Evaluation of six cold-season turfgrasses responses to lead phytotoxicity for screening tolerant species

[Research]

Evaluation of six cold-season turfgrasses responses to lead phytotoxicity for screening tolerant species

K. Gholamipour Fard¹, M. Ghasemnezhad¹*, H. Zakizadeh¹, M. Kafi², F. Rejali³

1- Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

2- Department of Horticultural Sciences, Faculty ofAgriculture and Natural Resources, University of Tehran, Karaj, Iran

3- Department of Soil Science, Soil and Water Research Institute, Karaj, Iran

* Corresponding author’s E-mail: Ghasemnezhad@guilan.ac.ir

ABSTRACT

Lead (Pb) is one of the most hazardous heavy metal that caused serious problems for ecosystem at recent years. In this study, the effect of Pb on seed germination, incipient growth and antioxidant enzymes of six common used turfgrasses was investigated. The results showed that after implementation of different concentrations (0, 1000, 2000 and 3000 µM) of Pb, Agropyron elongatum exhibited the highest germination percentage (90%) under 3000 µM Pb, followed by Lollium perenne L. (85.56%). The lowest germination parameters including germination vigor, rate, index and germination percentage was found in Festuca ovina, which means it is the most sensitive species to Pb than the other studied turfgrasses. In contrast, F. rubra, F. ovina, A. elongatum and L. perenne had the most significant aerial growth and the lowest chlorophyll degradation even at high lead concentration. An increase in L. perenne and A. elongatum antioxidant enzymes activities as well as a decrease in F. ovina observed after exposure to 3000 µM Pb which correlated to their tolerance and sensitivity to Pb respectively. However, such increase in antioxidant activity was also found in F. Rubra and Poa pratensis but could not inhibit chlorophyll degradation and seedling growth retardation. It might support that there is suggesting the probable existence of a lead-injury mechanism rather than the oxidative stress. Overall, A. elongatum is introduced as the most tolerant turfgrass among studied ones, which could be cultivated in Pb-polluted sites.

INTRODUCTION

Heavy metal-contaminated soil and water are serious worldwide problems at recent years that progressively increase. Lead (Pb) is the most common heavy metal contaminant which has been caused a great concern because of its wide spread occurrence, toxic nature, and long life in biological system. It originates from both natural sources and anthropogenic activities and causes noticeably annual reduction of crops productivity (Duruibe et al. 2007). Lead toxicity results in several damages from interception of germination to retardation of plant growth in herbages (Lamhamdi et al. 2011) and tree species  (Lin et al. 2005). Perturbations in cellular metabolism (Pandey & Sharma 2002), alteration in membrane permeability (Lukatkin et al. 2013), decrease in electron transport activity (Heyno et al. 2008), enzymes activities enhancement or inhibition(Shi & Zhu 2008), and particularly the elevated production of reactive oxygen species (ROS) (Steffens 2014) have been reported as consequences of plant exposure to excessive heavy metals. Moreover, seed germination, the initial event in the plant life, has been considerably influenced by the heavy metal stresses (Moosavi et al. 2012). Turf, a plant with high irrigation requirement, is commonly used in landscapes at widespread scales throughout the world (Romero & Dukes 2014). Several factors such as water deficiency, water pollution, and heavy metal-contaminated soils have strongly restricted the plant germination and even subsequent growth and development.  Bidar et al. (2007) Reported that L. perenne was sensitive to heavy metal toxicity than Trifolium repens. The tolerance of Poa annua to high concentrations of Cu in soil was shown by Brun et al. (2003). Vamerali et al. (2010) Considered Festuca spp. and Lollium spp. for phytoremediation of heavy metals-polluted soils. Moreover, Gąbka & Wolski (2011) showed that F. arundinacea Schreb. cv. Spirit increased its biomass yield under elevated Pb levels. However, they did not biochemically examine on them, nor introduced the most tolerant cultivar to the heavy metals.

The cessation of seed germination and the growth retardation have been illustrated as a response to elevated concentrations of  lead in L. perenne (Bidar et al. 2007), Elsholtzia argyi (Islam et al. 2007), Leucaena leucocephala (Shafiq et al. 2008), Triticum aestivum L. (Lamhamdi et al. 2013), Cicer arietenum (Goswami et al. 2013) and Sesamum indicum (Rao & Lakshmi 2014). However, the studies about the effects of lead on the seed germination of turfgrasses species are little.

Moreover, it has not been well understood to reveal the antioxidant enzymes profiles as determining factors in response of cold-season turfgrasses to various concentrations of lead. Bidar et al. (2007) evaluated only the superoxide dismutase (SOD) activity in L. perenne as affected by Cd, Zn, and Pb. Unfortunately, it has not been reported, to date, the analogy of tolerance/sensitivity of six commonly used cold-season turfgrasses, perennial ryegrass, tall fescue, blue sheep fescue, red fescue, kentucky bluegrass and agropyron, to lead-induced stress. Therefore, the present study was accomplished to identify the most sustainable vegetation cover under toxic levels of lead for the employing of not only heavy metal-contaminated soils, but also wastewater in cultivation and irrigation purposes of the grasses. So, the germination and incipient growth parameters, as well as the biochemical analyses of the six types of turfgrass were evaluated and monitored under lead-induced stress.

