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Effects of exogenous application of ascorbic acid on genotoxicity of Pb in Vicia faba roots.

Byline: Cui-Mei Yu Fu-Di Xie and Lian-Ju Ma

Abstract

Effects of ascorbic acid (AsA) were investigated on Vicia faba roots exposed to different concentration of Pb (NO3)2. Under all the Pb concentrations (10 20 40 M) treatment micronucleus (MN) frequency was higher when compared to the control. Furthermore chromosomal aberration (CA) frequency increased but not of mitotic index (P less than 0.05). The application of the AsA (0 0.5 1 2 4 mM) decreased markedly MN frequency and CA frequency and increased mitotic indexes under Pb treatments. The results indicated that exogenous application of AsA exerted a positive role on V. faba roots exposed to Pb stress. Copyright 2014 Friends Science Publishers.

Keywords: Vicia faba; Chromosomal aberratins; Mitotic activity; Ascorbic acid; Pb stress.

Introduction

Lead (Pb) is a toxic heavy metal which can be taken up by plant roots from soil and impact on human health through food chain (Shotyk and Le Roux 2005). Pb has no biological function and induces a broad range of deterioration effects on plant morphological physiological and biochemical processes due to strongly phytotoxic during plant growth and development. For example excess Pb causes plant growth retardation inhibits root elongation seed germination photosynthesis and damages membrane structure (Islam et al. 2008; Li et al. 2012). Moreover increasing evidence indicates that Pb toxicity is attributed to its oxidative damage (Pourrut et al. 2011). Superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidases (APX) were employed to mitigate and repair the ROS damages (Edreva 2005). Antioxidants such as reduced glutathione vitamin E and ascorbic acid (AsA) are also employed to counter ROS (Das et al. 2012).Ascorbic acid (AsA) is known to be a low molecular weight and an important water-soluble antioxidant molecule for enzymatic detoxification. Ascorbic acid is also as an ascorbate peroxidase substrate to scavenge H2O2 in the chloroplast stroma (Gadallah 2000; Shigeoka et al. 2002) and protects or regenerates oxidized carotenes and a- tocopherols (Noctor and Foyer 1998). Ascorbic acid plays a regulatory role in the plant growth by modulating the synthesis of plant hormones such as ethylene GA and ABA (Nambara and Marion-Poll 2003; Pastori et al. 2003) and also plays important roles under abiotic stress i.e. exogenous application of AsA enhanced plant tolerance tosalinity stress heavy metal Cd chill and drought stress (Shalata and Neumann 2001; Guo et al. 2005; Sajid and Aftab 2009; Chao and Kao 2010).Although many studies have been conducted on roles of exogenous application of AsA under abiotic stress conditions the function of exogenously applied AsA on Vicia faba root tip cells exposed to Pb stress has not been evaluated. It is reported that Pb interfered with the mitosis process and inhibited cell division of plant root tip cells (Shahid et al. 2011). Accordingly in this study we investigated effects of AsA on V. faba roots exposed to Pb stress.

Materials and Methods

Materials and Treatments

Some V. faba seeds were surface sterilized in 0.5% (v/v) sodium hypochlorite and washed with distilled water for 3 times. Seeds were then soaked in different concentrations Pb (NO3)2 solutions (0 5 10 20 40 M) for 24 h and transferred to Petri dishes for germination. Germinated seeds were cultivated hydroponically on Hoagland's nutrient solution consisting of 1 mM MgSO4 5 mM Ca (NO3)2 0.2 mM KH2PO4 50 M H3BO3 0.1 mM (NH4)6Mo7O24 4.5M MnCl2 3.8 M ZnSO4 10 M Fe-EDTA 5 mM KNO3and 0.3 M CuSO4 at pH 6.The nutrient solution wasrenewed every 3 days. Root length was measured afterculturing 7 days.Some seeds were soaked in the AsA (0 0.5 1 2 4 mM) solutions for 24 h then transferred to Petri dishes and incubated. Distilled water was renewed once at 12 h intervals. Growing about 1 cm the roots were cultured in different Pb(NO3)2 concentrations (0 10 20 40 M) for 6 h respectively. The other seeds were soaked in different Pb(NO3)2 concentrations (0 10 20 40 M) for 6 h 12 h and 24 h respectively. After Pb(NO3)2 solutions treatment roots were washed with distilled water reculturing for 24 h.

Slide Preparation Staining and Scoring

During cell division crest-time the primary root tips were cut about 1 cm from recultured seeds placed in Carnoy's fixation fluid for 24 h and stored in 70% ethanol. The root tips were first washed in distilled water for 5 min and hydrolyzed with 1 M HCl for 13 min at 60C. After HCl treatment the root tips were soaked in distilled water for 1 min. Finally the root tips stained with Feulgen for 30 min in dark. The root cap was removed and meristematic tissues were squashed on slides. The MN mitotic and CA were counted microscopically. The MN had a chromatin structure similar to the main nucleus and appeared within the cytoplasm. The MN diameter did not exceed 1/3 of the main nucleus and was on the same plane focus. Chromosomal aberration (CA) consisted of micronucleus lagging chromosomes chromosome fragment chromosome bridge etc.

