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To view a copy of this licence, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/. Akpinar and Cansev BMC Plant Biology (2024) 24:977 https://doi.org/10.1186/s12870-024-05653-w BMC Plant Biology *Correspondence: Ayşegül Akpinar aysegulakpinar@uludag.edu.tr 1Department of Park and Horticulture, Vocational School of Technical Sciences, Bursa Uludag University, Bursa 16059, Turkey 2Faculty of Agriculture, Horticulture Department, Bursa Uludag University, Bursa 16059, Turkey Abstract Sustainable plant production in soil polluted with heavy metals requires that novel strategies are developed for the benefit of humans and other living things. Cadmium (Cd) is a common heavy metal pollutant for plants, and there is limited information on the use of exogenous bio-regulators to reduce the accumulation and toxic effects of Cd pollution in plants. Choline is an endogenous quertarnary amine that is known to improve stress tolerance in plants, while its mechanism of action in certain conditions is yet to be determined. This study investigated the effects of foliar choline supplementation (10 mM) on Solanum lycopersicum seedlings exposed to Cd application (50 mg/L in soil). The seedlings were randomized to five groups: Control (E1), Cd stress (E2), Choline supplementation after Cd stress (E3), Choline (E4), and Choline supplementation before Cd stress (E5). Following the applications, the Cd content, growth and development parameters (chlorophyll content, fresh and dry weight), oxidative stress parameters (H2O2 and MDA contents), as well as antioxidative defense system (SOD, GSH, AsA, and TPC contents) were analyzed. Choline supplementation after Cd stress reduced the enhanced Cd content in roots by 38% but did not alter it in leaves (p > 0.05) compared to the Cd group. Choline supplementation before Cd stress decreased Cd content both in roots by 87.5% and in leaves by 50%. Choline supplementation after and before Cd stress increased fresh and dry weights in both roots and leaves. While the Cd group (E2) increased the H2O2 level and SOD activity, no remarkable change was observed in H2O2 levels in all choline supplementations (E3, E4, E5). Therefore, lipid peroxidation (MDA) was not observed in choline supplementation before Cd stress (E5), however, when the choline was applied after Cd stress (E3) MDA content was reduced by 40% compared with the Cd stress group (E2). Choline supplementations after and before Cd stress (E3, E5) increased AsA content by 30%, while the Cd group (E2) decreased it by 60% compared with the control group (E1). Choline supplementations before Cd stress (E5) increased TPC by 33%, while the Cd group (E2) decreased it by 18%, moreover, when choline was applied after Cd stress (E3), no change was observed compared to the control group. These data suggest that choline prevents inhibition of plant growth due to Cd toxicity by reducing Cd uptake. The results provided in the present study are likely to enhance the quality and efficiency of crop production in heavy metal-polluted areas. Keywords  Choline chloride, Heavy metal pollution, Cadmium uptake, Solanum lycopersicum Choline supplementation reduces cadmium uptake and alleviates cadmium toxicity in Solanum lycopersicum seedlings Ayşegül Akpinar1* and Asuman Cansev2 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1186/s12870-024-05653-w&domain=pdf&date_stamp=2024-10-10 Page 2 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 Introduction Choline is the precursor component of glycine beta- ine (GB) and is natural, biodegradable, water-soluble, and has no toxic effects [1]. Choline in plant cells is oxi- dized to betaine aldehyde by ferredoxin-dependent cho- line monooxygenase (CMO), and then betaine aldehyde is converted to GB in chloroplasts by betaine aldehyde dehydrogenase (BADH) [2]. GB regulates the osmotic balance within plant cells and tissues and acts as an osmoprotector [3]. GB is considered an osmoprotec- tant-regulator and a neutral pH, naturally accumulating against drought, cold, hot, and salty conditions in plants. External GB applied to roots and leaves in different plant species increases resistance to various stress conditions like salinity and drought [4]. It is noted in the literature that GB is utilized in agriculture to mitigate the impacts of abiotic stress factors. For