​Antioxidant Responses of Ricebean [Vigna umbellata (Thunb.) Ohwi and Ohashi] Seedling under Iso-osmotic Potential of Salinity and Drought Stress

Salinity stress can affect plants initially by creating an osmotic stress then it induces ion toxicity that lead to cyto-toxicity, metabolic impairment, nutrient imbalance and finally death of the plant. Initially, the presence of salts in high concentration makes very difficult for plants to withdraw water from soil due to very low osmotic potential. Salinity stresses during initial phases decreases water absorption capacity of root systems and loss of water from leaves is accelerated due to osmotic stress of high salt accumulation in soil and plants and therefore it is also considered as hyper-osmotic stress (Munns, 2005). In effect, the plants suffer from a sort of osmotic stress which causes yield reduction. At the later stages of stress, due to the absorption of sodium and chloride ions in high concentration plants suffer from cyto-toxicity which result in reduction of growth, leaf burn and plant death. The presence of high concentration of Na⁺ and Cl^- ion also reduces the availability of other ions like K⁺, Ca²⁺, Mg²⁺, thus, causing nutritional disorders (Marschner, 1995). Drought is a meteorological term and defined as a period without significant rainfall. It normally occurs under depleting soil moisture and the intensity of drought increases under atmospheric conditions conducive to increased water loss by transpiration and/ or evaporation. Crop growth and yield unfavourably affected by water deficit, which is one of the major abiotic stress. Drought stress is characterized by reduction of water content, diminished leaf water potential and turgor loss, closure of stomata and decrease in cell enlargement and growth. Severe water stress may result in the arrest of photosynthesis, disturbance of metabolism and finally the death of plant (Jaleel et al., 2008). The reduction in fresh and dry biomass (Farooq et al., 2009) is a major adverse effect of water stress on different crops.
       
Plant produces reactive oxygen species (ROS) continuously as bi-products of various metabolic pathways in different cellular compartments like chloroplast, mitochondria and peroxisome. Reactive oxygen species (ROS) are routinely produced during various physiological processes of the plants and it causes oxidative damage to membrane lipids, proteins, DNA and RNA. To protect the cells from oxidative damage, cells have evolved antioxidant defence which neutralizes, scavenges or dismutase ROS (Singh et al., 2015). The equilibrium between the production and the scavenging of ROS may be perturbed by various biotic and abiotic stress factors, such as salinity, drought, heavy metals, UV radiation, pathogen attacks etc. These disturbances in equilibrium lead to sudden increase in intracellular levels of ROS leading to oxidative stress which can cause significant damage to cell structure. To protect themselves against these toxic oxygen intermediates, plant cells contain both enzymatic and non-enzymatic components. Among the enzymatic antioxidants, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and non-enzymatic low molecular metabolites, such as ASH, GSH, a-tocopherol, carotenoids and flavonoids (Gill et al., 2011 and Mittler et al., 2004) have been found to be very important. In addition, proline can now be added to an elite list of non-enzymatic antioxidants that microbes, animals and plants need to counteract the inhibitory effects of ROS (Chen and Dickman 2005).
       
The present experiment has been designed to study the effects of different levels of salinity and drought stress on some biochemical frame work of ricebean during seedling stage.

Plant material
 
Seeds of ricebean [Vigna umbellata (Thunb) Ohwi and Ohashi] variety Bidhan-1 were used in the experiment. The seeds were collected from AICRP on Forage Crops, Kalyani Centre.
 
Experimental site
 
The experiment was done in the laboratory of Department Plant Physiology, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Mohanpur, Nadia and West Bengal in the year 2017-18 and 2018-19.
 
Plant culture
 
For studying the effect of salinity and drought stress on ricebean the experiment was conducted in sand culture using modified Hoagland solution (Epstein, 1972) under laboratory condition of diffused light, at around 80±1% relative humidity (R.H.) and at a temperature of 28±1°C.  For this purpose, the seeds of ricebean cultivar Bidhan-1 were surface sterilized with 0.1% (w/v) HgCl₂ for 3 minutes. The pre-germinated seeds (five seeds) were then transferred to plastic beakers of capacity one litre containing neutral sand. The seedlings were grown with supplement of full strength Hoagland solution (Epstein, 1972) maintaining the pH 6.3 at 3 days interval for 14 days.
 
