The soil obtained from the experimental plots had a lower stickiness and exhibited a dark gray color. The absorption, accumulation, and distribution of metals in different parts of the plant are significantly influenced by both the plant itself and the characteristics of the soil. The pH level and organic matter content of the soil play crucial roles in regulating the availability of metals to plant species. When the pH level and organic matter increase, the movement of heavy metals decreases due to the precipitation of hydroxides and carbonates, along with the formation of insoluble organic complexes (Adamczyk-Szabela and Wolf, 2022). Conversely, metals exhibit higher mobility in soils with a pH lower than 7 and lower organic matter content. In the present study, the pH of the experimental soil was determined to be 6.6 ± 0.13, while the organic matter content was found to be 1.37 ± 0.03%. This finding aligns with the work of Srivastava et al. (2017) and Li et al. (2022), who also emphasized that the mobilization of heavy metals in soil is influenced by pH and organic matter. Furthermore, the concentration of heavy metals in the experimental soil was found to be within the normal range.
Plants, being inherently immobile, frequently encounter various forms of environmental stress, one of which is the presence of heavy metals. PTHMs like Cr, Pb, Cd do not serve any essential metabolic function in plants, and in fact, disturb various physiological and biochemical process, ultimately reducing crop yield (Sarma et al., 2023). To combat the toxic effects of different PTHMs on various plants, including rice, numerous substances such as silicon, GB, hydrogen sulfide, selenium, and melatonin have been extensively studied (Dotaniya et al., 2014; Kumar et al., 2019). In this study, we aimed to investigate the effects of exogenous GB, an organic osmolyte known for enhancing physiological and biochemical processes, on Indian rice plants cultivated under stress from a mixture of PTHMs.
4.1 Change in plant growth parameters
Roots serve as the primary entry point for PTHMs and are typically the first organ to experience the toxic effects of these substances. In the current investigation, a significant reduction in growth, both in terms of RL, SL, HI and over all biomass production (FW, DW) were observed in rice plants subjected to heavy metal stress. These findings are consistent with previous studies that have reported similar outcomes (AbdElgawad et al., 2020; Tang et al., 2023). However, numerous studies have demonstrated that the external application of GB can alleviate the impacts of PTHMs and enhance the morphological characteristics of cultivated plants (Kumar et al., 2019). The present study further confirms that the application of exogenous GB effectively reduces the detrimental effects of a combination of PTHMs on the RL, SL, FW, DW and HI. GB has been known to enhance plant growth even in the presence of heavy metal stress. This could be attributed to the improved development of nutrient absorption and gas exchange qualities in plants when GB is applied. Scientists have reported similar findings in rice plants experiencing abiotic drought stress (Chaum and Kirdmanee, 2010). Additionally, GB may have a protective effect on key enzymes involved in CO2 fixation, such as RuBisCo and RuBisCo activase, when plants are exposed to abiotic stress conditions. Consequently, this protection leads to an overall enhancement in plant growth. The reduction in stress caused by different heavy metals following the application of GB has also been documented in wheat, mungbean, and chickpea crops (Ali et al., 2015; Chen and Murata. 2011; Singh et al., 2022).
4.2 The impact of GB treatment on proline, oxidative stress, and electrolyte leakage (EL) in plants under PTHMs stress
Exposure to PTHMs in the surroundings has a direct correlation with an increased likelihood of experiencing oxidative stress. In order to assess this relationship, we measured stress biomarkers, specifically proline level, malondialdehyde (MDA) and electrolyte leakage (EL%). An evident rise in the accumulation of proline was noticed primarily in the leaves of rice plants when subjected to PTHMs stress as compare to the control group (TC). Moreover, the application of GB resulted in a moderate increase in proline accumulation in rice plants, when compared to the treatment involving only PTHMs. Proline is a basic amino acid found in proteins, and free proline plays a crucial role in plants during biotic and abiotic stress conditions. The molecular mechanism underlying increase proline level under heavy metal stress are yet to be identified, but one hypothesis suggests that protein is broken down into amino acid and converted into proline for storage. Singh et al. (2022) reported that soil heavy metal increased the proline content of chick pea plant, and high heavy metal (Cr) with GB application resulted in a marked increases in proline content.
