences in photosynthetic characteristics between chlorsulfuron- and/or Cd-treated maize seedlings and control seedlings. The results of two-way ANOVA (Table 1) indicated that chlorsulfuron had a significant effect onFv/Fm,Fv′/Fm′, NPQ,qP, and Rfd(P＜0.05). Cd had a significant effect onFv/Fm, NPQ,qP, and Rfd (P＜0.05), but the effect onFv′/Fm′ was not significant(P=0.943). This result also indicated that there was an interaction between chlorsulfuron and Cd on NPQ,qP, and Rfd (P＜0.05), however, there was no interaction onFv/FmandFv′/Fm′ (P=0.824, 0.943).

Chlorsulfuron affected Chl fluorescence parametersCompared with control, the leaf of maize seedlings in chlorsulfuron-treatment had lower Chl activity (Fig. 4). The average values ofFv/Fm,Fv′/Fm′,qP, and Rfd decreased in the treatment group (Table 2). NPQ activity was higher in the treatment group than in the control.

Cd affected Chl fluorescence parametersThe images of Chl fluorescence did not show larger discrepancies in Cd-treated plants than in the control (Fig. 4). No changes in the values ofFv/FmandFv′/Fm′ were apparent, but NPQ activity was higher in the Cd-treatment group than in the control group (Table 2).

The combination of chlorsulfuron and CdThe combination of chlorsulfuron and Cd showed more areas with lowered activity levels in Chl fluorescence imaging(Fig. 4). The average values ofFv/Fm,Fv′/Fm′,qP, and Rfd decreased, but NPQ activity increased (Table 2).

Compared with the chlorsulfuron-treated group, the addition of Cd did not significantly affectFv/FmandFv′/Fm′,but significantly increased the upward trend of NPQ, and significant alleviated the downward trend ofqPand Rfd.

Fig. 4 Chlorophyll fluorescence images of Fv′/Fm′, nonphotochemical fluorescence quenching (NPQ), photochemical quenching (qP), and chlorophyll fluorescence decrease ratio(Rfd) in maize seedling leaves. Images are representative of control, chlorsulfuron treatment, cadmium (Cd) treatment, and combination treatment groups, respectively. CK, control check.

4. Discussion

A previous study, reported that chlorsulfuron caused reductiong of root growth ofPhaseolus vulgaris,Pisum sativum, andVicia faba(Fayeaz and Kristen 1996). In the present study, we investigated shoot length, root length, and wet weight of maize seedlings after exposed to chlorsulfuron and Cd. Chlorsulfuron was the main factor inhibiting the growth of maize seedlings, especially the growth of their roots. Moreover, there was an interaction between chlorsulfuron and Cd. This finding suggested that small amounts of chlorsulfuron residue in the soil likely affect maize seedlings by inhibiting root growth, leading to growth retardation at later developmental stages.

Some previous studies have reported that Cd can reduce the numbers of grana and thylakoids, destroying the chloroplast structure (Penget al. 1991; Su and Wang 2004; Paglianoet al. 2006). Other studies still reported that Cd reduced the content of chlorophylla, chlorophyllb, and total chlorophyll in pak choi, mustard, tomato, and gram leaves (Shafiand Agnihotri 2010; Chenet al. 2011; Boreket al. 2013; Haouariet al. 2012). Chl is a pigment that plays an important role in light energy absorption, transmission,and conversion to chemical energy during photosynthesis.It dynamically adjusts the ratio among these processes,regulates the reasonable distribution of energy, and ensures the normal operation of the photosynthetic system. In the present study, chlorsulfuron and Cd caused chlorophyll to decrease in maize seedlings. This may be due to more ROS being produced, which damaged the photosynthetic organs, eventually degrading the photosynthetic pigment(Dayet al. 1996).

