Multifaceted role of  osmoprotectant glycine betaine to ameliorate Cd and As toxicity by modulation of growth and biochemical indices in wheat plants

doi:10.21203/rs.3.rs-5340734/v1

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Multifaceted role of osmoprotectant glycine betaine to ameliorate Cd and As toxicity by modulation of growth and biochemical indices in wheat plants

https://doi.org/10.21203/rs.3.rs-5340734/v1

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Arsenic (As) and Cadmium (Cd) contamination is a pressing global issue spanning environmental, agricultural, and health domains, primarily due to their highly toxic and carcinogenic nature. Exposure of plants to Cd and As stress can induce numerous morphological, physiological, and biochemical changes. Glycine betaine (GB) plays a vital role in alleviating abiotic stresses by acting as a potent compatible solute. This study evaluated the effectiveness of GB (5 mM) treatment in wheat plants (HD2967 and HD3086) in mitigating the toxicity caused by Cd (1100 ppm) and As (225 ppm). Cd and As toxicity markedly suppressed growth parameters such as root-shoot length, plant biomass, and biochemical attributes (total protein and carbohydrate). It also decreases the germination rate and seed viability. Simultaneously, Cd and As stress conditions significantly elevated the concentrations of stress-specific parameters (phenol, MDA, and proline Content) in wheat plants. Conversely, the external application of GB alleviated the detrimental impacts of Cd and As stress by enhancing the growth characteristics, biomass, germination rate, and biochemical attributes. Additionally, GB treatment reduces the concentration of stress-specific parameters and suggests GB acts as a stress resilience. In conclusion, data revealed that applying GB can mitigate or lessen the toxic effects induced by Cd and As stress in both varieties, leading to positive modulation of plant growth performance and reduction of oxidative stress by reducing the concentration of the stress-specific biomarker. Thus, the current research suggests that GB application may benefit crops vulnerable to Cd and As toxicity by enhancing growth and biochemical attributes, ultimately resulting in yield.

Biochemical

Germination

Glycine betaine

Growth

Seed vigor index

Anthropogenic activities, such as urbanization, fast population expansion, industrial discharge, and agricultural fertilizer application, contribute to environmental pollution. Among these pollutants, heavy metals (HMs) significantly threaten plant growth, animal growth and development, and human health (Badawy et al. 2024; Sun et al. 2022). Heavy metals include chromium (Cr), cadmium (Cd), arsenic (As), cobalt (Co), iron (Fe), lead (Pb), zinc (Zn), mercury (Hg), nickel (Ni), and silver (Ag) (Badawy et al. 2024). The presence of certain HMs, such as Cu and Zn can enhance the efficiency of enzyme reactions. In contrast, others, such as As, Pb, Cd and Hg, can cause growth retardation and apoptosis (Ewais et al. 2015). Numerous activities emit HMs, such as mining metals, burning fossil fuels or coal, battery effluents, smelting ore, exhausting cars, using gasoline, paint, excessive pesticides, wastewater, and fertilizing agricultural lands give significant emitters of As and Cd (Agnihotri and Seth 2020; Sharma et al. 2023).

Arsenic (As) is a non-essential metalloid hazardous to all life forms, including plants and animals. It ranks as the 20th most abundant element and is widely distributed in the lithosphere (Chandrakar et al. 2016). It predominantly exists in the inorganic forms, i.e., As(III) and As(V) (Tripathi et al. 2007). Plants primarily uptake As in the form of As(V). As a result of arsenic contamination, plants suffer a variety of physiological, morphological, and biochemical disorders, such as diminished root elongation, proliferation, and nodulation, stunted growth, necrosis of leaf blades, and decreased leaf area, wilting, curling, which hinders the accumulation of biomass and photosynthesis. It also affects stomatal conductance, transpiration rate, ATP synthesis, mineral content losses, and energy flow alteration, ultimately leading to poor yield (Chandrakar et al. 2016; Pandey et al. 2017; Vezza et al. 2021).

