Choline chloride is an essential metabolite for enhancing plant stress tolerance without secondary soil contamination [57, 58]. However, studies on foliar application of choline on heavy metal stress tolerance and accumulation in plants are lacking in the available literature. In the present study, we have elucidated the effects of choline chloride on Solanum lycopersicum seedlings in relation to Cd stress. Furthermore, the protective effects of choline were demonstrated separately when applied before and after cadmium stress to learn more about choline chloride. As summarized in the figures and discussed in detail below, our study is the first to demonstrate that the improved Cd tolerance in S. lycopersicum by foliar application of choline is associated with its involvement in maintaining membrane stability and its effective role in strengthening the antioxidant defense system and causing a decrease in Cd accumulation.
Cd concentrations in leaves and roots of S. lycopersicum seedlings treated with choline before and after Cd stress are shown in Fig. 2. It is known that Cd accumulation occurs more in the roots of S. lycopersicum seedlings compared to other parts [48, 59]. Our results also show that Cd accumulation occurs in both the root and leaf tissues of S. lycopersicum seedlings, with the accumulation in the roots being at least four times higher than that in the leaves (p < 0.05). In general, foliar applications of choline reduced the accumulation of Cd in Solanum lycopersicum. The most effective results in reducing Cd accumulation in both roots and leaves of seedlings were obtained with choline applications before Cd stress (p < 0.05). For leaves, no change was observed with choline application after Cd stress. However, Cd accumulation in roots was reduced (p < 0.05). Diffusion of Cd toward the root occur with their proton excretion mechanism by maintain a negative gradient across their plasma membrane via Cation exchange capacity (CEC). The ion concentration at the root surface is low and is facilitated by the absorption of Cd by root cells. On the other way, Cd ions travels toward the root at the same rate as the water the plant absorbs by transpiration [60]. Betaine and phosphatidylcholine are the major metabolites of choline chloride in plants [61] and the polar phosphatidylcholine groups in the cell membrane combining with water molecules to prevent water loss from the membrane and thus could regulate cellular water potential and enhanced cellular dehydration.
Moreover, foliar choline treatment alone increased the leaves fresh weight of Solanum lycopersicum due to the polar phosphatidylcholine groups in the cell membrane (Fig. 3, p < 0.05). Choline application before Cd stress caused also similar increase in leaves compared to after Cd stress (p < 0.05). Moreover, the fresh weight of roots was increased when choline was applied before Cd stress compared to after Cd stress (p < 0.05). Hu et al. [57] concluded that choline applied with together fertilizer had a similar effect on salinity, is one of abiotic stress, thus improving the quality and yield of tomato.
At the same time, choline applications also increased the dry weight of both leaves and roots (p < 0.05, Fig. 3). The greatest increase in the dry weight of the leaves was observed in particular with the choline applications before the Cd stress (p < 0.05). As for the dry weight of the roots, it was found that the values obtained before and after Cd stress were statistically similar (p < 0.05). The results obtained show that choline applications have also impressive role on plant growth and development against Cd stress, as with other abiotic stress factors [58].
The buildup of dry matter intimately linked to chlorophyll metabolism as well as growth and development, and yield. Our results showed that Cd stress alone caused a significant decrease in chlorophyll content as determined by SPAD index, whereas choline application alleviated these changes (Fig. 4). The reduction of chlorophyll under Cd stress has been observed in different plant species [62–64]. Reactive oxygen species (ROS) are formed in excess by plants under Cd stress as a result of increased electron leakage in the Mehler reaction during photosynthesis and decreased electron transport in the Calvin cycle. ROS lead to the decrease of photosynthetic efficiency together with the destruction of cell structure and function [65]. But, it is observed in this study that exogenous choline chloride application is maintain ROS levels by enhancing cell membrane (Fig. 4, p < 0.05). The data showed that choline application alone resulted in the highest value of chlorophyll content in Solanum lycopersicum (p < 0.05). Moreover, when applied before Cd stress, chlorophyll levels are maintained, whereas choline application after Cd stress decreased chlorophyll levels (p < 0.05). These results were consistent with some evidence suggesting that choline affects the localization of plasma membrane transporters and consequently inhibits Cd uptake and long-distance transport [23]. Exogenous application of various osmoprotectants, phytohormones and organic acids is an environmentally appropriate way to eliminate heavy metal toxicity as well also heavy metal accumulation in plant tissues [66, 67]. As the effects of each are different, mechanisms which ensure heavy metal tolerance need to be elucidated. Therefore, our results suggest that choline, an osmoprotectant, prevents the inhibition of plant growth by reducing Cd uptake.
