Indole Acetic Acid (IAA) Mediated Amelioration of Lead (Pb) Stress- Physiological Indices of Mung Bean [Vigna radiata (L.) Wilczek]

Rapid increase in trends of urbanization and industrialization, has lead to more heavy metals in the environment during the past decades (Ashraf et  al., 2019). The origins of these heavy metals are natural or anthropogenic activities (Pichtel, 2016). The uses of agricultural fertilizers and pesticides, sewage sludge, mining and smelting of metal ores and fossil burnings are the main sources of heavy metals. Plant growth hormones are the phytochemicals which can compensate the adverse effects of heavy metals on plants. Auxins are the phytohormones which have their effects on different phenomenon of plant life like cell elongation, cell division, cell differentiation and expansion of leaf. Auxins induce antioxidant enzymes activities such as catalase and peroxidase. The major hormone among the auxins which is present in plant is Indole acetic acid (Jutta, 2000). Plant hormones have been reported to increase plant yield (Savita et al., 2011) and stomatal movement (Petri et al., 2010). Plant growth regulators have become commercialized in many countries like EU, USA and Japan due to their positive influence on plant growth and yield (Jahan et al., 2019). Mung bean [Vigna radiata (L.) R. Wilczek] has its origin in 1,500 BC in subcontinent and was thereafter introduced to the continents of Asia, Africa, Australia and America. In Pakistan, Mung bean is cultivated on an area of 141,000 hectares with mean annual yield of 93,000 tons (Pakistan Economic Survey, 2012). Keeping in view the importance of mash bean, hazardous effects of heavy metals and role of IAA, the present experiment was designed with objective to find out the ameliorating potential of IAA for Pb toxicity on mash bean.

The experiment was designed to find the ameliorating potential of indole acetic acid (IAA) for stress effects of rhizospheric lead (Pb) toxicity on two mung bean [Vigna radiata (L.) Wickzek] varieties. The pot culture experiment was conducted in Bahauddine Zakariya University, Multan Pakistan during 2016-17. Effluents hazards free sandy loam soil was selected for the experiment. Soil analysis revealed that the Ec of soil was 1.5 mM, pH 6.5 and saturation percentage was 56%. Earthern pots of 30 cm diameter were filled with 6 kg soil after mixing it well. Mung bean seeds of two varieties i-e M-8 and MN-92 varieties were obtained from Ayub Agriculture Research Institute, Faisalabad (Pakistan). Lead nitrate salt and IAA of Sigma Aldrich, Japan were purchased. Pots after filling were irrigated and left till getting of field capacity moisture contents. After germination normal agronomic practices like irrigation and two times pesticide spray of 5% Thiodon were performed. Arrangement of pots was complete randomization by design. To develop the metal stress, amounts of lead nitrate was added after calculation to develop lead @10 and 20 mg/kg soil. Control pots were left without addition of salt. Solutions of IAA (100.0 mM) were sprayed at the age of fifteen and thirty days of age.
 
Treatment plan consisted of following six treatments.
 
T₁ = Normal soil (Without addition of metal salts) +Distilled water foliar spray.
T₂ = Normal soil (Without addition of metal salts) + 100.0 mM IAA foliar spray.
T₃ = Lead (10.0 mg/kg soil) + Distilled water foliar spray.
T₄ = Lead (20.0mg/kg soil) + Distilled water foliar spray.
T₅ = Lead (10.0 mg/kg soil) + 100.0 mM IAA foliar spray.
T₆ = Lead (20.0mg/kg soil) + 100.0 mM IAA foliar spray.
 
There were five pots for each treatment. After thinning, three plants were maintained in each pot. Gas exchange parameters of leaf such as photosynthetic rate (A), internal CO² (Ci) concentration, stomatal conductance (gs), water use efficiency (A/E) and transpiration rate (E) were measured from youngest fully expanded leaf specified from the top of each plant which has a mean leaf area of 4.72 cm². For this, an open system LCA-4 ADC portable InfraRed gas analyzer (IRGA) of Analytical Development Company, Hoddesdon, England was used. Timing of measurements were from 11.00 a.m to 1.00 p.m. Followings specifications were adjusted: Molar flow of air was 403.3 mmol m^-2s^-1 per unit leaf area, 99.9 kg Pa atmospheric pressure, 6.0 to 8.9 mbar water vapor pressure of chamber, photon flux density at the surface of was 1711 µmol/m²/s, leaf temperature range was 28.4-32.4°C and external CO₂ concentration was 370 mmol/ mol.; Biomass production as stem, root and leaf dry weight were evaluated after 10 days of PGRs spray completion (40 days old plants). Biomass was determined with electrical balance after drying samples for 48 hours at 80°C. The data of experiment were analyzed statistically using software of COSTAT computer package. Keeping significance level of 5%, Duncan’s multiple range test was applied comparing mean values (Duncan, 1955). MSTAT-C Computer Statistical Programm was used. Wherever, F values were significant for testing of means using LSD tests.

The analysis of variance (Table 1) related to photosynthetic rate showed that significant differences were found among different treatments and varieties. Responses of varieties to treatment showed non-significant differences separately. IAA treatment caused an increase in photosynthetic rate (23.18%). Low and high lead doses decreased the photosynthetic rate by a value of 24.61 and 55.54% respectively. IAA ameliorated the lead stress so growth was reduced by a value of 17.78 and 27.35% in 10 and 20 mg lead treated plants (Table 2).
 