Another phytotoxic metal, Cd, has no biological activity in plants or animals. This substance impedes plant development, decreases biomass yield, lowers photosynthesis efficiency, and interferes with membrane penetrability. Thus, it affects plant processes such as water absorption and nutrient distribution (Rizwan et al. 2012; Rehman et al. 2015; Badawy et al. 2024). Cd isn't a redox-active element; it triggers oxidative stress in plants by generating reactive oxygen species. This process causes physiological disturbances, ultimately resulting in decreased growth and biomass. Furthermore, it adversely affects enzymatic functions, disturbs the electron transport pathway, and could potentially trigger changes in DNA (Choppala et al. 2014). A recent study by Sun et al. (2022) revealed notable proline accumulation in the cucumber seedlings' roots, stems, and leaves exposed to Cd toxicity. Additionally, Zhang et al. (2020) reported that Cd supplementation in the soil reduced super black waxy maize's morphological growth indices and protein contents. Cd toxicity produces excessive reactive oxygen species (ROS), damaging plant membranes and degrading cell biomolecules and organelles (Abbas et al. 2018). Cd also diminishes plants' Fe and Zn uptake, leading to leaf chlorosis. It also disrupts the transport and absorption of P, Ca, K, Mg, and Mn (Xu et al. 2017; Nazar et al. 2012).

Heavy metals (HMs) are toxic in soil and plants, which can be mitigated through numerous treatment methods (Sharma et al. 2023). A promising approach involves employing compatible solutes, which can potentially reduce the detrimental effects of HMs on plants. Compatible solutes are harmless substances characterized by their small molecular size and excellent solubility (Sharma et al. 2024 a). They assist in shielding plants from stress through various mechanisms, such as scavenging reactive oxygen species (ROS), stabilizing proteins, controlling cell osmotic pressure, and maintaining membrane integrity (Badawy et al. 2024).

Glycine betaine (GB) is a widely used compatible solute to mitigate and adapt to challenging situations (Jain et al. 2021; Sharma et al. 2024 b). This compound, which is colorless, odorless, water-soluble, and metabolically stable, functions exceptionally well as an osmoprotectant (Mäkelä et al. 1998; Sharma et al 2024c). Under various types of abiotic stresses, the concentration of GB in plants markedly rises (Aamer et al. 2018). GB functions as a robust compatible solute, synthesized in chloroplasts via oxidation, thereby aiding in stabilizing the structure and ensuring the functionality of photosystem II (PSII) under diverse stressful environments (Jabeen et al. 2016). Multiple studies have highlighted GB's effectiveness in shielding plants from various stressors, including temperature fluctuations, drought, salinity, and metal toxicity (Zhang et al. 2022; Wang et al. 2019; Sofy et al. 2020; Zulfiqar et al. 2019). In addition to scavenging reactive species, GB boosts both enzymatic and non-enzymatic antioxidants, thus protecting cells against oxidative damage (Rasheed et al. 2014). Some plant species cannot produce GB under either normal or stress conditions. Consequently, the external application of GB has proven effective in mitigating the harmful effects of various abiotic stresses (Kumar 2021; Sharma et al. 2024 b). The efficacy of externally applied glycine betaine depends on multiple factors, such as the plant species, glycine betaine concentration, and the developmental stage of the plant at the time of application (Ashraf and foolad 2007).

Wheat is a vital cereal, serving as a staple food in diverse regions globally (Giraldo et al. 2019). However, the yield of wheat crops is susceptible to various stresses, including Cd and As stress (Rasheed et al. 2014; Saeed et al. 2021). Currently, scant literature explores the relationship between externally applied GB and Cd and As stress tolerance in wheat. Thus, our study aims to elucidate how GB might mitigate Cd and As toxicity in wheat plant by influencing various cellular processes.

2.1 Experimental details

The current experimental study was performed on two wheat varieties (HD2967 and HD3086) under Cd and As exposures and exogenous GB treatments in a completely randomized design in natural conditions. Seeds of wheat varieties were procured from CCS HAU, Hisar, Haryana, India. Seeds were planted in earthen pots (10 x 12 in) filled with 6 kg of sandy soil. Six seeds per pot have been planted, after which they were thinned down to three plants per pot to facilitate optimum growth. There were ten replicates for each treatment. The nutrient solution (Hogland and Arnon [1957]) was given weekly to nourish a healthy establishment. After two weeks of planting, treatments were given for Cd (1100 ppm of cadmium chloride ) and As (225 ppm of sodium arsenate). GB (5 mM) treatment was given as a soil solution twice (25^th and 50^th days of sowing).

The treaments include: T0: Control, T1: Glycine betaine (GB), T2: Cd stress, T3: Cd + GB, T4: As Stress, T5: As + GB.

