Cadmium and Lead Differentially Affect Growth and Metal Accumulation in Guar (Cyamopsis Tetragonoloba L.) Varieties

Phytoremediation is a strategy to employ plants to recover high quantities of metals in the soil into the harvestable parts, such as shoots and roots. High levels of Cd and lead (Pb) in the soil cause several stress symptoms in plants, including a decrease in growth, reduced root growth, and carbohydrate metabolism. In this study, Saravan and HGS-867 as local landrace and Indian guar variety were selected to investigate the effect of the application of Pb (0, 40, 150, and 200 mg/l) and the cadmium (0, 25, 50, and 100 mg/l) on phonological, yield parameters and phytoremediation assessment. The results showed that Pb translocation factor (TF) was signicant in Pb×Cd and Pb×Cd×G (genotype) treatments at 1% and in Pb×G at 5%. Pb bioconcentration factor (BCF) was signicant in all treatments except Cd and Cd×G at 1%. Mean comparison of the data showed that the number of owers, leaves, and clusters in plant decreased signicantly with increasing Pb content. With increasing Cd concentration, the number of branches, height, the number of seeds, clusters, and leaves for each plant decreased signicantly at the level of 1%. The maximum TF was observed in Pb at 40 mg/l in the HG-867 variety. Also, the Saravan landrace in Cd (100 mg/l) showed the highest amount of BCF (Cd). With increasing the concentrations of Pb and Cd, the gum percentage decreased signicantly. The results of Pearson's correlation analysis indicated that plant height, number of pods/plant, root length, biomass, and pod length had a positive correlation with seed yield and a negative correlation with TF (Pb) and BCF (Pb). The results suggest that according to TF, BCF, and BAC, C. tetragonoloba L. can be effectively used as a good accumulator. The TF Cd Pb plant in the BCF the ratio of Cd Pb in the root to its level in the soil. The bioaccumulation coecient (BAC) was dened as the Pb and Cd ratio in the shoot to that in soil.


Introduction
Heavy metals, like Cadmium (Cd) and Lead (Pb) are particularly toxic to the majority of plants and animals even at low concentrations (Shukla et al., 2007). Heavy metals at their toxic concentration inhibit various stages of plant development, especially seed formation and physiological and biochemical processes. Pb is the second most dangerous substance, after arsenic, considering the occurrence frequency, toxic effects, and the potential for human exposure. Its transfer from polluted soils to plants has been extremely evaluated, particularly regarding food quality and use in phytoremediation (Uzu et al. 2009). Contamination of the soil with toxic trace metals as well as their accumulation in soil affect agricultural production because of the adverse impacts on crop growth; for example, metal phytotoxicity and soil microorganisms (Nagajyoti et al., 2010). Pb may exit in in soil as a free metal ion and as complexes with inorganic constituents (like CO 3 2− , HCO 3 − , SO 4 2− , and Cl − ), can exist as organic ligands (such as amino acids and humic acids), or can be adsorbed on particle surfaces (like biological material, peroxides, clay particles, and organic matter) (Uzu et al. 2009). Phytoremediation is a strategy to employ plants to recover high quantities of metals in the soil into the harvestable segments, such as roots and shoots (Mahmood, 2010). Harvesting of the biomass can be safely done using composting, drying, ashing, and stra ng at a land ll, pure plant oil manufacturing, anaerobic digestion, microbial, physical, or other chemical means (Ginneken et al., 2007).
High levels of Cd in the soil can elicit different stress symptoms in plants, like a decrease in growth, particularly root growth, impairments in mineral nutrition, and carbohydrate metabolism (Moya et al. 2004), leading to a reduction in biomass production.
In some earlier reports by Stoyanova and Tchakalova (1999) Cd at toxic levels disturbed the chloroplast envelope as well as the integrity of the membrane system resulting in an increase in plastoglobule number, alteration in the lipid composition, and the levels of the major structural constituents of thylakoid membranes.
Various plant species belonging to the Fabaceae family show phytoremediation potential (Ginneken & Gawronski, 2007;Anjum et al., 2014). Legumes are rich in phytoremediation and provide additional N-compounds to the soil leading to an improvement in soil fertility and capacity to support biological development (Hao et al., 2014). Cyamopsis tetragonoloba L. Guar (Cyamopsis tetragonoloba L.) is a low-input and annual fast-growing legume species that is almost cultivated in arid and semi-arid areas (Meftahizade et al., 2019). Guar has been recently widely considered because of the general tendency toward the application of polysaccharides from plants in different industrial applications. Guar is a multipurpose crop with economic values cultured for forage, vegetable (green pods), and seed gum that is widely used in various industries, such as food processing (as a novel natural additive), paper manufacturing (as a ber de occulants and beater additive), textile and carpet printing, mining explosive, drilling, pharmaceutical, cosmetics, agrochemistry, and oil and gas

Estimation of photosynthetic pigments
The chlorophyll was extracted based on the method reported by Arnon (1949). In brief, fresh leaves (1g) were ground with 80% acetone (20-40 mL) and the mixture was subjected to centrifugation (10000 rpm / 5 min). The solution absorbance was read at 645 nm and 663 nm against the blank (acetone). Total For determining gum%, the guar our dry weight achieved following the milling process was determined at 90°C.