instance, numerous studies have been carried out to explore the protective mecha- nisms of plants against drought and salt stress (sunflow- ers [5]; olive [6, 7]; tomato [8]; new world fruit [9]; squash [10]; corn [11]; tobacco [12]). Although there are many studies on GB, there are very few on choline in plants. It has been reported that choline increases stress tolerance by altering various biological and physical processes in plants [13]. These studies show that choline can be taken up from foliar and thus effectively alleviate the effects of stress [14], thus increasing plant growth, biomass, root- ing, and root growth under some abiotic stresses [1, 15–18]. Like an osmoprotectant, it can stabilize proteins and enzymes, maintain membrane integrity, and detoxify ROS [19]. External choline supplementation can amelio- rate the adverse effects of cellular stress and significantly improve plant growth [20]. However, whether and how choline supplementation affects metabolic processes involved in the regulation of heavy metal tolerance has not been investigated. Cadmium (Cd), among heavy metals, stands out as one of the major environmental threats in today’s conditions of increasing industrialization [21]. Phenotypic, bio- chemical and physiological changes occur in the plants due to the exposure to cadmium. These physiological disturbances inhibit plant development, reduce chloro- phyll content, and alter plasma membrane permeability [22–24]. Many metabolic and physiological distortions induced by Cd stress start with the increased amount of reactive oxygen species (ROS) at the cellular level [25, 26]. Cd ions enter the cytoplasm and produce ROS at high levels, leading to oxidative stress [27]. The cells’ anti- oxidant defense system ultimately limits elevated levels of ROS resulting in the protection of the plant metabolism. Cd can also accumulate in plants to varying degrees, depending on the amount of heavy metals in the envi- ronment and the characteristics of the species. Numer- ous studies have documented that plants can absorb and translocate harmful metals throughout their tissues [28–31]. There are several applications in the literature to reduce Cd uptake by plants and indirectly mitigate Cd toxicity. These studies include the application of exog- enous bio-regulators such as salicylic acid (SA) [32–34], abscisic acid (ABA) [35], sulfate [36, 37], and nitric oxide (NO) [38], which may serve as an effective management tool to reduce Cd accumulation in plants. There is only one study in the literature on the applica- tion of choline to plants against heavy metal stress [39]. However, it was conducted by first applying choline to Cr-contaminated soils and then looking at heavy metal uptake and tolerance of spinach from seed to seedling in this contaminated soil applied by choline. However, it is known that choline forms chelate with heavy metals in the soil, which ensures that heavy metals are retained in the soil and result in heavy metals not being taken up by plants [40, 41]. Therefore, the explanation by Hussain et al. [39] mentioning whether choline provides plants with tolerance to heavy metals in the study is not certain. Therefore, more evidence is needed to show that choline supplementations affect heavy metal uptake and toxic- ity. Consequently, the elucidation of the effects of exog- enous treatments with osmoprotectants such as choline has become the focus of both industrial and scientific interest. Tomato (Solanum lycopersicum L.) is a widely grown vegetable crop of considerable economic value. However, heavy metal contamination adversely affects its yield and quality because it is relatively sensitive to heavy metal stress [42]. This study aims to find out the effects of exog- enous choline chloride supplementations in Solanum lycopersicum seedlings against Cd stress and, in particu- lar, the effects of choline when applied before and after Cd stress. In addition, choline supplementations will be made as foliar applications to prevent it from forming compounds with heavy metals in soil. Materials and methods Plant material Five-week-old Solanum lycopersicum cv. H2274 seedlings were planted in pots (14 × 12 cm) containing a mixture of peat/perlite (1:1) and grown in plant growth chambers (16 h photoperiod, 1200  lx at 24  °C / 20  °C [day/night]) for 4 weeks. Actagro (7-7-7) nutrient