Treatment application
 
14 day old seedlings were subjected to salinity and drought treatments. For this purpose, the appropriate amounts of NaCl and PEG 6000 calculated as per Sosa et al., (2005) (Table 1) to obtain the osmotic potential (Ψ) of -0.2, -0.4 and -0.8 MPa were mixed with modified Hoagland nutrient solution and the pH was adjusted to 6.3. Thus, the drought and salinity stress with iso-osmotic potentials were created in the present experiment. A control set having Ψ_s equivalent to 0.0 Mpa osmotic potential without containing NaCl or PEG. Observations on different growth and biochemical parameters were recorded on 9 days after treatment application.
 

 
Estimation of biochemical characters
 
Proline content was estimated using the standard protocol (Mohanty and Sridhar, 1982). Proline was extracted from 0.25 g fresh leaf tissue of both stressed and non-stressed seedlings. The absorbance was measured at 520 nm and proline content was calculated from the standard curve of L- proline.
       
GPOX activity was calculated as per Siegel and Galston (1967). GPOX was extracted from 0.25 g fresh leaf tissue of both stressed and unstressed seedlings. The absorbance was measured at 470 nm and GPOX activity was calculated from the standard curve.
       
APX activity was calculated as per Nakano and Asada (1981). APX was extracted from 0.25 g fresh leaf tissue of both stressed and non-stressed seedlings. The absorbance was measured at 290 nm and APX activity was calculated from the standard curve of H₂O₂.
       
Catalase activity was calculated as per Cakmak et al., (1993). Catalase was extracted from 0.25 g fresh leaf tissue of both stressed and control seedlings. The absorbance was measured at 240 nm and Catalase activity was calculated from the standard curve of H₂O₂.
 
Statistical analysis
 
The mean data in all the cases were subjected to statistical analysis following completely randomised design using INDOSTAT version 7.1 Software. The mean values were statistically compared by least significant difference (LSD) at P≤0.05.

Proline (Pro) is the most important and efficient compatible organic solute (Tang et al., 2015), which act as an osmo-protectant. Along with its antioxidant properties, proline can act as a molecular chaperone to protect the structure of biological macromolecules during dehydration, thus conferring plant tolerance to environmental stresses (Kundur et al., 2016; Tang et al., 2015; Ashraf and Fooland, 2007). Apart from being an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures (e.g., membranes and proteins), scavenging free radicals and buffering cellular redox potential under stress conditions.
 
It may also act as protein compatible hydrotrope, alleviating cytoplasmic acidosis and maintaining appropriate NADP+/ NADPH balances compatible with metabolism (Strizov et al., 1997). The leaf proline content at 50 mM NaCl and PEG 10% (-2 MPa osmotic stress) solution increased significantly over that of control (Fig 1). This result was in accordance with the findings of Dar et al., (2007), El-Sayed (2011) and Aniat-ul-Haq et al., (2012) under salinity stress and with study of Mohammadkhani and Heidari (2008) and Bhardwaj and Yadav (2012) under drought stress. The increased level of proline in leaf might attribute for osmotic adjustment under low levels of osmotic shock induced by NaCl and PEG. The level of proline content decreases as the osmotic potential of the growing medium decreased more. Earlier, Verma et al., (2012) also observed a decrease in leaf proline at high level of NaCl stress (200 mM). The leaf proline content registered almost similar adverse effects at iso-osmotic potentials of high intensity of salinity and drought stress. Here, ricebean seedling recorded 16.50% reduction over that of control for both in leaf proline content for both 200 mM NaCl and 18% PEG solution at osmotic potential of -0.8 MPa. The salinity and drought at high intensity was found to produce similar adverse effects on content of leaf proline in ricebean in the present experiment. So it cannot be concluded that increase proline content is indicative of resistance against osmotic stress but it indicates certainly that increment in proline content at moderate stress level promotes the defensive mechanism against drought and salinity stress.
 

 
The guaiacol peroxidase (GPOX) was also involved in the catabolism of H₂O₂ in the cytosol, vacuole and cell wall (Asada, 1992). Carrasco-Ríos and Pinto (2014) showed in maize seedlings that GPOX levels increased significantly under saline conditions at different osmotic potential. The leaf GPOX activity of ricebean seedling significantly increased under all the treatments of salinity and drought stress over that of unstressed control (Fig 2) although the level decreased with increased stress intensity in all the cases. The variety Bidhan 1 recorded 28.60% and 75.53% increase in content of leaf GPOX activity in 200 mM NaCl and 18% PEG solution producing an osmotic potential of -0.8 MPa, respectively, over that of control. The findings corroborated well the early reports of Livingstone et al., (1992) and Shi et al., (2006). Shi et al., (2006) showed in maize a linear and significant increase in GPOX activity under water stress, but PEG at highest osmotic potential the GPOX activity decreased which is similar to the present experiment. Finally, the leaf GPOX content registered more induction under drought stress in comparison with salinity stress at iso-osmotic potentials.
 