PTHMs stress significantly enhances the levels of MDA and EL (%) in rice plants compared to the control group. However, when the PTHMs combined with GB treatment was applied, the levels of MDA and EL (%) in rice plants showed a decrease compared to the PTHMs-treated plants without GB application. The elevated MDA and EL percentage suggest that there is an excess generation of intracellular free radicals, which leads to membrane damage and cytotoxicity through the production of malondialdehyde, a byproduct of lipid peroxidation. Similar findings were observed by Singh et al. (2015), where they reported an increase in MDA levels in various parts of plants under mixture of heavy metal stress. Additionally, Bhargava et al. (2008) conducted a study on the Indian mustard plant (Brassica nigra L.), which also concluded that exposure to a mixture of heavy metals resulted in elevated levels of MDA in the plant's leaves.
4.3 Variation in pigments under PTHMs with or without GB
PTHMs toxicity has a significant negative impact on the pigment system of plants. This toxicity disrupts the photosynthetic system by causing heavy metals to enter the leaf tissue, potentially damaging the chloroplast's laminar membrane (Wang et al., 2009). The present study demonstrates that a high concentration of PTHMs in plant tissues leads to a decrease in chlorophyll content compare to control, which ultimately hinders plant growth and affects biochemical traits. Under the PTHMs exposure, rice plants exhibited a noticeable reduction in levels of Chlorophyll a, b, and carotenoid. Moreover, when PTHMs in elevated levels, metals exhibit a detrimental effect on chlorophyll production. Take the PTHMs Cd and Cr, for instance, which hampers the creation of the photoactive protochlorophyll reductase enzyme complex and disrupts the synthesis of aminolevulinic acid, both crucial steps in chlorophyll biosynthesis. Heavy metals like Cd, Cr, Cu accomplishes this by obstructing the sulfhydryl group of the enzyme, which is essential for its function, through the formation of a complex with active thiol groups (Hossain et al., 2012). The application of GB promotes shoot elongation by augmenting cell expansion and division. This mechanism may explain GB's positive impact on growth performance when plants are exposed to heavy metal stress. A comparable effect has been previously observed in sweet pepper and chick pea plants (Wang et al., 2016; Singh et al., 2022). Furthermore, the enhanced accumulation of antioxidant enzymes and pigment levels may have contributed to the improved growth performance in rice plants when treated with a combination of heavy metals and GB, compared to treatment with PTHMs alone.
4.4 Distribution of Antioxidant enzyme activity
Plant body is equipped with a range of scavenging machinery. Superoxide Dismutase (SOD), APX and Catalase (CAT) are considered primary antioxidants which are involved in direct scavenging of ROS. SOD catalyses the decomposition of superoxide as well as play important role in detoxification of anion to radical oxygen, hydrogen peroxide and finally convert to oxygen and water. SOD usually depends on different metals as cofactor. Further, CAT act as a primary indicator for removal of hydrogen peroxide under metal stress in peroxisomes. On other hand APX also involves in detoxifying H2O2 to H20 in plant cell via bate-glutathione cycle. The results of the present investigation revealed an amelioration of mixed heavy metals toxicity in rice plant exposed to GB. This amelioration might be resulted due to reduction in heavy metal uptake on GB application. It means there was no disturbance to stress machinery of the plants which turn maintains proper stomatal conductance, chloroplast ultrastructure, photosynthetic capacity and proper nutrient uptake. All these resulted in an increase the tolerance capacity of the plant. GB further increase the activity of antioxidant activity which in turns prevent plant to oxidative damage caused by free radicles generation due to stress condition that might also be the reason for enhancing amelioration process in rice plant. Kumar et al. (2021) demonstrated similar effect on GB application in sorghum plant under Cr stress, as were observed during the present investigation. However, the mechanism involved in the enhancement of amelioration behavior of the plant by GB application is still not clear.
In this study, SOD (superoxide dismutase) and CAT increased by 17.5–38.4% and 25.5–50% in Indian rice plants treated PTHMs in combination with Glycine Betain. Conversely, a small rise was observed in APX (ascorbate peroxidase) in all heavy metal treatments combined with GB. Our finding reveals that PTHMs in mixed condition increase the indices of oxidative stress parameters. However exogenous application of GB significantly enhances the antioxidant system which in turns reduced the indices of oxidative stress parameters under mixed heavy metal stress.
4.5 Uptake, Accumulation and distribution of PTHMs in different part of plant with or without GB
Heavy metal contamination in arable land is a pressing issue associated with industrialization globally. It has detrimental effects on plant growth and yield, leading to a significant concern for agriculture. Numerous studies have highlighted the hazardous impact of heavy metal toxicity on crop plants (Bharagava et al., 2008; Singh et al., 2015; Kumar et al., 2021; Singh et al., 2022). While there are reports suggesting the use of osmolytes such as GB to alleviate heavy metal toxicity, most of these studies focus on a single metal contamination scenario. Various crop plants have distinct mechanisms for accumulating and distributing metals in different parts of the plant such as the roots, shoots, fruits, or seeds. This study demonstrates that plants exposed to mixed concentrations of metals exhibit unique patterns of metal accumulation. However, uptake and distribution of metals in plants are influenced by factors such as the availability of metals, plant metabolism, and microbial interactions.