solution was used to irrigate the growing seedlings. Experimental design and treatments An experiment plan was performed to investigate the effectiveness of choline chloride supplementations under Cd stress by a random plot arrangement with three repli- cates, containing 3 pots for each replicate. In our prelimi- nary studies, CdCl2 applications were tried at different concentrations, and the optimum concentration that is Page 3 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 not lethal but can cause Cd accumulation was selected on the other side. Similarly, in determining choline concen- trations, choline supplementations were made at various concentrations in preliminary studies, and the ideal dose for choline was determined as 10 mM according to the growth and development parameters of Solanum lycop- ersicum seedlings. While Cd applications were with irri- gated water at once a week, exogenous choline chloride (Sigma Aldrich C7017) treatments (10 mM) were applied as foliar for 4 weeks at 2-day intervals. An experimental plan was performed from five differ- ent groups; E1. Control group: no application. E2. Cd stress group: Only 50 mg/L Cd was applied to the seedlings from the soil. E3. Choline supp. After Cd stress: 50 mg/L Cd was applied to the seedlings from the soil and then 10 mM choline chloride was supplemented as foliar. E4. Choline supp.: 10 mM choline chloride supplemented to seedlings as foliar; E5. Choline supp. Before Cd stress: 10 mM choline chloride was supplemented to seedlings as foliar and then 50 mg/L Cd was applied from the soil. The experimental design is shown in Fig. 1. At the end of the experiment, chlorophyll content and biomass as fresh and dry weight were measured. Plants were then harvested, some dried in a lyophilizer for determination of Cd content, and stored at + 4  °C. Oth- ers are frozen in liquid nitrogen and stored at -80 °C until further analysis or used fresh. Determination of cd content in tissues The Cd contents in leaves and roots of S. lycopersicum were determined using an ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer, Perkin Elmer 2100 USA) emission spectrometer. The samples dried in a lyophilizer were digested by microwave irradiation using an Anton Paar Multiwave Go Microwave Burner, which has a rotor with 12 sample chambers and polyethylene Teflon-coated containers. In the Anton Paar Multiwave Go microwave burner system, polyethylene Teflon- coated containers were disinfected in a 10% HNO3 (67% v/v) bath, then cleaned in ultrapure water and dried in an oven at 40 °C. All solutions were prepared using analytical grade ultrapure water (18 MΩ cm resistivity) with the TKA Ultra Pacific and Genpura water purification system. 67% HNO3 was obtained from Merck (Darmstadt, Germany). Argon (99.9995% purity, Linde, Türkiye) was used as car- rier gas. Standard stock solutions (1000 mg/L) were used to prepare Cadmium Absolute Standard (USA) calibra- tion standards for each element. Standard solutions were prepared daily using 0.3% HNO3. Fig. 1  The study’s experimental design involved choline chloride (10 mM), which was applied before and after 50 mg/L Cd stress to Solanum lycopersi- cum seedlings. The 5-week-old seedlings were used. Experiments were made after growing in the plant growth chamber for 2 weeks. Both Cd stress and choline supplementations were continued for 4 weeks, two weeks each one Page 4 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 The plant samples were homogenized in a sterile porce- lain mortar before analysis. Each sample (0.5 g) was then digested in duplicate in a 50 mL borosilicate glass vessel using a mixture of HNO3 (6 mL) and H2O2 (1 mL). The samples were digested by microwave irradiation using an Anton Paar Multiwave Go Microwave Burner. The heat- ing program employed is presented in Table 1. The solutions were allowed to reach room temperature and then diluted to 25mL in polypropylene centrifuge tubes by filtering through 0.45  μm filters (hydrophilic PVDF Millipore Millex-HV). After the samples were completed to a certain volume at room temperature, they were determined using an ICP-OES (Perkin Elmer 2100 USA) emission spectrometer. The instrumental operating conditions are summarized in Table 2. Cadmium was prepared as an external standard solu- tion using six calibration solutions and a blank containing 0.3% HNO3, within the