2.2 Germination setup and assay

Germination percentages (%) was assessed in growth chamber conditions. Petri plates containing one layer of filter paper were used to germinate the seeds. The seeds underwent disinfection for fungicide resistance using a 0.5% sodium hypochlorite solution before planting, followed by three rinses with dH₂O. dH₂O (10 mL) was used as the control treatment. Additionally, 10 mL solutions of Cd (1100 ppm) and As (225 ppm,) along with GB (5mM), were applied, ensuring the filter paper remained moist until the end of the experiment. Each treatment consisted of three replicates containing five seeds subjected to a completely randomized design (CRD). The experiment was done in a growth chamber at 20°C with 50% relative humidity under 16-hour light and 8-hour dark photoperiods. Data was collected on the third and seventh day to assess germination rate and seed viability in terms of seed vigor index (SVI). For germinated seeds, the radicle length was measured at 2 mm (Alsaeedi et al. 2018). The percentage of seeds that germinated (%) and the Seed Vigor Index (SVI) were determined following the procedure outlined by Biju et al. (2017). In this instance, the following formulas were employed:

Germination percentage (%) = (n/N) × 100,

In this expression, 'n' signifies the count of germinated seeds, while 'N' represents the total quantity of seeds tested for germination.

SVI (%) = Germination (%) × means of RL.

Where RL is radicle length

2.3 Growth parameters

Three randomly selected plants from each variety per treatment were excavated to measure growth attributes. The following metrics were assessed: shoot-root length (in centimeters), fresh weight (FW) of the shoot, root, and leaves (in grams), and dry weight (DW) of the shoot, root, and leaves (in grams).

2.4 Biochemical parameters

To assess lipid peroxidation, we determined the malondialdehyde (MDA) concentration following the method outlined by Heath and Packer (1968). Leaf samples weighing 100 mg were crushed and blended with 2 mL of 0.1% TCA (trichloroacetic acid). The liquid above the sediment was collected after centrifuging at 10,000 rpm for 10 minutes. Subsequently, 1 mL of this liquid was combined with 4 mL of a solution containing 0.5% TBA (thiobarbituric acid) and 20% TCA. The mixture was then heated to 95°C for 30 minutes, cooled rapidly in an ice bath, and centrifuged once more at 10,000 rpm for 10 minutes. The optical density of the liquid was measured at 532 nm using a spectrophotometer. This value was subtracted from the non-specific absorption at 600nm. By using a 155mM^-1 cm^-1 extinction coefficient, MDA content was calculated.

To determine proline levels, we followed the procedure outlined by Bates et al. (1973). Fresh leaf samples weighing 100 mg were blended in 2 mL of aqueous sulphosalicylic acid (3%), and then centrifuged to collect the supernatants. Following this, 1 mL of glacial acetic acid and 1 mL of ninhydrin reagent were introduced to the supernatants. The mixture underwent a 10-minute heating in a water bath, followed by rapid cooling in an ice bath. Subsequently, 2 mL of toluene was added and vigorously agitated using a vortex shaker. The absorbance was determined using spectrophotometry at a wavelength of 520 nm.

Phenol levels were assessed employing the Folin-Ciocalteu reagent method (Aiyegoro & Okoh 2010). Fresh leaf samples (100 mg) were initially macerated in 2 ml of 80% methanol and centrifuged at 10,000 rpm for 15 minutes. The resultant extract was then combined with solutions of Folin-Ciocalteu reagent and Na₂CO₃, followed by measuring the absorbance at 650 nm using a spectrophotometer.

The method described by Yemm and Willis (1954) was employed to determine the total carbohydrate content. Initially, fresh leaves weighing 100 mg were crushed in 80% ethanol and then subjected to centrifugation. A 0.2 mL aliquot of the resultant extract was diluted with distilled water to achieve a final volume of 1 mL. Subsequently, 4 mL of anthrone reagent was introduced, and the mixture underwent heating in a water bath for 8-10 minutes, followed by rapid cooling in an ice bath. The absorbance was measured at 630 nm, with anthrone used as the blank solution.

The total protein concentration in leaves was assessed via the Lowry method (1951). Fresh leaf samples (100 mg) were initially immersed in distilled water and then centrifuged. A 0.2 mL portion of the resulting solution was diluted to 1 mL and combined with a mixture consisting of 2% Na₂CO₃ in 0.1 N NaOH and 0.5% CuSO₄ in 1% NaKTa at a ratio of 50:1. Following this, FCR was added, followed by vigorous agitation and incubation in darkness. The absorbance was subsequently determined at 660 nm using a spectrophotometer.

