With rapid industrialization and urbanization, heavy-metal contamination of soil has become a serious environmental issue [26]. Anthropogenic activities such as agricultural production, industrial activities, mining and domestic waste discharge resulted in higher metal contamination. In previous studies, it was observed that heavy metals such as Cd, Pb, Hg, Cr, Zn, Cu, Cs, As and Se enter into the food chain and causes a deleterious effect on human health [27].
The current study revealed the high phytoremediation potential of Z. mays and P. hysterophorus grown in industrial areas and irrigated with wastewater containing heavy metals such as Zn, Ni, Co, Fe, Cr, Pb and Cd of 214 mg/L, 126 mg/L, 1807 mg/L, 29 mg/L, 18.02 mg/L, 14.44 mg/L, respectively. Previous studies also reported plant species that can accumulate two or more metals from contaminated soil [28, 29]. Maize plants can be used to eliminate heavy metals from soil due to their high biomass [30]. Due to fast growth, stress tolerance and inedible nature which inhibits the entry of heavy metals in food chain, P. hyterophorus is considered to be a useful phytoremediator [31]. In metal contaminated soil, hyperaccumulators showed a slow growth rate and little biomass [32].
In literature, at least 45 families are found that contain hyperaccumulator plants [33]. Asteraceae, Violaceae, Brassicaceae, Caryophyllaceae, Fabaceae, Poaceae, Lamiaceae, Cyperaceae and Euphorbiaceae are the most leading families for hyperaccumulation of heavy metals and around 400 plants have been reported for this purpose included [34]. In the present study plants selected for phytoremediation are Z. mays and P. hysterophorus belonging to Poaceae and Asteraceae family, respectively because these families are dominant in hyperaccumulation of heavy metals. It is reported that all parts of plants can accumulate heavy metals in their different parts [35].
The first concerned plant was Z. mays that plays a vital role in cleaning up the soils stressed by Cd due to their high biomass, large number of roots and an ability to accumulate cadmium [36]. It is proposed that maize having high biomass and least metal storage in its harvestable parts, can be used to eliminate heavy metals from soil. Metal removal from top layer of soil was more substantial for Zn with the decline of 0.4–0.7 mg/kg-1 per year but very less for Cd and Pb [37]. It was concluded that the leaning of As deliberation in the four parts of maize plants was as leaves > stems > bracts > kernels and in these four tissues eleven QTLs were detected for As deliberation [38]. The second plant under study was P. hysterophorus which is a robust tolerant specie that is capable of growing in extreme conditions over large areas and can produce a heavy biomass [34]. P. hysterophorus are more applicable in contaminated soils for phytoremediation as it shows high tolerance and accumulation for different heavy metals and are capable of wide distribution and adaptability [34]. Due to fast growth, stress tolerance and inedible nature which inhibits the entry of heavy metals in food chain, P. hyterophorus is considered as a good phytoremediator. It is stated by Baker and Brooks that metal hyper-accumulator are the plants that contains Cu, Cd, Cr, Pb, Ni, Co \(\ge\)0.1% i.e more than 1000mg/g or Mn or Zn 1% (> 10,000 mg/g) by dry matter and it is > 1% in dry weight for Cd and infrequent metals [39].
Hyper-accumulators have been detected for their altered genes (selected) expression after getting polluted by increased concentration of heavy metals with least harmful symptoms in plant morphology and physiology [18]. Identification and characterization of genes involved in heavy metal stress tolerance may enable us to understand the molecular adaptation of plants. In the present study the expression of expansins, Proteases and protease inhibitors plays a key role in response to heavy metals stress expansins, cell wall proteins play a significant role in extensibility of cell wall. In maize, β-expansins has been detected for its wall relaxing activity. In the current research increased expression of β-expansins were observed in maize and parthenium plants that were irrigated by wastewater i.e 186.79 ng in maize plant (MT1) collected in December and 298.15 ng in (MT2) maize plant collected in January as compared to control plants irrigated by the simple tap water i.e 135.34 ng in maize control 1 (M1), 141.96 ng in maize control 2 M2. Abiotic stress may cause the inhibition of cell expansion e.g. at low water potential increased activity of expansin and cell wall extension properties were reported in maize [40].
Whenever plants are facing abiotic stress like salinity, drought, heavy metal toxicity etc., chloroplast produces high amount of reactive oxygen species (ROS) that often attacked the proteins, causes changes in protein structure and ultimately jeopardize their functions [41]. Plants encounter oxidative stress by utilizing its antioxidant defense machinery which in turn enables them to keep cellular homeostasis for proper functioning. Plant proteolytic enzymes degraded frequently misfolded and aggregated proteins under abiotic stress. Metacaspases are the proteases that are considered as an important regulators of Programmed cell death (PCD) when plants are exposed to some sort of stress [42]. In our study, the expression of metacaspase showed a tremendous change in maize and Parthenium plants irrigated by wastewater i.e 210.4348 ng in maize plant (MT1) collected in December and 242.20 ng in (MT2) maize plant collected in January than that of a control i.e 66.28 ng in maize control 1 (M1), 76.22 ng in maize control 2 (M2).
In plant systems protease inhibitors may cease the active cellular metabolism in PCD initiated by H2O2 (Solomon et al., 1999). Cystatins (cysteine protease inhibitor) is one of the defense program of plant that plays a central role in modulating stress (salt, drought, cold and oxidation) and PCD responses [43]. In Z. mays and P. hysterophorus, the cystatins genes were expressed more in plants watered by wastewater 128.25 ng in maize plant collected in December (MT1) and 135.37 ng in maize plant collected in January (MT2) than those irrigated by simple tap water i.e 57.85 ng in maize control 1 (M1) and 76.22 ng in maize control 2 (M2) showed that these genes were part of the defense machinery. In stress situation, when protease inhibitors might not be overexpressed, massive amount of proteases might be produced that engulf the useful proteins. Cystatins plays an important role in such stress condition to break the undesirable proteins using protease and then rearranging the amino acids to form the required protein.
Decline in biomass of plants above ground parts should not occur and should have high tolerance to heavy metal toxicity according to the characteristics of hyper-accumulating plants. All these typical features of hyper-accumulators have been exhibited by P. hysterophorus. Additionally, it has been previously reported that P. hysterophorus was used to investigate the extent and effect of automobile related Pb+ 2 & Cd2+ metal pollution in soil [44]. In industrially polluted soil, Parthenium being inedible weed showed a great potential for phytoremediation [45, 46]. P. hysterophorus was also successfully used to eliminate methylene blue dye from an aqueous solution [47].
Identification and characterization of genes involved in heavy metal stress tolerance may enable understanding the molecular adaptation of plants to stresses [48]. In the present study, increased expression of β-expansins was observed in maize and Parthenium plants that were irrigated with wastewater as compared to control plants irrigated with tap water. This might be attributed to the fact that abiotic stress may cause the inhibition of cell expansion e.g. at low water potential, increased activity of expansin and cell wall extension properties were reported in maize [49]. Additionally, the expression of metacaspase also exhibited a tremendous change in maize and Parthenium plants irrigated with wastewater in the current study. Metacaspases importantly regulate programmed cell death (PCD) when plants are exposed to environmental stresses [50].