range of 0.05-2 mg/l. Calibration curve was drawn linearly with 6 standard solutions. In addition, detection (LOD), and quantification (LOQ) limits, were also carried out and presented in Table 3. Chlorophyll content, dry and fresh weight The chlorophyll content of S. lycopersicum seedlings was analyzed using a portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan) and expressed as SPAD values. Fully inflated leaves were used for measure- ments, which were averaged over ten measurements per plant. Fresh weight is the measurement made after har- vesting the plants (FW, g). The dry weight (DW, g) was determined after oven drying the samples at 80ºC until the weight remained constant. Hydrogen peroxide (H2O2) content H2O2 was detected spectrophotometrically as reported by Alexieva et al. [43]. Plant samples were contacted with the reaction mixture containing 0.1% trichloroacetic acid (TCA, 0.5 mL), 100mM K phosphate buffer (0.5 mL), and KI reagent (1 M KI w/v in fresh dd water, 2 mL). The blank consisted of 0.1% TCA in the absence of samples. After incubation in the dark for one hour, the absorbance of the mixtures was measured at 390 nm. The amount of H2O2 was determined using a standard curve. Malondialdehyde (MDA) content The thiobarbituric acid (TBA) reaction, modified from Heath and Packer [44], was used to determine the MDA content. After homogenization of plant samples in 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA), the mixture was centrifuged at 15.000 g for 10 min. After incubation of the samples at 95 °C for 30 min, the reaction mixture (0.5 mL of the supernatant and 1.5 mL of the combina- tion of 20% TCA and 0.5% TBA) was added. The samples were then centrifuged (15.000 g, 4 °C, 5 min). Absorbance was measured at 532 and 600 nm. The extinction coeffi- cient (Ɛ) used in the calculation was 155 mM1/cm [45]. Superoxide dismutase (SOD) activity The procedure of Ardıc et al. [46] was followed for plant extraction in the antioxidant enzymatic activity assay to determine SOD activity. Samples were homogenized in an ice bath using a buffer solution consisting of 50 Na- phosphate buffer [pH 7.8], 2% polyvinylpolypyrrolidone [PVP; w/v], and 1 mM EDTA. They were then centri- fuged at 14.000  g for 40  min at 4  °C. The supernatants were used for the determination of SOD activity. SOD activity was determined according to the method of Beauchamp and Fridovich [47] using bovine erythrocyte SOD standard (SOD S7446, Sigma-Aldrich, USA). This method is based on the inhibition of nitroblue tetrazo- lium at 560 nm. SOD activity was defined using the linear equation derived from the curve following the calcula- tion of the percentage inhibition and expressed as units per mg protein (U/mg protein). Glutathione (GSH) assay The method of Ellman [48] was used to determine glu- tathione (GSH) levels. The analysis was performed by homogenizing frozen plant samples (0.2 g) in ice-cold 5% (w/v) trichloroacetic acid and centrifuging the mixture at 15.000 g for 15 min. Then 0.2mL of the supernatant was mixed with 2.6 mL phosphate buffer (pH 7.7) and 0.2 mL DTNB (5,5ˈ-dithiobis (2-nitrobenzoic acid) (2.51  mg/ mL). Absorbance was measured at 412 nm after incuba- tion for 5 min at 30 °C. The GSH content was calculated from a standard curve. Table 1  Heating program in microwave digestion system Step Ramp (min) Temperature (ºC) Hold (min) 1 10:00 120 5:00 2 05:00 200 10:00 Table 2  Operating conditions for ICP-OES Instrument Perkin Elmer 2100 ICP-OES, Axial RF generator Frequency: 40 MHz, Power output 1300 W Plasma gas flow rate (l/min) 15.0 l/min Auxiliary gas flow rate 1.0 l/min Nebulizer as flow rate 0.5 l/min Integration mode Peak field Auxiliary flow rate 0.8 l/min Wavelength Cd 228.502 nm Table 3  Performance characteristic of the method Element Cadmium MLOD (mg/kg) 0.15 MLOQ (mg/kg) 0.5 Page 5 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 Ascorbic acid (AsA) content The AsA contents (reduced AsA [AsA] and total ascor- bates [AsA + DASA]) were quantified according to the method of Çakmak and Marschner [49]. Plant samples (1 g) were extracted with 10 mL of 5% meta-phosphoric acid and centrifuged at 22.000  g for 15  min. For total ascorbates, 0.2 mL of plant extract was added to 150 mm phosphate buffer (pH 7.4) containing 10 mM DTT and 5 mm EDTA. Samples were incubated for 10 min at room temperature and 