Cd toxicity generates oxidative stress by disrupting ROS stability and limiting cellular metabolism, then induces plant antioxidant defences [15, 68]. Increased ROS levels are responsible for oxidative damage, which firstly affects lipids and then other different biomolecules such as DNA/RNA, proteins and sugars [69, 70]. In plants that are exposed to heavy metal stress, the membrane, which is the first biological barrier, plays an important role in protecting the cells from the damage and toxicity of heavy metals [71]. Therefore, it is necessary to investigate lipid metabolism, which plays a key role in regulating plant tolerance to abiotic stress [72, 73]. Therefore, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were analyzed as oxidative injury and stress markers in the present study (Fig. 5 and Fig. 6). Cd stress induced a significant increase in H2O2 levels (p < 0.05, Fig. 5). However, choline application alone didn't cause any remarkable change in H2O2 levels, as also in choline applications before and after Cd stress (p < 0.05, Fig. 5). In addition, it was found that there was a similarity in the degree of peroxidation of the membrane lipids that was detected with the MDA levels. Figure 6 shows that cadmium stress alone caused extensive MDA accumulation, whereas both choline applications were beneficial for the reduction of lipid peroxidation (p < 0.05), especially choline application before cadmium stress is more effective. Studies have shown that membrane remodeling under abiotic stresses such as heavy metals and salinity changes phospholipid, glycolipid and sterol content, composition and saturation levels in the membrane [74–76]. Choline is the precursor of phospholipids and glycerolipids, which are important for the maintenance of membrane structure and function [77, 78]. Therefore, choline application before Cd stress may have altered lipid metabolism in S. lycopersicum in a way that heavy metal tolerance developed.
To clarify the function of choline, there are also investigated that role of enzymatic and non-enzymatic antioxidant mechanisms effective in protection from oxidative damage during Cd stress. SOD activity, one of the primary enzymes of the antioxidant defense system, was measured (Fig. 7). SOD activity increased significantly to reduce ROS levels in Cd application alone (p < 0.05). However, all choline treatments maintained SOD activity at levels similar to control due to be maintained the ROS levels (p < 0.05). So, the antioxidative enzymatic responses also indicated similarly that choline has the function of protecting cells from Cd-induced oxidative injury. Similar responses in SOD activities with choline treatment have been reported in various plants under drought stress [44]. However, our results suggest that choline treatments in Solanum lycopersicum may also improve heavy metal tolerance by maintaining membrane stability and integrity.
In addition to enzymatic antioxidants, non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH) also have an important role in the tolerance of plants to heavy metal stress (Sobrino-Plata et al., 2014). AsA is a potent antioxidant in plant cells that may scavenge ROS directly [79]. As shown in Fig. 8, Cd stress caused a significant decrease in AsA content compared to the control (p < 0.05), due to Cd induced ROS generation leading to the depletion of non-enzymatic antioxidants. The findings were consistent with those reported by Anjum et al. [21], Wang et al. [80] and Ahmad et al. [81]. Similarly, certain studies have documented that various abiotic factors lead to a decrease in AsA content of sensitive cultivars [82, 83, 84]. The biosynthetic capacity of AsA is impaired under stress conditions because the AsA pool is generally determined by its rates of not only regeneration but also synthesis [85]. There have been reports of insufficient AsA regeneration under stress or of lower AsA synthesis than AsA catabolism [82, 86]. However, all choline treatments resulted in increased AsA levels (p < 0.05, Fig. 8). These results demonstrated that exogenous choline applications increased the AsA level and contribute to improve Cd tolerance of Solanum lycopersicum seedlings.
GSH is also a precursor for phytochelatin (PC) biosynthesis, which is associated with the ability of plants to tolerate metal toxicity by forming PC-Cd complexes [87]. Our results showed that exposure to Cd resulted in a significant increase in the amount of GSH compared to the control (p < 0.05, Fig. 8). As highlighted in the literature, it is worth mentioning that the GSH alleviates Cd toxicity by converting it into PC-Cd complexes and limiting the circulation of free Cd ions in the cytosol [88]. As shown in Fig. 8, supplementation of exogenous choline to Solanum lycopersicum seedlings under Cd stress further increased the amount of GSH (p < 0.05), suggesting that choline can promote GSH synthesis. Although defense against stress circumstances can occasionally occur regardless of the GSH concentration, the increased level of GSH pool is widely regarded as a protective response against oxidative stress [89].
The phenolic content of plants has an antioxidant potential and has been linked to their ability to chelate metal ions, which are associated with the production of free radicals under heavy metal stress [90, 91]. However, the level of many phenolic compounds in cells is linked by their rate of degradation in addition to regulation at the biosynthetic level. When the antioxidant system breaks down in the presence of heavy metals, the biosynthesis of new phenolic compounds is slowed down and phenolic content may decrease [92]. As shown in Fig. 9, Cd stress at 50mg/L decreased the total phenolic content (TPC) in Solanum lycopersicum (p < 0.05), which is similar to the findings of Kisa et al. [93]. This is a further indication that Cd stress at 50mg/L causes oxidative damage to the cells, in addition to our results on the MDA content. However, choline treatments extinguished this decrease in TPC of Solanum lycopersicum (p < 0.05, Fig. 9) by preventing oxidative damage. Thus, in addition to their participating in ROS scavenging, TPC in Solanum lycopersicum were also inhibit new radical production, like that presented also in few studies [94].
In Fig. 10, a Pearson's correlation coefficient has been illustrated if there is a correlation between the parameters analyzed in Solanum lycopersicum seedlings exposed to choline chloride (10mM) applications before and after 50mg/L Cd stress. Cd concentration in roots has a positive linear correlation with Cd concentration in leaves, SOD activity, H2O2 and MDA content, while negative correlation with TPC, GSH and AsA content, fresh and dry weight, chlorophyll content (p < 0.05). Furthermore, the heatmap in Fig. 11 reflects the degree of similarity between the groups. It was used to determine if there were any underlying relationships between the choline applications before and after Cd stress. While choline applications before Cd stress are found to be closely related to the choline application alone, the control group and choline application after Cd stress are similar. Cd stress application is completely independent from other groups.