Statistical Analysis

Ten tips (2000 cells in per tip) were utilized in each different treatment. The results were expressed as the values standard deviation (SD).All Data were analyzed with thestatistical package SPSS11.0.

Results

Several different Pb2+ concentrations (0 5 10 20 and 40M) were checked to research circumstance of Pb2+ damage on V. faba root length. The root length of 5 M Pb2+ treatment was the longest in all treatments which was much longer when compared to control (Fig. 1). However other root length here in tested was pronouncedly shorter than control. The higher Pb2+ concentrations inhibited the growth of V. faba roots.

Based on the analysis of V. faba root growth 10 20 and 40 M Pb2+ concentrations were used to investigate the relationship between genotoxicity and Pb concentration. Micronucleus (MN) analysis was performed to estimate genetic effects of chemicals during mitotic activity on V. faba. It was observed that MN frequency was significantly higher than control after treatment with distinct concentrations of Pb (Table 1) and also relatively increased with increasing Pb concentration. MN induction was significantly detected (Pless than 0.01) after Pb treatment 6 h and MN frequency relatively increased with prolongation of Pb treatment time. Application of AsA decreased markedly (Pless than 0.05) MN frequency of V. faba root tip cells exposed to different Pb concentrations (Table 2). There were the lowest values on inhibiting MN frequency at 1 mM AsA. Namely MN production decreased with AsA concentration changing from 0.5 mM to1 mM but relatively increased from1 mM to 4 mM.

The genotoxic activity of Pb stress also showed chromosomal aberration which concomitantly occurred with MN. With increasing Pb concentration and

Table 1: Effects of different concentration Pb in V. faba root tip cells

Pb2+###Micronucleus Frequency###Chromosome Aberration Frequency###Mitotic Index

M###S

###x###S%

###x###S%

###x

###6h###12h###24h###6h###12h###24h###6h###12h###24h

0###1.03###1.18###0.96###0.22###0.19###0.24###6.31###6.36###6.79

###0.25###0.14###0.22###0.08###0.03###0.15###1.02###2.27###1.49

10###7.97###9.24###13.41###8.65###12.91###17.42###4.96###4.31###3.82

###2.13###1.96###3.07###1.59###2.32###2.23###1.84###2.18###0.85

20###11.36###14.81###17.32###11.37###14.45###19.31###4.27###3.84###3.14

###1.98###3.21###2.11###2.21###2.53###1.58###1.47###1.24###1.02

40###13.25###14.22###14.54###10.45###13.15###12.64###2.85###2.61###1.94

###1.53###2.75###2.63###1.84###1.74###3.53###0.79###0.66###0.76

Table 2: Effects of different concentration of AsA and Pb in V. faba root tip cells

###AsA###Pb2+

###Micronucleus Frequency###Chromosome aberration frequency###Mitotic index

###mM###M

###S

###x###S

###x###S

###x

###0###10###7.97 2.13###8.65 1.59###4.96 1.84

###0.5###10###5.431.01###6.24 1.37###6.78 2.32

###1###10###4.362.21###3.96 0.83###8.27 2.4

###2###10###4.691.37###5.381.1###7.93 1.2

###4###10###4.882.26###6.03 2.03###6.45 2.13

###0###20###11.36 1.98###11.37 2.21###4.27 1.47

###0.5###20###8.54 1.43###8.79 0.87###5.69 1.3

###1###20###6.42 2.36###6.01 1.64###7.46 2.71

###2###20###6.971.66###6.65 2.2###7.52 1.84

###4###20###7.34 2.03###5.32 1.73###6.31 1.47

###0###40###13.25 1.53###10.45 1.84###2.85 0.79

###0.5###40###11.25 1.31###8.03 2.09###3.25 0.63

###1###40###9.71 1.97###7.72 2.03###4.02 1.6

###2###40###9.53 2.27###7.26 1.38###3.76 0.95

###4###40###11.65 1.56###9.54 1.57###3.19 2.1

prolongation of Pb treatment time CA frequency gradually increased (Table 1). Different type of chromosomal aberrations was induced in the whole cell cycle such as the presence of micronucleus (Fig. 2a b) alignment chromosomes (Fig. 2c) laggard chromosomes (Fig. 2d f) chromosome fragment (Fig. 2e g h) and chromosome bridge (Fig. 2i j k l) etc. A significant difference between application AsA and non-application AsA was observed in CA frequency (Table 2). The CA frequency gradually decreased when AsA concentration changed from 0.5 mM to 1 mM.1 mM AsA treatment had the lowest CA frequency under different Pb2+ concentration.

The mitotic indexes treated with different Pb concentrations significantly decreased (P less than 0.05) in comparison with control (Table 1). Moreover mitotic indexes decreased with increasing Pb concentration and with prolongation of Pb treatment time. The exogenous application of AsA had increased the mitotic indexes than that of Pb alone treatment (Table 2). The mitotic indexes of 1 mM AsA treatment was the highest under different Pb2+ concentration. However when treatment concentration was 4 mM AsA the mitotic indexes began to decrease.

Discussion

It is well known that Pb stress induces many deterioration effects on plant morphological physiological and biochemical processes. However little information is about effects of Pb stress on plant genotoxicity. In the present study MN frequency and CA frequency relatively increased with increasing Pb concentration and prolongation of Pb treatment time. This indicated a direct correlation between Pb dose and MN and CA frequency which was consistent with some studies (Rucinska et al. 2004; Shahid et al. 2011). Increasing Pb concentrations strengthened the cell toxicity and seriously disturbed normal metabolic activity. Different type of chromosomal aberrations induced in the whole cell cycle such as the presence of micronucleus lagging chromosomes chromosome fragment chromosome bridge etc. The results indicated Pb stress directly affected DNA duplication disturbed RNA transcription and protein synthesis involved in the cell cycle. Shahid et al. (2011) reported that Pb also broke single and double strands of DNA and affected horizontal DNADNA or DNAprotein links. However several literatures reported that Pb genotoxicity was due to oxidative stress which was mediated by ROS and/or MDA (Rucinska et al. 2004; Pourrut et al. 2008; Shahid et al. 2013). Mitotic index is an important indicator of cell division frequency and root growth rate. The mitotic indexes decreased with increasing Pb concentration (Table 1). High concentration Pb affected root tip cells division and prolonged interphase in cell division thus lengthened whole cell cycle and made mitotic indexes decrease. And consequently root growth was inhibited (Fig. 1). The mitotic indexes also decreased with prolongation of Pb treatment time. The longer Pb treatment time was the more root tip cells were hurt.AsA is as an ascorbate peroxidase substrate to scavenge H2O2 produced by ROS and plays important roles in plants under abiotic stress tolerance (Gadallah 2000; Shigeoka et al. 2002). Application of AsA decreased markedly (P less than 0.05) MN frequency under different Pb concentrations here in tested. Pb-induced MN formation may be due to potential role of oxidative stress which played a major role in damaging DNA. AsA can function as antioxidant to directly detoxify ROS. In addition AsA may be as an enzyme cofactor to indirectly detoxify ROS. Reduction of ROS led to decrease DNA-damage induction. So MN frequency decreased. A significant difference between application AsA and non-application AsA was also observed in CA frequency (Table 2). Dhir et al. (1993) reported that AsA can reduce sister-chromatid exchange caused by lead in mouse bone marrow. Exogenous application of AsA increased the protein content to NaCl- treated potatoes (Sajid and Aftab 2009). That CA frequency decreased with exogenous application of AsA may be due to activate a complex network of proteins including of both DNA-repair proteins and antioxidant enzymes removing ROS (Wierzbicka 1998; Balestrazzi et al. 2011). The exogenous application of AsA had increased the mitotic indexes than that of Pb treatment alone (Table 2). Citterio et al. (1994) found that AsA enhanced cell division effectiveness of competent cells. AsA content increased in pumpkin roots exposed to 50 M aluminum sulfate stress (Dipierro et al. 2005). It was also reported that exogenously applied AsA enhanced V. faba plant seeds germination and root growth under NaCl and mannitol stress (Younis et al.2010) and promoted seed germination in halophytes exposed to sea salt (Khan et al. 2006). In our experiment we also found that root growth was improved with application of AsA (Data not shown). Therefore AsA may highly alleviate genetic damage of interior cells and promote root to grow.In conclusion Pb genotoxicity was clearly noted on V. faba root cells micronucleus test in the present study. Reason of Pb genotoxicity may be due to Pb directly damaging DNA and also be due to potential role of oxidative stress. Application of AsA effectively inhibited MN and CA production and enhanced the mitotic indexes of V. faba root cells exposed to Pb stress. Ascorbic acid also promoted V. faba root growth and exerted a positive role on the V. faba tip cells following exposure to Pb stress. Muchof the AsA is essential to resist abiotic stress during plant growth and development.

Acknowledgments

The National Natural Science Foundation of China (31171570) supported this work.

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Author:Yu, Cui-Mei; Xie, Fu-Di; Ma, Lian-Ju
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Aug 31, 2014
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