       
Ascorbate peroxidase (APX) is one of the fine regulators of intracellular ROS level and the enzyme detoxify H₂O₂ by using ascorbate for reduction in the ascorbate glutathione cycle (Noctor and Foyer, 1998). The APX has higher affinity for hydrogen peroxide and this enzyme is present in almost every ROS generating subcellular compartment. The activity of APX in leaf significantly increased over control at mild and moderate intensity of drought and salinity while it ultimately decreased under severe stress (Fig 3). Previously, the adverse effects of salinity stress (Dar et al., 2007) and drought stress (Bhardwaj and Yadav, 2012; Jiang-Jing Long et al., 2013) on APX in legumes were reported by different researchers. Jiang-Jing Long et al., (2013) showed in grass pea and garden pea seedlings stressed with 20% polyethylene glycol 6000 (PEG) that PEG caused a significantly greater increase of ascorbate peroxidase (APX). Here, the variety of ricebean recorded 65.30% and 57.14% reduction in APX activity for both 200 mM NaCl and 18% PEG solution producing an osmotic potential of -0.8 MPa, respectively, over that of control.
 

       
Catalase (CAT) is a main enzyme to eliminate H₂O₂ in the mitochondrion and microbody (Shigeoka et al., 2002) and thus help in ameliorating the detrimental effects of oxidative stress (Armand et al., 2016). It converts hydrogen peroxide to water and molecular oxygen (Willekens et al., 1995). Catalase (CAT) activity increased gradually over control along with increase in NaCl concentration upto 100 mM followed by slight decline at the highest concentration, while in case of PEG, it only registered higher value over control at 10% PEG (Table 2), but then decreased substantially with increase in PEG concentration (Fig 4). Thus, it might be concluded that higher intensity of drought stress produced negative effects on CAT activity in this variety of ricebean. De Azevedo Neto et al., (2006) also found higher CAT activity in two maize cultivars differing in salt tolerance. There are other reports are available on the effect of salt stress on CAT activity in several plant species. CAT activity has been found to increase under salt stress in blackgram (Sivakumar and Jaya Priya, 2021), soybean (Comba et al., 1998), tobacco (Bueno et al., 1998). Pratap and Sharma (2010) showed in the seedling of blackgram under drought stress using PEG-6000 at various osmotic potentials (-2, -5 and -10 bars) that the catalase (CAT) activity was increased at different osmotic gradients in comparison to control. This finding also conformed to early reports of Jiang-Jing Long et al., (2013). Bhardwaj and Yadav (2012) on an experiment of horsegram showed that antioxidant enzymes, likely Catalase (CAT) showed significant increase in the tolerant variety than the sensitive one under drought stress. PEG caused a significantly greater increase of catalase (CAT) showed by Jiang-Jing Long et al., (2013) in grass pea and garden pea seedlings stressed with 20% polyethylene glycol 6000 (PEG). In term of leaf catalase activity drought stress was found to produce more adverse effects than salinity stress.
 

 

From the present study, it might be concluded that drought and salinity are both important environmental challenges that reduces crop productivity by hampering balance between ROS generation and its safe detoxification by antioxidants. Though both the stresses ultimately induce oxidative stress to plants but drought stress was found to register more drastic effects on seedling growth as compared to iso-osmotic potential of salinity stress (200 mM NaCl and 18%PEG) in ricebean cultivar Bidhan 1. The drought stress also exhibited more negative effects on GPOX and CAT activity. While the APX activity was found to be more adversely affected by salinity stress. The leaf proline content at the highest intensity of both the salinity and drought stress have found similar effects. Differential sensitivity and affinity of catalase (CAT) and ascorbate peroxidase (APX) to ROS under drought and salinity stress is suggestive for their differential role in ROS scavenging.

The authors acknowledge the assistance extended by AICRP on Forage Crops, Kalyani Centre, for supplying plant materials and Department of Plant Physiology, Faculty of Agriculture, BCKV for providing with all the facilities and support.