0.5  N-ethylmaleimide (0.1 mL) was added to remove excess DTT. For AsA, 0.5 mL 150 mm phosphate buffer (pH 7.4) and 0.2 mL water were com- bined with 0.2 mL plant extract. Total polyphenol content (TPC) Plant samples (1.5 g) were extracted five times with meth- anol (5 mL). The extracts were then mixed, the methanol was evaporated and the residue was dissolved in metha- nol/water (8/2) (5 mL) and stored at -18 °C after filtration through a 0.45  m filter (hydrophilic PVDF Millex-HV; Millipore, Bedford, MA, USA). All the extracts obtained were used for the determination of total phenolic con- tent. The amount of total phenolic in the extracts was determined using the Folin-Ciocalteu reagent according to the method described by Apak et al. [50]. Diluted plant extracts (1 mL) were combined with 0.5 mL Folin-Ciocal- teu reagent and 5 mL dd water. After vortexing, the mix- ture was allowed to stand for three minutes. At the end of this period, 1mL of 7.5% Na2CO3 was added and the mixture was shaken sporadically for 1 h at room temper- ature in a dark environment. Absorbance was measured at 750  nm. The gallic acid standard curve was used to calculate the total phenolic content and the results were expressed as mg gallic acid equivalents (GAE)/gram fresh weight. Statistical analysis Data were expressed as mean ± standard deviation of mean. IBM SPSS software 24.0 for Windows was used for statistical analysis. Differences between Cd applica- tions and choline supplementations were compared using ANOVA and the significance level was set at p < 0.05. Multiple comparisons of means were compared using the significant Tukey’s HSD post-hoc test. In addition, the Pearson correlation coefficients between the measured variables of S. lycopersicum were calculated using SRplot, and a heat map was created at the same time. Results and discussion Choline chloride is an essential metabolite that enhances plant stress tolerance without secondary soil contamina- tion [51, 52]. However, there is a lack of studies on foliar choline supplementation for heavy metal stress tolerance and accumulation in plants in the available literature. In our study, we investigated the effects of choline chloride supplementation on Solanum lycopersicum seedlings under Cd stress. Additionally, we separately demon- strated the protective effects of choline when applied before and after Cd stress. Our study is the first to show that the enhanced Cd tolerance in S. lycopersicum by foliar choline supplementation is associated with its role in maintaining membrane stability, strengthening the antioxidant defense system, and causing a decrease in Cd accumulation. Figure 2 illustrates the Cd concentrations in the leaves and roots of S. lycopersicum seedlings treated with Fig. 2  Cadmium (Cd) accumulation (mg/kg dry weight) on leaves and roots of Solanum lycopersicum seedlings with choline chloride (10 mM) supple- mentations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA followed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 6 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 choline before and after Cd stress. Our results indicate that Cd accumulation occurred in both root and leaf tis- sues of S. lycopersicum seedlings, with the accumulation in the roots being at least four times higher than in the leaves (E2) (p < 0.05). It is known that Cd accumulation occurs more in the roots of S. lycopersicum seedlings than in other parts [42, 53]. When choline was applied after Cd stress (E3), Cd accumulation decreased in the root parts of S. lycopersicum, while there was no change in Cd accumulation in the leaves (p < 0.05). This may be related to the fact that choline applied to the leaves is metabolized by the plant and affects the stability of the root membranes where Cd is taken up. Cd ions move towards the root at the same rate as the water absorbed by the plant through transpiration [51]. Betaine and phosphatidylcholine are the main metabolites of choline chloride in plants [52]. Polar phosphatidylcholine groups in the cell membrane can regulate cellular water poten- tial and increase cellular dehydration by combining with water molecules to prevent water loss from the mem- brane. Thus, it can be said that choline affects root mem- branes in the uptake of heavy metals, which is one of the novel findings in this study. Furthermore, in choline