Phonological parameters
Phonological parameters, like days to 50% owering, days to 50% podding, and time to start pod harvesting were estimated for two landraces and variety (Patil, 2014).

Yield and yield components
Pod length, the number of pods for each plant and each cluster, the number of seeds for each pod and each plant, seed yield, and weight of 100 seeds were determined at harvesting time.

Evaluation of phytoremediation e ciency (Cd, Pb, BCF, BAC, and TF)
That plants were oven-dried at 80°C for obtaining a constant weight and their dry weights were noted. To determine Cd and Pb accumulation in different plant tissues (roots and shoots), harvested plant parts were rinsed completely using tap water, and deionized (DI) water for cleaning the adhered components of soil followed by oven-drying at 80°C until reaching a steady weight. Pb concentration after extraction of samples with 2N hydrochloric acid was read using atomic absorption spectrometry. To measure the concentration of Cd in the shoots and roots of the plant, a sample of the dried plant weighing 2 g was poured into a porcelain crucible and placed in an oven at 30°C. The oven temperature was gradually raised to 550°C within two hours. After holding for 4 to 12 hours at 550°C, the oven was turned off and the crucibles were removed from the oven. After cooling, the ash was soaked in a little water and covered with a watch glass, and at the same time, 10 ml of 2 M hydrochloric acid was slowly added. The cruises were then placed in a water bath to release the rst white vapors at 80°C. The contents of the crucible were ltered through Whatman No.1 lter paper into a 100 ml jogger balloon, and after rinsing the crucible, the contents of the jogger balloon were measured. The extract was used to read the concentration of Cd by atomic absorption spectrometer. For quantifying the Pb content in the soil, a specimen of 1 g soil was digested using a wet acid digestion approach through HNO3 and HCl (3:1) followed by heating on a hot plate for 2 h until observing a clear solution. Following cooling, the volume was completed to 50 mL through the addition of distilled water. The solution was ltered through Whatman's lter paper and Pb contents were examined with Atomic Absorption Spectrophotometer.
To evaluate the uptake of Cd and Pb in cultivated plants, indices, such as TF and bioconcentration factor (BCF) were estimated (Monni et al., 2000). The TF was determined as a ratio of Cd and Pb concentration in plant shoot to that in plant root and the BCF was obtained from the ratio of Cd and Pb concentration in the root to its level in the soil. The bioaccumulation coe cient (BAC) was de ned as the Pb and Cd ratio in the shoot to that in soil.

Data analysis
The normality of data was assessed by Skewness and Kurtosis test prior to analysis of variance (ANOVA). Differences between treatments were determined using a two-way analysis of variance (ANOVA) by SAS software. The mean analysis was done by Duncan's Multiple Range Test (DMRT). The signi cant differences were calculated at p < 0.01. The principal component analysis (PCA) was applied for assessing possible relationships between the treatments by SPSS 10 software. Cluster analysis (UPGMA) using Euclidean distance and scatter plot diagram was done by the PAST software.

Results And Discussion
According to the ANOVA, plant height was affected by all treatments except Cd and Pb interaction at a signi cance level of 1%. The number of branches per plant was not signi cant by genotype. But there was a signi cant difference using other treatments at the level of 1%. The number of owers and leaves was signi cant by all treatments, except G and the interaction of G (genotype) in Cd at the level of 1%. The number of clusters per plant was signi cant in all treatments, but no signi cant difference was found between the two genotypes at the level of 1%. Chlorophyll content was not signi cantly affected by Cd and number of pods/plant and seed yield were also signed by the Pb, G, Pb in G, Pb in Cd, and Pb in Cd in G at the level of 1%. Also, 50% of owering was not affected by any of the treatments. However, 50% of pods showed a signi cant difference by all treatments except genotype at the level of 1%. The weight of 100 seeds using Pb, Cd, and Pb ×Cd was signi cantly different at the level of 1% ( The gum percentage was also affected by the concentration of Pb. In Pb (100 mg/l) treatment, the gum was 32.2% (Table 2). A reduction in gum in the treatment of high concentration of Pb may be due to the release of polysaccharides in the pericellular space that largely determines the course of allelopathic processes of sorption, desorption, ion exchange, and cell protection from extreme in uences (Lombardi and Vieira, 1999).
The total level of metals accumulated in the shoots is regarded as the most important factor for evaluating the potential of phytoextraction in plants (Zaier et al., 2010). The highest level of BAC (Pb) was observed in Pb (25 mg/l). BCF (Pb) increased with increasing the concentration of Pb. In Pb (100 mg/l) treatment, the BAC (Pb) was 4.11. The mobility, as well as availability of heavy metals in the soil, are commonly low, particularly when the soil is high in pH, clay and organic matter (Rosselli et al., 2003). Thus, it seems that at the low concentration of these heavy metals, their concentration in soil is so high. The highest Pb TF from root to shoot was 2.9, 3.7, and 3.54, respectively. This result emphasis that guar can accumulate Pb in root and can be a good source of Pb phytoremediation. It seems that Pb accumulation will occur in the apoplast cell of the root. Kopittke et al. (2007) reported that Pb reduces the fresh weight of buds in Vigna unguiculata. Also, their examinations using TEM microscopy showed that most of the root Pb accumulates in the apoplast of the outer cell layers of the root. Smaller amounts of Pb were also observed in the central cylinder apoplast. It was also observed that the amount of Pb accumulated in the roots is 10-50 times higher than in the buds (Kopittke et al., 2007).
Mean comparison of different levels of Cd in the studied traits showed that with increasing Cd concentration, height, the number of branches, clusters, seeds, and leaves per plant decreased signi cantly at the level of 1%. Also, the increase in Cd levels has caused the number of days to 50% of pods to be increased. However, the maximum days to 50% pods maturity was observed in the Cd (40 mg/l) (51.21 days) ( Table 3). Root depth and gum percentage also showed a signi cant decrease with increasing Cd concentration at the level of 1%. Although in Cd (40 and 150 mg/l) treatment, the gum percentage compared with the control showed a signi cant increase, at 250 mg/l, this rate decreased (31.19%). Bioaccumulation coe cient (Cd) increased signi cantly with increasing Cd levels, at 200 mg/l Cd and the BAC (Cd) was 3.97 (Table 3) The mean comparison on the studied variety and landrace showed that HG-867 showed superiority to Saravan landrace in terms of plant height, the number of owers, seed/ plant, root depth, and BCF (Cd and Pb). However, gum percentage and biomass in the Saravan landrace were higher than the other variety (36.6% gum vs. 33.6%) ( Table 4). The current results showed that HG-867 has a stronger accumulator rather than Saravan in the case of Pb and Cd accumulation in roots.