2.5 Data analysis

Results were presented as mean and standard error values (3 replicates for analysis). The statistical significance of the differences in treatments and varieties was determined by analysis of variance (2-way ANOVA) and Tukey's HSD posthoc test at 5% probability (p < 0.05). An Origin PRO® 2024 program was used to plot the graphs and correlation (Origin Lab Co., Northampton, USA).

3.1 Impact of GB treatment on germination percentage, seed vigor index (SVI), and radicle length under Cd and As stress conditions

The results demonstrate that Cd and As toxicity significantly decreased the germination rate (%),SVI, and radicle length (Fig. 1). Cd toxicity reduced the germination percentage on 3^rd day by 34% and 23% in HD3086 and HD2967 varieties. Similarly, As toxicity reduced the germination percentage (3^rd day) by 41% and 24% in both (HD3086 and HD2967) varieties. The same trend was observed for 7^th day of germination. It decreased by up to 26% under Cd and As toxicity in HD2967. Similarly, 23% and 25% decreases were noted for the HD3086 variety under stress conditions. GB treatment alleviates the negative impact of Cd and As toxicity and improves the germination (%) by up to 34% in both varieties under Cd and As stress conditions.

Similarly, radicle length and SVI decrease under stress conditions. In the HD2967 variety, radicle length decreased by 36% and 48% under Cd and As toxicity. Similarly, in the HD3086 variety, it decreased by 44% under Cd and As stress conditions. However, GB treatments improve the radicle in both varieties under Cd and As stress conditions. In the HD2967 variety, the radicle length was increased by 29% and 32% under Cd and As toxicity. In the HD3086 variety, a 33% increase was observed under Cd and As stress conditions. SVI decreased by up to 53% and 58% under Cd stress, and 61% and 57% decreases was observed under As stress in both wheat varieties. On the other hand, GB treatment increases the SVI by reducing the negative impact of Cd and As toxicity in both varieties by improving the germination rate and radicle length (Fig. 1D).

3.2 Effect of GB treatment on morphological attributes (root-shoot length) under Cd and As stress conditions

When comparing the root-shoot length of control plants with those treated with Cd ans As, significant reductions was observed in root and shoot length for both HD2967 and HD3086 varieties (Fig. 2). Cd treatment led to a decrease of 36% in shoot length, 42% in root length for the HD2967 variety, and a reduction of 27% in shoot length and 39% in root length for the HD3086 variety. Similarly, As treatment led to a decrease of 38 % in shoot length, 43% in root length for the HD2967 variety, and a reduction of 42 % in shoot length and 41% in root length for the HD3086 variety. However, GB treatment increased the shoot-root length of the HD2967 variety by 28%, 24%, 29%, and 35%, respectively, and of the HD3086 variety by 23%, 26%, and 32%, respectively, compared to Cd and As stress treatments alone (Fig. 2). Hence, GB treatment increases the growth indices (root-shoot length) in both varieties by reducing the negative impact of Cd and As stress.

3.3 Effect of GB treatment on fresh and dry weight under Cd and As stress conditions in wheat plant

Cd and As stress decreases the FW and DW of wheat plant in both varieties (Fig. 3, 4 and 5). As compared to control treatment the decrease in FW of root was 38%, and 39 % in HD2967 variety under Cd and As stress. Similiarly, in HD3086 variety, the decrease in root FWwas 41% (Cd stress) and 44% (As stress), respectively. Cd and As treated plants in combination with GB treatments exhibited 44% (Cd+GB) and 47% (As+GB) in HD2967 variety and 46% (Cd+GB) and 49% (As+GB) increase in FW of root as compared to the respective treatments without GB. Similar pattern was observed for DW of roots. In HD2967 variety, the decrease in DW of roots was 45% (Cd treatment) and 48 % (As treatment) and in HD3086 variety, the dcerease was 50% (Cd treatment) and 49% (As treatment). GB addition under Cd and As stress increases the DW of both varieties and improves the root growth.