 

       
The analysis of variance of transpiration rate (Table 1) showed that treatments differed non significantly and separate response of both variety to each treatment differed non significantly. While significant differences were observed between two varieties. Increase in transpiration rate by the application of IAA was (8.02%). Lead toxicity of low and high levels decreased the transpiration rate 2.87 and 14.12% respectively. IAA ameliorates the lead toxicity and metal effects on transpiration rate were reduced to values of 1.73 and 7.28% respectively (Table 3).
 

       
The analysis of variance (Table 1) showed that stomatal conductance significantly affected by different treatments. Results revealed that varieties and treatments were differed highly significantly. Separate responses of varieties to treatments revealed non-significant differences. Stomatal conductance was increased by exogenous IAA spray as 1.96%. Low and high lead treatments decreased the stomatal conductance 0.56 and 3.37% respectively. IAA application decreased the harmful effect of lead therefore effects of metal on stomatal conductance were lowered to values of 0.28 and 1.68% respectively (Table 4).
 
  
 
The analysis of variance of sub stomatal CO₂ concentration (Table 1) showed that varieties, treatment response of each variety to treatments differed non significantly. Low and high lead treatments decreased the sub stomatal CO₂ concentration 1.70 and 5.98% respectively. IAA treatment increased the sub stomatal CO₂ concentration (1.70%). IAA alleviated the toxic effect of lead and CO₂ concentration reductions were 0.56 and 3.41% respectively under low and high levels of stresses (Table 5).
 

       
Reduction in photosynthetic rate, CO₂ assimilation and transpiration rate might be due to decline in water potential (Atteya, 2002). Water contents in metal treated plants are decreased (Poschenrieder et al., 1989) due to increasing resistance in water flow (Barcelo et al., 1988) or alteration of cell wall properties by metal (Poschenrieder et al., 1989). Conduction and transport of water is influenced in root by toxic metals (Barcelo and Poschenreider, 1990). Photosynthetic reduction might be due to chlorophyll decrease by metal. Hampp et al., (1974) have shown that enzymes of chlorophyll biosynthesis like 6-amino laevulinic acid dehydratase and porphobilinogenase are affected in by metal treatment. Lead is also found to depress the rate of photosynthesis (Carlson et al., 1975). Decrease in activities of many enzymes of CO₂ fixation (Barcelo et al., 1988); changes in the thylakoid structure (Fodor et al., 1996) may contribute to reduction in photosyntheticactivity and growth. Reduction in plant growth perhaps is conducive to Reactive oxygen species (ROS) production as heavy metals toxicity as an efficient generator of toxic ROS inhibits photosynthetic ETC (Kappus, 1985).
       
The data related to analysis of variance of stem dry weight (Table 1) showed that varieties, treatments and response of individual variety to every treatment differed non significantly. Foliar application of IAA non significantly increased the stem dry weight in two varieties (8.74%). Low and high lead treatments decreased the stem dry weight by values of 27.01 and 39.57% respectively. The lead toxicity was decreased due to foliar application of IAA and dry weight reductions were by values of 12.88 and 24.75% respectively (Table 6).
 

       
The analysis or variance of root dry weight (Table 1) showed that treatments, varieties and separate response of varieties to treatment differed non significantly. Application of IAA increased the root dry weight (8.53%). Low and high lead doses decreased the root dry weight 14.07and 22.61% respectively (Table 7). The analysis of variance of leaf dry weight (Table 1) revealed that varieties, treatments and individual response of all varieties to treatment were different to non significantly degree. Indole Acetic Acid application increased the leaf dry weight upto 4.88% (Table 8). Low and high lead treatments decreased the leaf dry weight 14.09 and 23.35% respectively. Application of IAA alleviated the lead stress. Therefore, leaf dry weight reductions were up to 6.45 and 15.92% respectively (Table 8). Biomass of plant organs decreased due to metal stress (Table 6-8). Such type of findings are reported also by Ouariti et al., (1997) and Fengxiang et al., (2003). Plant growth and plant water contents have relations with plant weights and biomass.  Decline in biomass might be due to Inhibition of both cell elongation and division by heavy metals (Arduini et al., 1994) or due to reduction in nitrogen contents (Andrews et al., 1999). Decrease in dry mass production may also be due to decreased cytokinin as a result of limited nutrients supply (Van der Werf and Nagel, 1996). Reduced amount of cytokinin decreases leaf expansion in plants treated with metal (Gadallah and El-Enany, 1999). Decreased cytokinin contents also affect cell division and cell expansion (Downes and Crowell, 1998).  IAA when applied on plants increases net photosynthesis rate and synthesizes more C: N ratio which results in growth enhancement (Sudadi, 2012).
 

 

From the results of experiment it can be infered that foliar application of 100 mM indole acetic acid (IAA) affected the photosynthetic rate and stomatal conductance to statistically significant level. The ameliorative effects of IAA on transpiraton rate and internal CO₂ concentration were statistically non significant. Similarly, IAA effects for mitigation of Pb stress in term of dry biomasses were to a non significant level.