supplementation before cad- mium stress (E5), Cd accumulated in the root and leaf parts of S. lycopersicum seedlings compared to the con- trol group (E1), but the accumulation rate was consider- ably lower than the group that was only applied with Cd for the same time (E2) (p < 0.05). This shows that cho- line changes the membrane lipid structure, as reported in various studies [17]. Cd uptake to plants occurs via the proton transfer mechanism, maintaining a negative gradient across plasma membranes through the cation exchange capacity (CEC). The fact that choline provides cellular ion balance and maintains membrane stability has limited this uptake. The results of fresh and dry weight of S. lycopersicum seedlings before and after Cd stress given in Fig. 3 show that choline is efficient in maintaining intracellular sta- bility by affecting the composition of cell membranes. Choline supplementation (E4) increased the leaves’ fresh weight of S. lycopersicum (Fig.  3, p < 0.05). Choline sup- plementation before Cd stress (E5) similarly increased the fresh weight of leaves (p < 0.05). When choline was applied after Cd stress (E3), there was no statistically significant difference in the fresh weight of the leaves. In roots, an increase in fresh weight was observed in choline supplementations before and after Cd stress (E3 and E5, respectively) compared to the control group (p < 0.05). This situation indicates the existence of choline-depen- dent regulations, especially in root cells where Cd is taken up, due to the addition of choline to polar phosphatidyl- choline groups in the cell membrane. Hu et al. [54] con- cluded that the choline supplementation with fertilizer had a similar effect on salinity, one of the abiotic stresses, thus improving the quality and yield of tomatoes. At the same time, choline supplementations also increased the dry weight of both leaves and roots (p < 0.05, Fig. 3). The buildup of dry matter is intimately linked to growth, development, and yield. The greatest increase in the dry weight of the leaves was observed in particular with the choline supplementations before the Cd stress (E5) (p < 0.05). As for the dry weight of the roots, it was found that the values obtained after and Fig. 3  Dry and fresh weight (g) belong to leaves and roots of Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA fol- lowed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 7 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 before Cd stress were statistically similar (E3 and E5, respectively) (p < 0.05). The results indicate that choline supplementations significantly promote plant growth and development under Cd stress, similar to other abiotic stress factors [55]. Our results showed that Cd stress alone (E2) caused a significant decrease in chlorophyll content as deter- mined by the SPAD index, however, choline supplemen- tation after Cd stress (E3) did not alleviate these changes (Fig.  4). The data showed that choline supplementation alone (E4) resulted in the highest value of chlorophyll content in S. lycopersicum (p < 0.05). However, in choline supplementation before Cd stress (E5), chlorophyll levels maintained the same value as the control group (p < 0.05). The reduction of chlorophyll under Cd stress has been observed in various plant species [56–58]. Cd toxicity often disrupts chlorophyll synthesis and causes struc- tural degradation. As a result, affected plants may exhibit symptoms such as yellowing of leaves and decreased overall vigor. Due to increased electron leakage in the Mahler reaction during photosynthesis and reduced elec- tron transport in the Calvin cycle, excessive amounts of ROS can accumulate in plants under Cd stress. In this scenario, ROS results in decreased photosynthetic effi- ciency, along with the deterioration of cell structure and function [59]. Furthermore, the inability to absorb micro- nutrients such as Mn, Fe, and Mg in the presence of Cd is a main factor contributing to structural damage to chlo- rophyll [60]. Disruption in ROS stability source from Cd toxicity generates oxidative stress by limiting cellular metabolism, and later induces plant antioxidant defenses [14, 61]. Otherwise, oxidative damage occurs with high ROS levels [62, 63]. 