MDA is the final decomposition product of lipid peroxidation, and the amount of MDA in plants can reflect the degree to which they are suffering from stress. A previous study reported that 5.0 mg kg–1Cd did not cause the accumulation of MDA (Yan and Lu 2013). In the present study, low-level chlorsulfuron and Cd did not increase MDA.Moreover, there was no interaction between chlorsulfuron and Cd on MDA content.

Table 2 Summary of chlorophyll fluorescence parameters1)

GST has a very important effect on detoxification of exogenous compounds. It catalyzes the binding reaction between nucleophilic glutathione and various electrophilic exogenous chemicals (Pascalet al. 1998; Yinet al. 2008;Cuiet al. 2010). The activity level of GST is an important index for the resistance of maize seedlings to herbicides(Edwardset al. 2000; Robertet al. 2000). Cd caused GST to decrease, indicating that GST can play a role in detoxification of exogenous compounds (Yan and Lu 2013).

Chl fluorescence imaging was a useful approach to evaluate the photosynthetic performance of plants under the toxic stresses of chlorsulfuron and/or Cd. Exposure to chlorsulfuron generated fluorescence responses in maize leaves, including obvious declines in the values ofFv/Fm,Fv′/Fm′,qP, and Rfd and a significant increase in NPQ.The decrease inFv/Fmsuggested that the photochemical capacity of PSII diminished, whileFv′/Fm′ provided important information about the maximum efficiency of PSII (B?czek-Kwintaet al. 2011b). Changes in the values of these parameters may be associated with alterations in the amount of chlorophyll, described earlier (Fig. 2), and suggest that chlorsulfuron inhibited PSII activity. NPQ alterations implied that the nonphotochemical quenching of maximal Chl fluorescence was increased by chlorsulfuron treatment,which allowed excessive energy within the photosystems to dissipate (Maxwell and Johnson 2000; Sofoet al. 2009;Munizet al. 2014).

The changes in NPQ andqPin the Cd treatment group might be associated with the amount of Chl and the decreased activity of CAT. The decrease of CAT activity might lead to excessive O2-·, which could destroy the structure and function of the cell membrane (Luoet al. 2011),ultimately affecting photosynthesis. The increase of Rfd in the combination chlorsulfuron and Cd treatment indicated that Cd enhanced the resistance of maize seedlings to chlorsulfuron.

The results of Chl fluorescence imaging showed that, compared with chlorsulfuron treatment alone, the combination treatment caused the dissipation of excess energy, as shown by the significantly increased value of NPQ. NPQ is one of the most important photoprotective mechanisms (Horton and Hague 1984; Mülleret al.2001; Moustakaet al. 2015), and it seems to respond to combination treatment. The increased NPQ appeared to serve as a protective mechanism for maize seedlings under combination treatment. Meanwhile, the value of Rfd significant increased in maize seedlings after exposed to combination treatment.

5. Conclusion

This study revealed that chlorsulfuron is a main limiting factor in growth of maize seedlings, and the addition of Cd aggravated the inhibitory effect of chlorsulfuron.Moreover, we found that chlorsulfuron inhibited PSII activity,accompanying the decreases in Chl and Rfd, and increase of NPQ. The addition of Cd alleviated the downward trend of Chl and Rfd in chlorsulfuron treatment, but aggravated the upward trend of NPQ in chlorsulfuron treatment. This result indicated an interaction of chlorsulfuron and Cd, but this interaction could be positive or negative depending on different indexes.

Our results also indicated that Chl fluorescence imaging can identify crops with abnormal metabolic activity in early stages without causing stress or damage to the plants.Furthermore, the results also suggest that the parameters of Chl fluorescence in PSII in maize seedlings could be a good indicator for the early, rapid detection of toxic effects from environmental pollutants.

Acknowledgements

This research was supported by grants from the National Natural Science Foundation of China (30740037), the Special Fund for Agro-scientific Research in the Public Interest,China (201103024), and the Foundation for Graduate Innovation, Shanxi University, China (011452901009).

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