Genotypic screening of wheat and their physiological responses under lead toxicity

Lead (Pb) is the second most harmful heavy metal contaminant in the environment and toxic for plant growth and development. Therefore, the identification and selection of plant genotypes tolerant to Pb stress are of great significance. In this study, twenty-six wheat lines (Triticum aestivum) were screened for Pb tolerance based on their morpho-physiological variations at the seedling stage with a rapid hydroponic technique using lead nitrate (Pb(NO3)2) at two concentrations (500 ?M and 1 mM) along with control. Wheat genotypes showed distinct variations in plant height, plant biomass and chlorophyll concentration in response to different concentrations of Pb. Considering all parameters, Akbar was found most tolerant (T) with minimum RS (2.97) to Pb stress, followed by BARI Gom-31 (3.45), Barkat (3.54) and Sufi (3.65), while BARI Gom-26 (10.14) was most sensitive (S) followed by Khude Gom (9.69), BARI Gom-30 (8.79), LalGom (8.76) and BARI Gom-32 respectively. More scores were seen in the remaining genotypes and were graded as moderately tolerant/resistant (MT) to Pb stress. Results showed that the resistant line had less damage to root and shoot characteristics along with chlorophyll score, thereby providing a hint about the Pb tolerance capacity of wheat genotypes at the seedling stage. Furthermore, findings indicate that Pb susceptibility in wheat is predominantly associated with a decrease in the Pb components of the root and shoot. We suggest Akbar as an elite genotype to cultivate or use in downstream studies on the basis of our findings to ensure an improved crop production relative to other varieties evaluated. These findings provide the necessary background for Pb cleansing and Pb-free wheat development for environment and health safety.


Introduction
Wheat is the second most important crop after rice as it fulfils the protein and caloric requirement of the world's one-third population (1). According to FAO estimate, world would require around 840 million tonnes of wheat by 2050 from its current production level of 642 million tonnes. Given the growing demand of wheat for human consumption, it is estimated to grow at 1.6% per year by 2020 (2). This target will be achieved only if global wheat production is increased by 2.5% per annum (3). Though, various abiotic stresses are responsible for poor wheat growth (up to 50%), such as drought, saline, poor soil fertility and heavy metals (4,5). Under stress conditions, morphological and physiological characteristics are affected (6).
The term "heavy metals" refers to the group of metals and metalloids of relatively high atomic mass (>4.5 g/cm 3 ) that can cause toxicity problems (7). Pollution of air, soil and water resources with heavy metals is a global environmental issue (8,9) because their contamination is harmful to humans, wildlife and agriculture (10). In addition, rapid industrialization and urbanization have caused pollution of the environment by heavy metals and their rates of mobilization and transport in the environment have greatly accelerated since the 1940s (11,12).
Contrasted to other heavy metals, lead (Pb) is the second most harmful pollutant after arsenic and listed as "the chemical of great concern" according to the new European REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) regulations (13) and its concentration in agricultural soil has rapidly increased and persists longer due to various anthropogenic inputs (14). Lead is not a bioessential ingredient, but it is readily consumed and stored in plants and not only effects development and production, it also reaches the food chain, creating human and animal health hazards (15)(16)(17). Ultrastructures of organs, tissues and subcellular components such as chloroplast, mitochondria, nucleus, cell wall and plant cell membrane can also be impaired. In addition, this disruption may result in the loss of organelle function and may potentially affect the normal physiological functions of plants, including photosynthesis, respiration, protein synthesis and division of cells (18,19). Lead may appear in soil as a free metal ion, complexed with inorganic constituents (HCO3 -, CO3 2-, SO4 2-, and Cl -), or as organic ligands (amino acids, fulvic acids, and humic acids); lead may alternatively be adsorbed on the surfaces of particles (Fe-oxides, biological material, organic matter and clay particles) (20)(21)(22)(23).
Techniques for heavy metal restoration are classified as biological (biodegradation by living organisms), chemical (chelators, chemical immobilization, oxidation) and physical (electrokinetic remediation, incineration technologies, soil washing, stabilization/solidification, thermal desorption), which are costly, timeconsuming and environmentally hazardous (24). Thus, their removal/immobilization requires successful cleanup to mitigate or eradicate toxicity (25). Plants were suggested as a low-cost, sustainable and ecologically sound solution for the remediation of heavy metal-contaminated land (26), especially by phytoextraction (27). Different processes (physical, chemical and biological) are developed to reduce total Pb concentration and bioavailability to mitigate Pb accumulation in the food chain (28,29). Lead (Pb) uptake is usually limited to roots, with only slight translocation to the shoots (30)(31)(32). Plants also respond to harmful effects of lead in a variety of ways, such as selective metal uptake, metal binding to the root surface, cell wall binding and antioxidants induction: non-protein thiol (NP-SH), cysteine, glutathione, ascorbic acid, proline and antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR). These responses vary with plant species, metal content and exposure conditions (13).
Hydroponic methods are effective in the rapid screening for heavy metal tolerance and accumulation in plants and have been widely used in evaluating the phytoremediation potential (33,34). The relative success of the hydroponically analyzed species is thus broadly comparable to that studied in the field (35). Wheat plants were chosen for this study because of their agricultural importance as a source of food whereas, 26 wheat genotypes were studied for their Pb tolerance based on morphological parameters as well as photosynthetic pigments content under hydroponics conditions. Thus, the present study was aimed at screening different wheat genotypes tolerant to Pb stress. A further purpose of this study was to establish the hydroponic method for screening wheat plants under Pb stress. (2), Na2MoO4.2H2O (0.5), CuSO4.5H2O (0.5)at 25 for 7 ℃ days. Under 10 hr of light and 14hr of darkness (550-560 μmol s -1 per μA), the environment was strictly controlled. By using NaOH or HCl, the pH was set to 6. For Pb treatment, the culture solution in the hydroponic system was supplemented with two concentrations of Pb(NO3)2 (500 μM and 1 mM) for seven days (37). Unstressed control plants were grown simultaneously and harvested. The treatments were arranged with three replicates in a completely randomized design.