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 dam- age and toxicity of heavy metals [64]. Therefore, it is nec- essary to investigate lipid metabolism, which plays a key role in regulating plant tolerance to abiotic stress [65, 66]. Malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were analyzed as oxidative injury and stress markers in this study (Figs. 5 and 6). Cd stress (E2) caused a significant increase in H2O2 levels (p < 0.05, Fig.  5). However, when choline was applied alone (E4), there didn’t occur any remarkable change in H2O2 levels according to the control group (E1), as in choline supple- mentations after and before Cd stress (E3 and E5, respec- tively) (p < 0.05, Fig. 5). In addition, Fig. 6 shows in S. lycopersicum the degree of peroxidation of the membrane lipids that were detected with the MDA levels. Cd stress alone (E2) caused extensive MDA accumulation, whereas choline supplementations both alone and before Cd stress (E4 and E5, respectively) did not bring forth lipid peroxida- tion (p < 0.05). In choline supplementation after Cd stress (E3), lipid peroxidation was reduced but different than the control group (p < 0.05). Choline is the precursor of phospholipids and glycerol, which are important for the maintenance of membrane structure and function and is effective in membrane remodeling by changing the phos- pholipid, glycolipid, and sterol content, composition, and saturation levels in the membrane under various abiotic Fig. 4  Chlorophyll contents (SPAD value) of Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 6). Comparisons between groups were made by ANOVA followed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 8 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 stresses [67–71]. In this study, the effect of choline on membrane lipids under heavy metal stress was demon- strated for the first time. It is thought that, especially in choline supplementation before Cd stress, cell membrane lipids in S. lycopersicum are protected from oxidative stress caused by Cd stress, and the structure and function of the cells are preserved, thus providing a basis for the development of heavy metal tolerance. To clarify the function of choline, the role of enzymatic and non-enzymatic antioxidant mechanisms effective in protection from oxidative damage during Cd stress are investigated. SOD activity, one of the primary enzymes of the antioxidant defense system, was measured in this study (Fig.  7). SOD activity increased significantly to reduce ROS levels in Cd application alone (E2) (p < 0.05). However, SOD activity in all choline supplementations was at levels similar to control due to maintaining the ROS levels (p < 0.05). So, the antioxidative enzymatic responses also indicated that choline has the function of protecting cells from Cd-induced oxidative injury. Simi- lar responses in SOD activities occurred with choline supplementation have been reported in various plants under drought stress [18]. However, our results presented a novelty that choline supplementations in S. lycopersi- cum may also improve heavy metal tolerance by main- taining membrane stability and integrity. While enzymatic antioxidative responses are effective in short-term stress, non-enzymatic antioxidants such as Fig. 6  Malondialdehyde (MDA) contents (mg/g fresh weight) in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA fol- lowed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Fig. 5  Hydrogen peroxide (H2O2) amounts (µmol/g fresh weight) in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementa- tions before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA followed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 9 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 ascorbic acid (AsA) and glutathione (GSH) are taken into account in the tolerance of plants to heavy metal stress [72]. AsA is a potent antioxidant in plant cells that may scavenge ROS directly [73]. As shown in Fig. 8, Cd appli- cation alone (E2) caused a significant decrease in AsA content compared to the control group (p < 0.05). This situation may be due to the depletion of non-enzymatic antioxidants caused by Cd-induced ROS production. The findings were consistent with those reported by Anjum et al. [74], Wang et al. [75] and Ahmad et al. [76]. The biosynthetic capacity of AsA is impaired under various abiotic stress conditions, especially in sensitive cultivars [77–79]. AsA pool is generally determined by its rates of not