As compared to control treatment the decrease in FW of shoot was 49%, and 50% in HD2967 variety under Cd and As stress, respectively. Similiarly, in HD3086 variety, the decrease in root FW was 49% (Cd stress) and 52% (As stress), respectively. Cd and As treated plants in combination with GB treatments exhibited 38% (Cd+GB) and 40% (As+GB) in HD2967 variety and 34% (Cd+GB) and 46% (As+GB) increase in FW of shoot as compared to the respective treatments without GB. A significant reduction in DW of shoot was observed under Cd and As stress conditions, respectively. Compared to Control plants, the decrease in DW of shoot was 40% (Cd stress) and 45% (As stress) in HD2967 variety. Similar pattern was observed for DW of shoot in HD3086 variety under stress treatments (Fig. 3 B). Plants with GB treatments increased the DW of shoot by up to 41% in both varieties under Cd and As toxicity.

A significant decrease in fresh and dry weight of leaves was observed in both varieties under Cd and As toxicity compared to the control treatment (Fig. 5). FW of leaves was reduced by 45% (Cd treatment) and 46% (As treatment) in HD2967 variety and 44% (Cd treatment) and 46% (As treatment in HD3086 variety, respectively. Similarly, the DW of leaves of both varieties decreased under Cd and As stress treatments. The decrease in DW of leaves was 35% and 37% in both (HD2967 and HD3086) varieties under Cd toxicity. Similarly, As toxicity decreases the DW by 32% and 38% in both varieties, respectively. The fresh and dry weight increased remarkedly in the leaves of both varieties by GB treatment in combination with Cd and As stress conditions. Compared with the Cd treatment, FW increased by 51% and 54% in both varieties by GB addition. Similarly, As treatment with GB addition increases the FW of leaves by 55% and 56% in both wheat varieties, respectively. GB treatment observed a similar pattern for DW of leaves compared to stress conditions alone (Fig. 5 B). In conclusion, the GB addition under stress increases the plant biomass and improves the growth characteristics of both varieties.

3.3 Effect of GB treatment on stress-specific parameters (total proline, total phenol, and MDA content) under Cd and As stress conditions

Cd and As toxicity caused a significant increase in stress-specific biomarkers (total proline, total phenol, and MDA content) in both varieties (Fig. 6). Data revealed that the total proline content of leaves increased under Cd and As stress. Meanwhile, GB treatment decreases the proline level in both varieties and alleviates the negative impact of stress conditions. GB treatment decreases the proline content by 17% (Cd+ GB) and 19% (As+GB) in the HD2967 variety and 23% (Cd+ GB) and 33% (As+GB) in the HD3086 variety, respectively.

The total phenol content of leaves also increased under stress conditions in both wheat varieties as compared to control plants (Fig. 6 A). The increase in total phenol content was 21% and 24% under Cd and As toxicity in the HD2967 variety. Similarly, in the HD3086 variety, phenol content rise by 23% and 27% under Cd and As toxicity. However, GB addition decreases the phenol content of both varieties as compared to Cd and As stress alone. GB addition decreases the total phenol content by up to 24% under Cd and As stress conditions in the HD2967 variety. Similarly, in the HD3086 variety, GB treatment decreases the phenol content by up to 22% under Cd+GB and As+GB treatment, respectively.

Cd exposure increases the MDA content (lipid peroxidation) in both varieties compared to the control plants (Fig. 6 C). Cd + GB treatment significantly decreased MDA content by 29% and 32% in both varieties, respectively, compared to Cd treatment alone. Similarly, data revealed that As toxicity also increases the MDA content in both wheat varieties. However, As+ GB treatment decreases the MDA content by 30% and 34% in both varieties. Hence, GB treatment mitigates the toxic impact of Cd and As stress by reducing the concentration of the stress-specific biomarker and providing a stress tolerance mechanism.

3.3 Effect of GB treatment on total carbohydrate and total protein content under Cd and As stress conditions

The result revealed that Cd and As exposure decreases the total carbohydrate and total protein in both varieties (Fig. 7). Total carbohydrate content was reduced by 23% (Cd treatment) and 28% (As treatment) in HD2967 variety and 39% (Cd treatment) and 38% (As treatment in HD3086 variety, respectively. Cd and As treated plants in combination with GB treatments exhibited 44% (Cd+GB) and 51% (As+GB) in HD2967 variety and 56% (Cd+GB) and 45% (As+GB) increase in total carbohydrate content as compared to the respective treatments without GB. Similarly, Cd treatment reduced the total protein content by 43% and 39% in both varieties (HD2967 and HD3086). A similar pattern was observed for As stress condition for total protein content. The decrease in total protein content was 40% (HD2967) and 46% (HD3086), respectively. Total protein content increased remarkedly in both varieties by GB treatment in combination with Cd and As stress conditions. Compared with the Cd treatment alone, protein content increased by 47% and 36% in both varieties by GB addition. Similiarly, As treatment with GB addition increases the protein content by 35% and 38% in both wheat varieties. Hence, GB addition under stress conditions alleviates the negative impact of Cd and As stress and increases the total cabohydrate and protein content in both varieties.

3.4 Pearson's Correlation Analysis between growth and biochemical attributes

Pearson's correlation analysis was performed to analyze the relationship between the two wheat varieties' diverse growth and biochemical attributes. In both varieties, all examined growth parameters exhibit positive correlations among themselves while displaying negative correlations with stress-specific parameters like phenol, proline, and MDA content at a statistically significant level (p < 0.05). However, parameters specific to stress (Proline, Phenol, and MDA content) display a positive correlation with each other and a negative correlation with carbohydrate and protein levels. On the contrary, protein and carbohydrate content positively interact with growth characteristics in both wheat varieties.

Several studies have explored the alarming issue of HM pollution, which adversely affects plant growth and development, contributing to worsened environmental degradation. Studies have revealed that increased levels of HMs hinder seed germination, impede plant growth, and disrupt various physiological and biochemical processes (Farooq et al. 2016; Bharwana et al. 2014). Elevated concentrations of HM ions can additionally hinder photosynthetic processes and the breakdown of protein synthesis. They disrupt the antioxidative defense mechanisms of plants by impacting enzymes like SOD, CAT, APX, and POD, consequently reducing plant tolerance and resistance to both abiotic and biotic stresses (Bharwana et al. 2014). In recent years, multiple researchers have documented the positive effects of GB on the growth and development of different plant species. However, to date, no study has explored the impacts of applying exogenous GB on Cd and As stress tolerance. To our knowledge, this study marks the first investigation into the interaction between GB and Cd and As tolerance in wheat plants.

Seed germination and seedling development are pivotal stages for plant growth and productivity. These phases are notably sensitive to various environmental stresses, including HM stress (Seneviratne et al. 2019). Current study data revealed that germination percentage and SVI decreased under Cd and As stress conditions (Fig. 1). Seed reserves furnish metabolites for respiration and various anabolic processes during the germination of seeds. Starch stands as the most prevalent storage substance within seeds. As supported by available evidence, it is predominantly degraded through the amylolytic pathway during germination. α-Amylase serves as the primary enzyme in the initial breakdown of starch into more soluble forms during seed germination. The enzymes β-amylase and phosphorylase facilitate the conversion into free sugars, supplying vital nourishment for germination (Juliano and Varner 1969). A study by Liu et al. (2007) found that increased concentrations of Cd and As led to a notable decrease in α and β-amylase activities. Among these, α-amylase activity was susceptible to the elevated concentrations of Cd and As tested. The decrease in amylase activity could thus be the primary factor contributing to the inhibition of seed germination. These findings align with earlier findings, which reported Cd stress seed germination and vigor index (Ahmad et al. 2012; Asgharipour et al. 2011). Consistency with this, Chintey et al. (2022) observed that germination percentage of mungbean on the 3rd and 7th day was decreased with an increase in the As toxicity. The reason might be that AS, a heavy metal, may negatively impact enzymes like α-amylase, essential for seed germination, consequently reducing germination percentage. Similarly, Chintey et al. (2022) observed that As toxicity reduced the radicle length and seed viability. The inhibitory effect of As on growth could be due to a hindrance in cell growth. It becomes linked with the middle lamella within cells, enhancing cross-linking and consequently resulting in diminished cellular expansion and growth. Several researchers have noted that HMs may hinder root elongation by interfering with cell division processes, potentially inducing chromosomal aberrations and abnormal mitosis. These disruptions can hamper seedling growth and reduce seedling vigor index (Chintey et al. 2022; Liu et al. 2009). However, GB treatment shows a controversial effect on the germination percentage, vigor index, and radicle length. Study results show that GB treatment reduces Cd and As toxicity and increases the abovementioned attributes. Consistent with the current findings, Sharma et al. (2024 b) demonstrated that adding GB enhances germination percentage and vigor index under Pb stress conditions in barley plants. A possible explanation for this effect could be that GB stimulates the activation of enzymes that hydrolyze food reserves into carbohydrates, thereby facilitates seed germination (Zhang et al. 2020).

The current findings indicate that Cd and As toxicity significantly impacted root and shoot lengths (Fig. 2). The decrease in shoot-root length under Cd and As stress can be attributed to several factors. Cd and As affect plant cells by inhibiting their division, elongation, and differentiation, ultimately resulting in root and shoot reductions (Liu et al. 2013). Moreover, Cd and As ions disrupt the uptake and translocation of essential nutrients, such as phosphorus, nitrogen, and potassium, which are crucial for root and shoot development. Additionally, exposure to Cd and As induces the generation of ROS in plant tissues, leading to oxidative stress and damage to cell membranes, proteins, and DNA, which can hamper shoot and root growth (Rizwan et al. 2016). These collective factors inhibit root and shoot growth in plants subjected to Cd and As stress. Likewise, research on Brassica oleracea revealed that Cr stress leads to decreased plant root-shoot length, a phenomenon likely attributed to Cr's interference with nutrient uptake (Ahmad et al. 2020). However, present study data revealed that GB treatment increases the root and shoot length in both wheat varieties under Cd and As toxicity (Fig. 2). Kumar et al. (2019) observed in sorghum plants exposed to Cr (VI) toxicity that GB exhibited positive effects. This may be attributed to GB's ability to chelate Cr, preventing its uptake by plant tissues and reducing Cr stress levels, thereby enhancing overall plant growth. Moreover, GB protects key CO₂-fixing enzymes like RuBisCo and RuBisCo activase from abiotic stress damage, thereby improving photosynthesis and promoting better plant growth and development (Sharma et al. 2023). These results align with earlier research.

The current findings revealed that As and Cd toxicity decreases the FW and DW (root, shoot, and leaves) as compared to their respective control treatment (without stress) (Fig. 3-5). Cd and As exposure inhibits nutrient uptake, disrupts photosynthesis, induces oxidative stress, and alters hormone balance, all leading to impaired growth and biomass reduction (Hasanuzzaman et al., 2020; Rizwan et al., 2016). The notable decrease in these characteristics was significant across all wheat varieties under Cd and As stress, consistent with earlier findings under different types of metal stress (Bharwana et al. 2014; Ji et al. 2022; Ashraf et al. 2022). Contrarily, GB treatment reduced the negative impact of Cd and As stress and improved the plant's fresh and dry weight. A study by Rasheed et al. (2014) also found that GB supplementation enhanced spring wheat biomass by reducing Cd accumulation and translocation. In addition to its chelating properties, GB may enhance plant biomass under Cd and As toxicity by modulating antioxidant defense mechanisms (Hasanuzzaman et al. 2020). By scavenging ROS and shielding cellular components from oxidative damage, GB alleviates the oxidative stress triggered by HM stress, promoting better growth and biomass accumulation (Sharma et al. 2024 b).

In plants, the accumulation of proline and phenol is closely associated with stress injury. The increase in phenol and proline concentrations under Cd and As toxicity reflects the plants' osmotic imbalance induced by heavy metal exposure (Ahanger et al. 2020). In the present study the proline and phenol levels increase under Cd and As toxicity (Fig.6). Phenols act as antioxidants, scavenging ROS, while proline serves as an osmoprotectant, maintaining cellular integrity and hydration levels under stress conditions (Ahanger et al. 2020; Hasanuzzaman et al., 2020). Numerous studies have documented the increase in phenol and proline levels in response to Cd and As toxicity across various plant species. In rice (Oryza sativa), exposure to abiotic stress resulted in heightened phenol and proline accumulation, indicating a stress response mechanism (Li et al. 2018). Similarly, wheat (Triticum aestivum) subjected to Cd and As stress exhibited elevated levels of phenol and proline, reflecting stress injury (Kaur et al. 2019). However, the addition of GB decreases their level in plants subjected to HM toxicity and indicates stress resilience. Similarly, in the current study, GB treatment decreases the phenol and proline accumulation (Fig. 6). In consistency with the findings, Sharma et al. (2023) reported analogous findings in barley plants exposed to Pb toxicity. The specific mechanism by which glycine reduces phenol and proline accumulation under heavy metal toxicity is not extensively documented in the literature. However, a study by Gupta et al () proposed that GB supplementation under abiotic stress led to the upregulation of genes involved in antioxidant defense pathways, resulting in decreased oxidative stress and ultimately reducing phenol and proline accumulation. Although this study did not directly address the mechanism of GB action on phenol and proline metabolism, it suggests that the antioxidant properties of GB may play a role in modulating their levels. GB demonstrates significant benefits in restoring osmolyte levels and supporting optimal plant functioning. Cucumber seedlings consistently displayed similar outcomes under Cd stress after GB application (Sun et al. 2022).

The oxidative stress induced by heavy metal (HM) toxicity is evidenced by increased lipid peroxidation. Malondialdehyde (MDA) is a well-established marker of oxidative stress linked to lipid peroxidation in plant systems. HMs are primarily absorbed by plant cell membranes. They induce the generation of ROS through various pathways. Additionally, metals can disrupt the antioxidant defense system by inhibiting the activity of key antioxidant enzymes, thereby reducing the plant's ability to scavenge ROS (Gill and Tuteja 2010). In the present study, the MDA content is found to be increased under Cd and As stress, showing stress injury (Fig. 6). Similar findings have been observed in various crop plants, including barley (Sharma et al 2023), chickpea (Singh et al. 2022) and mungbean (Hossain et al. 2010). However, GB treatment decreased MDA levels in both cultivars under stress conditions (Fig. 6). This reduction suggests that GB mitigates oxidative harm induced by Pb toxicity by enhancing antioxidant enzyme functions. Correspondingly, Sharma et al. (2023) and Kumar et al. (2019) reported similar observations under Pb and Cr metal stress. The reduction in MDA content by GB application is attributed to its enhancement of antioxidant enzyme activities, direct scavenging of ROS, stabilization of cell membranes, and osmoprotective properties (Hasanuzzaman et al. 2020; Chen and Murata 2011).

The data of the current study revealed that Cd and As toxicity decreases the total carbohydrate and protein content in both wheat varieties (Fig. 7). Overproduction of ROS could be contributing to oxidative damage by inhibiting protein synthesis and catalyzing carbohydrate oxidation, resulting in decreased protein and carbohydrate content (Gill and Tuteja 2010). Increased protease activity may be responsible for the decrease in total soluble protein content under HM stress (Palma et al. 2002). In addition, carbohydrate levels are reduced as chlorophyll biosynthesis is inhibited (Gubrelay et al. 2013). However, the current finding suggests that the addition of GB under Cd and As stress conditions increases the protein and carbohydrate content in both varieties (Fig. 7). A positive effect of GB on plant growth and chloroplast function was shown in the present study, which might explain the observed increase in protein and carbohydrate content. The addition of GB resulted in similar outcomes in barley (Sharma et al. 2023), cotton (Bharwana et al. 2014), and Brassica chinensis L. (Ji et al. 2022) under various metal stresses. One hypothesis proposes that GB might safeguard proteins through chaperone-like mechanisms, aiding in proper protein folding, and also serve as a signaling molecule to facilitate stress tolerance responses (Rontein et al. 2001).

The current research underscores the significant ecological threat of Cd and As toxicity. The present study evaluates the effectiveness of GB as a compatible solute in alleviating the damaging effects of Cd and As on two wheat varieties. The external application of GB effectively alleviated the growth inhibition and toxicity induced by Cd and As. This alleviation was associated with significantly reduced MDA accumulation and increased carbohydrate and protein content. Additionally, GB supplementation led to a decrease in proline and phenol content. The decline in phenol and proline levels facilitated by GB improves plant growth. These findings underscore the potential of GB as a valuable tool for promoting plant resilience and productivity in the face of environmental challenges. However, there are substantial gaps in our understanding regarding how exogenous application of GB impacts metal uptake and stress tolerance mechanisms in different plant species. Conducting field trials in natural agricultural environments is essential to effectively utilizing GB as a stress mitigator.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interest

The authors declare that there is no competing interest.

Author's contribution

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Sandeep Kumar (SK), Jyoti Sharma (JS), Sunder Singh Arya (SSA), Anju Ahlawat (AA) and Kirpa Ram (KR) writing—review and editing—were carried out by JS, SK; SSA, AA, and KR statistical analysis-SK and JS; writing—original draft preparation—was performed by SK, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated or analysed during the current study are available from the corresponding author on reasonable request.

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Multifaceted role of osmoprotectant glycine betaine to ameliorate Cd and As toxicity by modulation of growth and biochemical indices in wheat plants

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