Measurement of morphological characters
Plants were carefully taken from the pot after 7 days of treatment and washed twice with purified water to extract excess nutrient and then dried easily with tissue papers and then recorded the different morphological parameters such as root length (RL) plant -1 , shoot height (SH) plant -1 , root dry weight (RDW) plant -1 and shoot dry weight (SDW) plant -1 . After incubation in an oven at 80 °C until constant weight was achieved, shot and root dry weights were determined (37).

Determination of chlorophyll score
A portable chlorophyll meter or SPAD meter (atLEAF CHL STD, Wilmington, Delaware, USA) was used to take chlorophyll contents or SPAD values from the uppermost fully expanded leaves on each plant at 7 th day of treatments. A total of three plants were measured in every plot and took average as the mean SPAD value of the leaf (38).

Statistical analysis
The experiment was conducted with three replicates in a completely randomized design. Variations within the Pb concentrations and among the genotypes were checked using the computer package IBM SPSS Statistics 20 and MSTAT-C2.10 software and graphs were done using GraphPad Prism 6 software, respectively. Finally, the mean of the different parameters was compared by Duncan's Multiple Range Test (DMRT). Statistical significance was identified at P≤0.05. Means ± standard deviations (SD) of three replicates for each treatment (Table 1 and Fig.1). For screening of Pb tolerant genotypes, a rank-sum (RS) was calculated by the following relationship (39): Rank sum (RS) = Rank mean (Ṝ) + Standard deviation of rank (SDR).

Results
The findings of the variance analysis for the characters: root length, shoot height, root dry weight, shoot dry weight, as well as chlorophyll content was presented in Table 1. The mean squares for the Pb treatment levels, genotypes and interaction between the treatments and genotypes (AxB) were highly significant (p<0.01) for all the characters suggesting the presence of considerable variations among the Pb treatments as well as genotypes.

Root length and shoot height
In Sonora-64, Sourav, LalGom, BARI Gom-26 and BARI Gom-27, root lengths were substantially reduced compared to controls for all Pb concentrations ( Table 2). In addition, when treated with plants with 500 μM Pb, Kheri, Kalyansona, Sonalika, Pavon-76, Gourab, Shatabdi, Bijoy, Prodip, BARI Gom-29, BARI Gom-31 and Balaka showed no substantial reduction in root lengths, but a major reduction in 1 mM Pb compared to controls. However, BARI Gom-32, BARI Gom-33, KhudeGom, BARI Gom-28 and BARI Gom-30 showed a significant reduction in root lengths started from 500 μM Pb in comparison with non-treated controls. Interestingly, no significant changes were found in the root lengths of Kanchan, Sufi, BARI Gom-25, Akbar and Barkat at all levels of Pb compared to control levels ( Table 2).