The results of this study will be very helpful in phytomanagement of metal-polluted soils, and provide a tendency to the employing of lead - contained wastewater for irrigation of the grass in low water regions.

MATERIALS AND METHODS

Plant material

In the present study, the seeds of six turfgrasses; perennial ryegrass (Lollium perenne), tall fescue (Festuca aurandinaceae), blue sheep fescue (Festuca ovina), red fescue (Festuca rubra), kentucky bluegrass (Poa pratensis) and agropyron (Agropyron elongatum) were purchased from Pakan Bazr Company, Esfahan, Iran.

Germination experiments

The firs seeds were surface-sterilized with 5% (v/v) sodium hypochlorite for 10 minutes to prevent any fungal contamination, thereafter seeds were thoroughly rinsed with deionized water to ensure that no residual Na adhered to the seed surface and allowed to imbibe for 3 h. After water imbibition, the seeds were placed into Petri dishes (90 mm diameter) containing sterile filter sheets (Whatman No. 42) moistened with either 6 ml of distilled water as a control or Pb solutions (1000, 2000, 3000 µM). The lead solutions were freshly prepared by dissolving Pb (NO₃)₂ in deionized water and adjusting their pH to 5.5 with HNO₃. There were four replicates per each test concentrations and control. Petri dishes were kept in an incubator at 25 ± 2.5˚C and 70% RH under a light irradiance of 300 mmol m^-2 s^-1 (12:12 h light: dark conditions), and the various parameters of germination and growth indices were recorded.

Germination and seedling growth indices

The number of seeds germinated was determined in each treatment every 24 h intervals for 14 days using radicle protrusion (i.e., appearance of a radicle ≥2 mm in length) as a criterion (Gill et al. 2003), and the seed germination percent (GP) was calculated as follows: (number of seeds germinated/total number of seeds) × 100. Day 14^th of experiment was considered as the final day, because no more germination was observed and root and shoot length was measurable for all dishes. Germination rate (GR) was determined  daily until the seedling stage(Ellis & Roberts 1980). Germination vigor (GV) and germination index (GI) were measured using following formulae: GV = seedling length (cm) × GP, and GI = (GP/DT), where GP is the germination percentage on the T day, and DT is the day of germination. The length of shoots and roots were also recorded after two weeks from culturing.

Biochemical analysis

Chlorophyll content

Weighed 0.5 g of leaf samples from controls and the different Pb treatments were homogenized in 20 ml 80% acetone. The absorbance of the supernatant measured at 663 and 645 nm using a Spectrophotometer (Ltd T80 + UV/VIS; PG Instruments, Leicestershire, UK). It was finally calculated according to Wellburn & Lichtenthaler (1984) and expressed as mg.g^-1 fresh weight (FW).

Preparations and assays of antioxidant enzymes

The leaves of the turfgrasses (200 mg) were homogenized in 1 ml ice-cold extraction buffer containing 50 mM phosphate buffer (pH 7), 1 mM EDTA and 1.5% (w/v) of PVP. The homogenate was centrifuged at 9000 g for 15 min. The supernatant was used as the crude extract for determination of enzyme activities (Zhang & Nan 2007).

The SOD activity was measured spectrophotometrically as described by Beyer & Fridovich (1987). The reaction solution (1 ml) contained 50 mM phosphate buffer (pH  7), 12 mM riboflavin, 13 mM methionine, 0.1 mM EDTA , 7 mM nitro blue tetrazolium (NBT) and 10 µL of extracted enzyme solution. A solution with no enzyme was used as the control. Test tubes were irradiated under fluorescent lights at 100 mM m^-2 s^-1 for 20 min.

The absorbance of each solution was measured at 560 nm using a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that would inhibit 50% of NBT photo reduction.

The catalase (CAT) activity was assayed as reported by (Brouwer & Brouwer 1998). The reaction solution (0.5 ml) contained 25 mM phosphate buffer (pH = 7), 10 mM H₂O₂ and 10 µL of extracted enzyme solution. The reaction was initiated by adding the enzyme solution. Changes in absorbance at 240 nm were read every 10s for 60s using a spectrophotometer. One unit of CAT activity was defined as the absorbance change of 0.01 units per minute. The peroxidase (POD) activity was determined by the method of Chance & Maehly (1955). POD activity in leaves was assayed by the oxidation of guaiacol in the presence of H₂O₂. The increase in absorbance was recorded at 470 nm.

The reaction mixture contained 100 µL of crude enzyme extract, 500 µL of 5 mM H₂O₂, 500 µL of 28 mM guaiacol, and 1900 µL of 50 mM potassium phosphate buffer (pH 7.0). POD activity of the extract was expressed as activity U/g FW min.

Statistical analysis

Statistical analysis was performed using SAS software (Version 9.0). The significance of the differences among treatments was tested by One-Way ANOVA at p ˂ 0.01. Values reported here are means of four replicates.