only regeneration but also synthesis [80]. There have been records of insufficient AsA regeneration appearing under stress or of lower AsA synthesis occurring than AsA catabolism [77, 81]. In this study, all choline supple- mentations (E3, E4, E5) resulted in increased AsA levels (p < 0.05, Fig.  8). These results demonstrated that exog- enous choline supplementations increased the AsA level in S. lycopersicum seedlings and contributed to improv- ing the heavy metal tolerance. GSH, one of the non-enzymatic antioxidants in plants exposed to heavy metals, plays a leading role in phy- tochelatin (PC) biosynthesis by forming PC-Cd com- plexes with heavy metal PC, thus limiting the circulation of free Cd ions in the cytosol and playing an effective role in providing tolerance to metal toxicity [82, 83]. As shown in Fig.  8, exogenous choline supplementations after and before Cd stress (E3, E5) like in alone choline supplementation (E4) increased the amount of GSH (p < 0.05), suggesting that choline triggers GSH synthesis. Although defense against abiotic stress conditions can sometimes occur independently of GSH concentration, the increased level of the GSH pool is widely accepted as a protective response against oxidative stress [84]. The phenolic content of plants possesses antioxidant potential and is associated with their ability to chelate metal ions, which are linked to the generation of free radicals under heavy metal stress [85, 86]. When the anti- oxidant system deteriorates in the presence of heavy met- als, the biosynthesis of new phenolic compounds slows, leading to a decrease in phenolic content [87]. As shown in Fig. 9, Cd application (E2) decreased the total pheno- lic content (TPC) in S. lycopersicum (p < 0.05), which is similar to the findings of Kisa et al. [88]. However, choline supplementations (E3, E4, E5) extinguished this decrease in TPC of S. lycopersicum (p < 0.05, Fig. 9). When choline was supplemented even after Cd stress (E3), TPC also inhibited new radical production, like that presented in a few studies in addition to their participation in ROS scavenging [89]. As a result, phenolic content seems to be effective in protecting S. lycopersicum from oxidative damage under Cd stress. A Pearson’s correlation coefficient in Fig.  10 has been illustrated if there is a correlation between the parame- ters analyzed in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50  mg/L Cd stress. Cd concentration in roots has a positive linear correlation with Cd concentration in Fig. 7  Superoxide dismutase (SOD) activity (U/mg protein) in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA fol- lowed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 10 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 leaves, SOD activity, H2O2, and MDA content, and a neg- ative correlation with TPC, GSH, and AsA content, fresh and dry weight, and chlorophyll content (p < 0.05). Fur- thermore, 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 cho- line supplementations before and after Cd stress. While choline supplementations before Cd stress (E5) are found to be closely related to the choline supplementation alone (E4), the control group (E1) and choline supplementation after Cd stress (E3) are similar. Cd stress supplementa- tion (E2) is completely independent of other groups. In summary, the results of the present study, which focused on reducing Cd accumulation and eliminating the nega- tive effects of Cd toxicity by choline supplementations, revealed that choline supplemented to S. lycopersicum seedlings under Cd stress significantly affected plant growth, photosynthetic pigments, antioxidant defense system, and Cd uptake. Fig. 8  Ascorbic acid (AsA) (µg/g fresh weight) and Glutathione (reduced GSH) (nmol/g fresh weight) contents in Solanum lycopersicum seedlings ex- posed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA followed by post-hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Page 11 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 Fig. 9  Total phenolic contents (mg/ 100 g GA) in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50mg/L Cd stress. Data points reflect means and standard deviations (n = 4). Comparisons between groups were made by ANOVA followed by post- hoc Tukey test (p < 0.05). Different lowercase letters indicate significant differences between groups. E1. