Effect of arsenic on growth, lipid peroxidation and antioxidant defence system in cowpea 

Arsenic (As) is the most toxic metalloid known for centuries. Arsenic is omnipresent in environments and highly lethal to all forms of life. As is a food chain contaminant and considered as a class I carcinogen (Zhao et al., 2010). The Comprehensive environmental response, compensation and liability act permanently listed As as no. 1 in its significant list of hazardous materials (ATSDR, 2007). Plants reportedly represent the initial path for arsenic to enters the food web (Hartley et al., 2001). Arsenic is known to inhibit the plant growth together with fresh and dry biomass (Stoeva et al., 2003) and causes physiological disorders (Wells et al., 1997) as well as decrease the crop productivity. (Stepanak et al., 1998). Plants usually take up arsenic predominantly in trivalent (As III) and pentavalent (As V) forms, which are well identified to hinder several metabolic pathways like reacting with sulfhydryl groups and substitution of phosphate from ATP (Shri et al., 2009). As have been reported to stimulate the production of free radicals, leading to oxidative stress (Singh et al., 2007). According to  Mishra et al., (2008) the generated free radicals may induce peroxidation of unsaturated fatty acids in the cell membrane resulting in formation of lipid peroxidation products like MDA. The generated Reactive oxygen species (ROS) as a consequence of As toxicity need to be scavenged for plants development. Reportedly, a natural detoxification mechanism in plants is the upregulation of enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX) (Dave et al., 2013; Sobrino-Plata et al., 2014). Under abiotic stress conditions, plant frequently accumulate osmoprotectant compound such as proline which helps in direct scavenging of free radicals and also act as membrane plasticizer (Yadu et al., 2016).
 
Among the most important cultivated legumes, cowpea [Vigna unguiculata (L.) Walp] has shown numerous environmental, agronomic and economic advantages. (Singh et al., 2014). Under the present changing climatic and ecological conditions cowpea is predictable to fulfil the growing requirements of dietary proteins for humans (Goncalves et al., 2016). However, the physiological and biochemical responses of cowpea to toxic As (V) are very scanty. With the above background the objective of this  experiment was to inspect the effect of As (V) on the growth, lipid peroxidation, proline accumulation and antioxidant response on the cowpea cultivar grown in Saudi Arabia.

The seeds of cowpea were obtained from the local market of Gizan from western Saudi Arabia. The seeds were surface sterilized and sown in soilrite. Three days after germination, the seedlings of cowpea were shifted to Hoagland’s solution (Hoagland and Arnon, 1950). Ten-day-old seedlings were exposed to five levels of As (V) in the form of (Na₂HAsO₄) Sodium arsenate viz., T1 = 0 µM (Na₂HAsO₄), T2=25 µM (Na₂HAsO₄), T3=50 µM (Na₂HAsO₄), T4=75 µM (Na₂HAsO₄), T5=100 µM (Na₂HAsO₄). Growth performance, enzymatic antioxidants and non-enzymatic antioxidants were studied after 7-days of As V treatment. All the experiments were conducted in three replicates.
       
Plant shoot and root length were assessed on an electronic balance (Model EBL-120-S, Shimadzu, Japan). Samples were oven dried for dry weight (DW) determination at 65°C ± 2°C for 72 h and independently weighed. Plant DW and FW were expressed in g. Plant length was determined by using metric scale and recorded in centimeter.
       
Malondialdehyde (MDA) content in the leaves was determined by the method of Heath and Packer (1968). The level of lipid peroxidation as MDA concentration was evaluated using an extinction (e) of 155 µM^-1 cm^-1.
       
Free proline content was determined by the method of Bates et al., (1973). The concentration of proline was estimated using calibration curve and denoted as mmol proline g^-1 FW.
       
For assays of CAT, SOD, APX and GR, leaf samples was homogenised in ice cold 50 mM sodium phosphate buffer (pH 6.8) containing 1mM EDTA and 2% (w/v) PVPP. The homogenate was centrifuged at 13,000 x g for 40 min at 4°C and the supernatant was used for enzyme assays.
       
SOD activity in the leaf sample was estimated by observing its ability to inhibit photochemical reduction of NBT at 560 nm (Beauchamp and Fridovich, 1971). The activity of SOD was expressed in enzyme unit (EU) mg^-1 protein h^-1.
       
Enzymatic activity of CAT was studied by using the method of Aebi (1984). The activity of CAT was estimated spectrophotometrically by observing the dissolution of H₂O₂, recording a decline in the absorbance at 240 nm and expressed as EU mg^-1 protein.
       
APX activity was measured by the method described by Nakano and Asada (1981). Activity of APX was determined by recording the reduction in ascorbate at A290 (ε = 2.8 mM^-1 cm^-1). One enzyme unit, measures the quantity of enzyme required to degrade 1 mmol ascorbate per mg of protein per min. The activity of APX was expressed as enzyme unit (EU) mg^-1 protein.
       
GR activity in the leaf samples was estimated by utilising the method of Foyer and Halliwell (1976). GR activity was measured by extinction coefficient of 6.2 mM^-1 cm^-1 and expressed as EU mg^-1 protein. Protein concentration was measured by using the method of  Bradford (1976).
       
All analyses were done on a completely randomized design. All the data were subjected to one-way analyses of variance (ANOVAS). All the data point was the mean of three replicates. Comparisons with P values < 0.05 were considered significantly different.

Growth parameters
 
Arsenic is reported to have phytotoxic effects on some plant species (Talukdar 2013). Data of plant shoot length, root length, plant fresh weight and dry weight of cowpea as influenced by various doses of As (V) treatments are presented in Table 1. In the present study As (V) proved toxic, in a dose dependant manner, causing significant reduction in the plant growth. Plant shoot length and root length was reduced up to 20-58% and 24-63% over control. Whereas reduction in plant fresh weight and dry weight was 24-62% and 30-83% respectively when compare to control. Corresponding to result of this study significant decrease in root and shoot length under arsenic stress has been reported in other plants also by many investigators (Liu et al., 2005; Shri et al., 2009; Talukdar, 2013).
 

 
Lipid peroxidation
 
A significant change was observed in the MDA content at all level of As (V) treatment. Maximum amount of the MDA (98%) was reported at T2 (25 µM) while the minimum level of MDA content (21%) was observed at T5 (100 µM) As (V) treatment (Fig 1) Yadav et al., (2015) also observed same trend in Zea mays under As stress. As lipid peroxidation is the indicator mostly ascribed to oxidative damage high level of MDA content indicates toxicity, whereas a lower MDA content indicates improved antioxidative system as reported by various authors (Rai et al., 2013; Upadhyay et al., 2016).
 

 
Proline accumulation
 
Among non-enzymatic antioxidants, proline is a well-documented osmolytic compound which accumulates in plants under stress condition. (Chandrakar et al., 2016, Begum et al., 2016). In the present study proline accumulation in the cowpea increased significantly with increasing levels of As (V) treatments, The percent increase in the proline accumulation was (52-161%) when compared to control (Fig 2). An increase in proline content upon As (V) exposure has also been reported in earlier works on Vigna mungo (Srivastava and Sharma 2013) and rice (Kumar et al., 2014) Soybean (Kamran et al., 2018).
 

 
Antioxidant defence system
 
Under As stress, plants can induce oxidative stress, which causes an imbalance between ROS generation and ROS scavenging (Abbas et al., 2018). Among the antioxidant enzymes, SOD is a major enzyme that constitutes the first line of defence against ROS in plants (Abbas et al., 2014). In the present study SOD activity showed variation at different levels of As (V) treatments (Fig 3). The highest activity of SOD was recorded at T5 (100 µM) (31-86%) when compared to control. As-induced SOD activities have been observed by many other investigators (Begum et al., 2016, Singh et al., 2017).
 

       
In the present study As (V) treatment induced the CAT activity in cowpea seedlings in dose dependant manner. Maximum induction (36-89%) was observed at T5 (100 µM) when compared to control (Fig 4). Similar to our findings increased CAT activity under As stress was also observed in P. vittata by Tiwari et al., (2017).
       

 
The vital role of APX in mitigating the harmful effects of H₂O₂ under As (V) stress, has been well documented (Singh et al., 2017). The activity of APX showed similar trend to that of SOD and CAT. The maximum APX activity (27-94%) was observed at T5 (100 µM) level of As (V) treatment when compared to control (Fig 5). Similar results of increased APX activity in response to As stress was reported in Zea maize and Oryza sativa by Shri et al., (2009).
 

       
Glutathione is an essential antioxidant involved in cellular defense mechanism against toxicants (Goldsbrough, 2000). In our study GR activity showed significant variation at different levels of  As (V) treatment but the maximum GR activity (34-79%) was observed at T5 (100 µM) over control. (Fig 6). Overproduction of GR, under As stress, has been reported by number of other investigators (Degola et al., 2015, Jozefczak et al., 2012).
 

Arsenic is considered as one of the most toxic metalloid threatening the plant productivity. The study showed that As (V) causes significant reduction in growth rate of cowpea in terms of fresh and dry biomass. As toxicity induced the oxidative stress in cowpea which is related to higher proline production. To combat arsenic-induced oxidative damage in cowpea enzymatic antioxidant proved to play a major role. Thus, the present study contributes to the understanding of biochemical strategies adapted by cowpea to avoid As toxicity. Further investigation on a molecular level is essential to comprehend the mechanism of free radicals induced by As stress.

The author would like to extend sincere thanks to King Saud University, Dean ship of Scientific Research, Research Centre for supporting this work.