The research findings indicate that the Indian rice plant has the highest accumulation of Fe, followed by Mn, Zn, Cr, Ni, Pb, Cd, and Cu in its roots. When subjected to PTHMs stress, the uptake of metals was significantly higher in the roots compared to the shoots and seeds, and this uptake increased with higher concentrations of PTHMs. The high accumulation of heavy metals in the roots may be attributed to the higher metabolic rate in this plant part, as suggested by Haddad et al. (2023). The levels of PTHMs in all plant parts increased as the PTHMs stress intensified, surpassing the permissible limits set by FAO/WHO (1984) for Cr, Pb, Ni, Cu, Cd, and Mn in terms of human consumption suitability (permissible limit, PL: mg/kg) Cr: 0.02; Pb: 0.43; Ni: 1.63; Cu: 3.0; Cd: 0.21; Mn: 2.0). However, the levels of Zn (PL: 27.4) and Fe (20.0 mg/kg) in the seeds of the rice plant were below the permissible limits. Hence, it may be advisable that this Indian rice plant treated with high concentration of heavy metals should not be taken as food by human beings and cattle because these metal rich plants may cause several clinical problems (Rattan et al., 2005).
Nonetheless, the application of GB externally led to a decrease in the levels of 8 heavy metals across different parts of the plants. The most substantial decrease in chromium (Cr) and lead (Pb) concentrations was noted in the roots, stems, and seeds of the treated plants with 400 mM GB. PTHMs like Cr, Pb, Cu are known to impede plant metabolism and growth factors, cause changes in chloroplasts and cell membranes, reduce photosynthetic pigments, induce chlorosis, disrupt the movement of water and minerals, and hinder enzymatic activity in plants (Haddad et al., 2023). Under T4 PTHMs stress, the Cr and Pb content in the roots, shoots, and seeds was decreased by as much as 33.3%, 36%, and 89.3% respectively, and 36.4%, 55%, and 80.5% respectively when treated with 400 mM GB (Fig. 4). The decrease in the concentration of heavy metals in plant samples could be attributed to the presence of GB, which helps in maintaining the integrity of cell membranes and protects cells from damage. This protective mechanism reduces the likelihood of heavy metals entering the cells. Additionally, the application of GB may also shield the cell membranes, thereby preventing the movement of heavy metals into the cells. Similar findings have been observed for Pb and Cd levels in mung beans, rice, and wheat. Studies by Kumar et al. (2021) and Karagiannidis and Hadjisavva (1998) have shown that the use of GB as an osmolyte, along with arbuscular mycorrhizal fungi (AMF) inoculation, can enhance nutrient uptake and inhibit the absorption of various heavy metals such as Cr, Mn, Fe, Co, Ni and Pb in beans and Durum wheat. It is suggested that the competition between essential nutrients and heavy metals for entry into the cells could be another reason for the reduced absorption of heavy metals with GB application.
Bioaccumulation and Translocation factor
The factor responsible for transporting metals within plants, known as the translocation factor (TF), plays a critical role in monitoring metal presence across different plant parts. Essentially, TF evaluates the movement of metals either from the roots to the shoots or from the shoots to the seeds in plants (Baker and Walker, 1990). Research has shown that TF values were consistently below 1 when metals were moved from roots to shoots and shoots to seeds across all tested metals. Notably, the TF values for root to shoot translocation increased significantly with higher levels of PTHMs, ranging from T1 to T4 concentrations. Intriguingly, essential metals such as Mn, Cu, Ni, Zn, and Fe exhibited higher TF values compared to toxic heavy metals like Cd, Cr, and Pb. This disparity may be attributed to the crucial role of these essential metals in protein and pigment synthesis within plants (Rotkittikhun et al., 2006). Generally, plants tend to avoid accumulating unnecessary heavy metals that do not contribute to their metabolic processes (Liu et al., 2007; Satpathy et al., 2014). TF values for metals like Cd, Ni, Pb, Cr, and Fe increased with PTHMs concentration, but were subsequently reduced following the application of a GB. On the whole, GB has been acknowledged as a beneficial natural compound that enhances plants' resilience against stress induced by heavy metals.