RESULTS

Seeds germination characteristics

The effect of Pb concentrations on seed germination characteristics of different turfgrasses are summarized in Table 1. Germination index (GI) of studied turfgrasses was significantly affected by Pb concentrations. GI was significantly decreased when Pb concentration increased in the solution except for A. elongatum. Among the lead concentrations, No significant difference was found for GI in A. elongatum. The lowest GI was observed in F. ovina when exposed to 3000µM Pb (Table 1). Increasing Pb concentration in the solution caused a significant decreasing in germination percentage (GP) in the all turfgrass species. However no significant decreasing was found among Pb concentrations in A. elongatum and L. perenne (Table 1). The GP of the seeds of F. ovina was considerably affected by Pb treatments, so as it reduced from 93.6% in control to 8.9% in plants treated by 3000 µM Pb concentration. A. elongatum and L. perenne had the maximum GP about 90 and 85.56% respectively, whereas F. ovina showed only 8.9% GP. As shown in Table 1, by increasing Pb concentration in the solution, germination vigor (GV) also decreased in all studied turfgrass species, however, no significant difference was found between Pb concentrations in A. elongatum. The maximum and minimum GV were observed in A. elongatum and F. ovina as compared to their controls, with 57% and 97% reduction, respectively. The germination rate (GR) of F. ovina was more sensitive than that of other studied turfgrasses in response to the high Pb concentration in the solution, as shown by the 97% reduction at 3000 µM Pb solution.

The maximum GR was observed in A. elongatum, therefore this species could be less sensitive to Pb (Table 1).

Seedling shoot and root growth

Pb significantly affected different turfgrasses seedling shoot and root length (Fig. 1a).

The root growth of all studied turfgrasses was significantly inhibited by Pb solutions. The highest suppressing effect was found in the F. ovina roots with 2 mm in length, while the lowest one was found in those of A. elongatum with 28mm in length (Fig 1a).

So that, it was suppressed in all turfgrasses as 50% as control at 1000 µM, except for F. ovina, which remained nearly unchanged.

This inhibition reached over 90% for the treatment with 3000 µM Pb. Unlike roots, the seedling shoots was less affected by Pb concentrations (Fig 1 b). No significant difference was found between 1000 µM Pb treated plants in comparison with control. But, the F. rubra and F. ovina shoots length significantly reduced after treatment with 2000 µM Pb solution. All turfgrass species (except for L. perenne) showed a significant reduction in their shoot length when exposure to 3000 µM Pb. The highest shoot growth was observed in A. elongatum with 96 mm in length comprising about 85% of control, whereas the minimum was in F. rubra with only 26 mm about 50% of control.

Chlorophyll content

The chlorophyll content of all turfgrass species was significantly decreased with increasing Pb concentration in the solution, except for A. elongatum (Table 2). However, no significant difference was found between control and 1000 µM Pb from point view of chlorophyll content. The lowest chlorophyll content was found in F. ovina, when treated with 3000 µM Pb. It was declined as 45.8% as control in F. ovina. Therefore, this species could be the most sensitive turfgrass to the Pb contamination with respect to chlorophyll content.

Antioxidant enzymes activities

Pb - induced oxidative stress was further confirmed by the dose-dependent increase in the antioxidant enzyme activities, as shown in Fig. 2A about the SOD activity in all Pb-treated seedlings, but F. ovina, tended to ascend when the lead concentration increased from 1000 to 3000 µM, in comparison with control. The SOD activity in F. ovina was significantly inhibited by the increasing of Pb concentration. The highest SOD activity was observed in L. Perenne with 263 U.g^-1 FW. As shown in Fig. 2B, except for F. ovina, the CAT activity continued to increase with the elevation of Pb concentration in all turfgrass species and peaked at the exposure of 3000 µM. So that, the maximum CAT activity was observed in L. perenne and P. pratensis (109 and 101 U.g^-1 FW, respectively), whereas the minimum enzyme activity was shown in F. ovina (20.2 U. g^-1 FW). Moreover, when Pb concentration reached 3000 µM, CAT activity in F. Rubra and A. elongatum became approximately 5.2 and 3.2-fold higher than in their controls respectively.

Effects of Pb on POD activity are shown in Fig. 2C.

Fig. 1. Percentage decrease in shoot length (A) and root length (B) of turfgrass seedlings at different concentrations of Pb (NO₃)₂ (1000, 2000 and 3000 µM). Bars indicated mean ± SE (n = 3).

Table 1. Germination index, percentage, vigor and rate of six turfgrass species in response to different concentrations of Pb.

Means in each row and column followed by different letters were significantly different (P< 0.01 level). Capital and small letters show statistical differences for data in columns and rows, respectively. 

Fig. 2. The activities of SOD (A), CAT (B) and POD (C) of six turfgrass seedlings treated with 0, 1000, 2000 and 3000 µM Pb (NO₃)₂. Data are expressed as mean ± SE (n = 4).

Table 2. Shoot chlorophyll content of six turfgrass species (mg.g^-1 fresh weight) when exposed to different Pb concentrations.

Values within the same column with different letters are significantly different at P<0.01.