Root dry weight and shoot dry weight
Compared to control, root dry weights were greatly reduced in Sourav and BARI Gom-26 supplemented with both Pb concentrations (Table 4). Furthermore, when plants were treated with 500 μM Pb, Kheri, Sonora-64, Pavon-76, Gourab, Bijoy and BARI Gom-29 displayed no substantial reduction in root dry weights but a major reduction in 1 mM Pb compared to controls (Table 4). However, BARI Gom-32, BARI Gom-33, LalGom, Balaka, BARI Gom-28 and BARI Gom-30 showed a significant decrease in root dry weight started from 500 μM Pb in comparison with non-treated controls. Interestingly, no significant changes in root dry weights were observed in Kalyansona, Sonalika, Kanchan, Shatabdi, Sufi, Prodip, BARI Gom-25, BARI Gom-31, KhudeGom, Akbar, Barkat and BARI Gom-27 subjected to all levels of Pb compared with controls (Table 4).
Shoot dry weights were significantly decreased only in BARI Gom-26 supplemented with all concentrations of Pb compared with control (Table  5). However, when plants were treated with 500 μM Pb, Kheri, Kalyansona, BARI Gom-29 and KhudeGom displayed no substantial reduction in shoot dry weights but a major reduction in 1 mM Pb compared to the controls (Table 5). Further, BARI Gom-32, BARI Gom-33, Balaka and BARI Gom-30 showed a significant decrease in shoot dry weights concentration started from 500 μM Pb in comparison with non-treated controls.

Chlorophyll concentrations in leaves
Total chlorophyll concentrations were measured in the leaves of wheat genotypes cultivated in the absence and presence Pb (Table 6). When plants were treated with 500 μM Pb, Kheri, Sonora-64, BARI Gom-31, BARI Gom-32, KhudeGom and BARI Gom-30 displayed no substantial reduction in chlorophyll score but a major reduction in 1 mM Pb compared to  Table 6).

Ranking of the genotypes
In order to find out the performance of genotypes for Pb tolerance, mean rank, the standard deviation of ranks and rank-sum were calculated, ranked and Table 2. Measurement of root length (cm) in various genotypes of wheat cultivated without (control) and with varying levels of Pb (500 μM and 1 mM Pb(NO3)2). Values are the means ± SD of three replicates (n=3). In accordance with the DMRT, various superscripted letters (a-c) within the row suggest statistically significant variations between the treatments (P<0.05). Data were taken at 7 th day of treatments.  Table 7. Scoring and ranking were conducted on the basis of the overall genotype performances on the statistical relevance of Pb treatments and controls. The lowest ranked sum reveals the best performing and the greater ranked sum indicates sensitive genotypes in response to Pb stress.

Genotypes
In response to Pb stress, the lowest ranked sum displays the best performance and the higher ranked Table 4. Measurement of root dry weight (gm) in various genotypes of wheat cultivated without (control) and with varying levels of Pb (500 μM and 1 mM Pb(NO3)2). Values are the means ± SD of three replicates (n=3). In accordance with the DMRT, various superscripted letters (a-c) within the row suggest statistically significant variations between the treatments (P<0.05). Data were taken at 7 th day of treatments. ) and BARI Gom-32 were the most sensitive (S) respectively. More scores were seen in the remaining genotypes, which we rated as moderately Pb tolerant (MT).