Interaction of Pb, Cd, and genotypes
The maximum TF (Pb) was observed in Pb (40 mg/l) in the HG-867 variety (Fig 4A). Also, the Saravan landrace in Cd (100 mg/l) treatment showed the highest amount of BCF (Cd) (Fig 4B). The HG-867 variety had the highest amount of BAC (Pb) compared with the Saravan landrace in Pb (40 mg/l) treatment (Fig 4C).
With increasing the levels of Cd, its BAC (Pb) did not increase (Fig 4C). Regarding BCF (Pb), the Saravan landrace in Pb treatment (200 mg/l) showed the highest BCF (Pb) (Fig 4D). Ouzounidou et al. (1995) declared that the uptake of heavy metals can cause chromosomal aberration and division of cells to become unusual that signi cantly reduced plant growth.
The interaction of Pb in Cd in terms of TF (Pb) showed that Cd (100 mg/l) and Pb (200 mg /l) had the highest TF (Fig 5A). Regarding the BCF (Pb), the results indicated that Cd (0) and Pb (200 mg /l), as well as Cd (25 mg/l) and Pb (200 mg/l), had the highest BCF (Pb) (Fig 5B). Also, as can be seen in Fig 5C, the highest amount of TF (Cd) was observed in Cd (50 mg/l) and Pb (40 mg/l) treatments (Fig 5C). in addition, with increasing Cd concentration, the BCF (Cd) increased. The interaction of Cd (100 mg/l) and Pb (0, 40 mg/l) showed the highest amount of BCF (Cd) (Fig 5D).
Regarding the effect of interaction treatments on root length, the results showed that interaction of Cd (25 mg/l) and Pb (control), Cd (50 mg/l), Pb (control), and Pb (150 mg/l) caused the highest root depth (27,26, and 25 cm, respectively) ( Fig 6A). The interactions of Pb and Cd regarding gum percentage showed that treatment with Cd (25 mg/l) and Pb (control) (40 and 150 mg/l) showed the highest gum percentage (38, 39, and 40%). However, with increasing the concentrations of Pb and Cd, the gum percentage decreased signi cantly (Fig 6B). Regarding biomass trait, with increasing Cd and Pb, the amount of biomass decreased signi cantly (Fig 6C). Regarding the interaction effect of Cd and Pb on seed yield per plant, in control treatments, Cd (25 mg/l) and Pb (150 mg/l) and Cd (100 mg/l) and Pb (150 mg/l) were found with the highest amount of seed per plant. By further increasing the concentration of Pb and Cd, the amount of seed yield in the plant decreased signi cantly (Fig 6D). Fouda and Arafa (2002) assessed soybeans and reported that treatment with high concentrations of Cd reduced plant height, number of leaves, and leaf area, while Pb also caused water stress, thereby reducing leaf area, photosynthesis, plant dry weight, and plant height and it was also effective in reducing the number of nodes.
As can be seen in Fig 3, the maximum weight of 100 seeds was observed in Pb (50 mg/l) and Cd (150 mg/l) treatments, while the number of branches was more in Pb (0) ×Cd (150 mg/l), Pb (0) ×Cd (25mg/l), and control treatments.
Chlorophyll content decreased by the interaction of Cd × Pb, especially at high concentrations. It seems that a reduction in protochlorophyllide reductase enzyme activity because of heavy metals toxicity is the main cause for less production of total chlorophyll. Cd can inhibit the photoactivation of photosystem-II, which is the main reason for less pigment generation in plants while growing in Cd contaminated soils (De Filippis and Pallaghy, 1994).
A TF greater than 1 indicates that the accumulation in shoots is more than roots and soil, respectively (Marrugo-Negrete et al., 2016). In the current research, TF in Cd×Pb treatment was more than 1, which means that guar is a good accumulator of Cd and Pb in the shoot. This phenomenon occurs at a high concentration of the used heavy metals. Table 5 lists the results of Pearson's correlation analysis. As shown in this table, plant height, number of pods/plant, root length, biomass, and pod length showed a positive correlation with seed yield, while TF (Pb) and BCF (Pb) were negative. A gum percentage was another main factor in guar. In the current research, plant height, weight of 100 seeds and pod number showed a positive correlation with gum percentage, while chlorophyll content was found with a negative correlation. there was a negative relationship between the effect of Cd and the speci c surface area of the leaf. Cd reduces water absorption and transpiration, too (Vassilev et al. 1997). Decreased water uptake in Cd-treated plants can be well justi ed by reducing root growth.

Multivariate analyses
We conducted principal component analysis (PCA) for nding trends in the collected dataset and de ning the multivariate relationships between the studied parameters (Table 6). In Tables 6 and 7, the individual values, the percentage of variance, the cumulative percentage, as well as the loading of the components are also given. Only the rst four major components (PC1, PC2, PC3, and PC4) were found with values greater than one (6.59, 2.2, 1.95, and 1.38, respectively); thus, data can be divided into four variables comprising 73.21% of the overall variance.
These high values in uence the distinction between landraces, and the high integrity of the relationships observed. In Table 7, the equation of the rst four components is shown. The coe cients in this table are ordinary, and thus their numerical values re ect their weight when the corresponding component is formed. The highest coe cients were for the rst part, plant height, branch number per plant, number of ower per plant, and number of clusters per plant (Fig.  1).
Cluster analysis (UPGMA) was also conducted through the Euclidean distance coe cient and the average method of linkage based on all the tested traits ( Fig.  2) for approximating the relationship between the used treatments. The clusters were divided into two primary clusters (I and II) and two sub-clusters (IA, IB, and IIA, IIB) for Cd ×Pb treatment (Fig. 2). For example, Pb 3 Cd 1 and Pb 3 Cd 2 were most closely clustered (Fig. 2).

Conclusions
The BCF, BAC, and TF values are used for identifying the effectiveness of plants for phytoremediation (phytoextraction or phytostabilization) through explaining the accumulation features and translocation behaviors of metals in plants. Plants with BCF, BAC, and TF values > 1 can be promising phytoextractors, appropriate for phytoextraction, while those with BCF and TF < 1 are regarded potential. It can be concluded that guar, especially HG-867 variety at high doses of Pb and Cd showed values of more than 1, which indicates that it is effective for translocation and phytostabilization at high concentrations of Pb and Cd.        Dendrogram of cluster analysis for the interaction of Pb and Cd on investigated traits.

Figure 3
The comparison of the impact of interaction Pb in Cd on weight of 100 seeds and number of branches.