Discussion
Lead tolerance was the primary criteria for assessing genotypes in our study. The metal resistance contrast between species within the same genus is a significant method in assessing whether a physiological parameter is correlated with the tolerance of metals (40). In addition, the performance of phytoextraction is directly associated to the plant's ability to withstand and absorb contaminants in its over-ground parts (18,27). In phytoremediation processes, suitable plant assortment is also an important factor (41). It was reported that hydroponic screening is a fast method of identifying of potential species of phytoremediation as an alternate option to expensive field studies (42). During our study, wheat plants display significant variation in growth characteristics, such as RL, SH, RDW, SDW and chlorophyll content under hydroponics conditions with applied Pb stress (Table  1-6).
Many research confirmed that the effect of the higher concentrations of phytotoxic metals on plant tissues was more hazardous than the effect of the lower concentrations by inhibiting the development of roots and aerial plant components (16,(43)(44). According to the studies reported (45, (46), low Pb concentrations can stimulate plant development, but plant growth can be prevented by concentrations above 0.5 μM. The harmful effect of Pb on plant growth is time and dosage dependent in most instances (47)(48)(49). Our findings similarly showed differential variations in Pb tolerance in responses to different doses of Pb in wheat. In this study, genotypes displayed reduced growth parameters by Pb concentrations (500 μM and 1 mM) compared to the control. Similar Pb toxicity symptoms were seen in earlier research (34,42,(50)(51)(52)(53)(54)(55)(56). The toxic effect of Pb may be caused by affecting a number of physiological and biochemical systems, including ion toxicity, enzyme activity, respiration and photosynthesis (57,58).
From our results, distinctly visible symptoms were observed in both root length and shoot height of wheat genotypes, but root growth was highly restricted by Pb (Fig. 1, Table 2), suggesting an increased number of secondary roots per unit root length, which were similar as of earlier findings (44). Roots are the first organ to come to Pb contact, providing the main path for metal ion penetration (37,59). However, most of the absorbed Pb (about 95% or greater) are stored or uptake is restricted to roots, only a few are passed to aerial plant component (30)(31)(32), which is a good indication for tolerance. Upon entering the root, lead passes predominantly through apoplast and bind the sources of water before it enters the endodermis (30,60). Balanced nutrients supply is vital for normal growth and development of the plant, while plants Table 6. Measurement of chlorophyll contents or SPAD values from the uppermost fully expanded leaves in different wheat genotypes grown without (control) and with different Pb concentrations (500 μM and 1 mM Pb(NO3)2). Values are the means ± SD of three replicates (n=3). In accordance with the DMRT, various superscripted letters (a-c) within the row suggest statistically significant variations between the treatments (P<0.05). Data were taken at 7 th day of treatments. For dry weight of plant parts, considerable reductions were observed under Pb treatment. Similar phenomena were also described in wheat and lentils (62), in Pisum sativum (63), in Plantago major (64), in Zea mays (65). Similarly, in tomato seedlings, fresh and dry biomass of roots, shoots and leaves were negatively affected by increasing Pb concentrations (66). These symptoms can be essentially attributed to a deficiency of macroelements.

Genotpes
The core components of photosynthesis and biomass production in plants are chloroplast pigments, Chlorophyll. In the wheat seedlings in our study, Chl contents were already significantly lowered at 500 μM Pb and this effect was even more pronounced at 1 mM Pb compared with the control (Table 6). Nevertheless, Pb-treated plants displayed decreased biosynthesis of Chl content and heightened deterioration of Chl due to increased chlorophyllase activity (67), the disturbance of the amount of chloroplastic stroma triggered by ROS (58,68) or a lack of availability of nutrients such as Mg and Fe (69). From the results in this study, high Chl score findings suggest a lower chlorophyll synthesis disruption and hence a higher lead resistance from this parameter. In addition, for screening several species, Chl has been used as a parameter in corn (70), in wheat (71), in sweet pepper (72) and in field peas (73).
Wheat genotypes exhibited significant variation in all growth parameters, indicating that these parameters could be used as a selection criterion for Pb tolerance in the hydroponic conditions. Noticeably, it is argumentative to recognize the Pb tolerant genotypes based on a single criterion. The results of our study are comforted with earlier works (74)(75)(76)(77). Also, similar ranking procedure was followed in screening rapeseed and mustard genotypes for salt tolerance (78), in Brassica varieties (79) for drought tolerance and in rice (80) for cadmium tolerance. Previously it was proved that the morphological score is well correlated to the physiological state of plants and used as rapid screening for stress tolerance (80). In all parameters, Akbar consistently displayed superior tolerance, followed by other genotypes. This genotype can be used in future breeding programmes to develop lead-tolerant wheat cultivars. We also suggest that farmers cultivate Akbar in Pb polluted soils in order to ensure improved crop yield relative to other varieties evaluated.

Conclusion
This study concluded that Pb stress may have severe effects on wheat yield and quality characters by changing its morpho-physiological traits and chlorophyll content and noteworthy genotypic differences were found. Among studied wheat genotypes Akbar proved tolerant to lead (Pb), Table 7. The Ranking of various genotypes of wheat owing to the Pb stress. Compared to controls based on morphological criteria, the numerical number shows the tolerance rating of genotypes complemented by all Pb concentrations (RL=Root length, SH=Shoot height, RDW=Root dry weight, SDW=Shoot dry weight and SPAD=Total chlorophyll). Here, RS= Rank sum; Ṝ= Rank mean; SDR=Standard deviation of rank. suggesting this genotype is prominent resource and could be used as genetic materials for the further breeding programme.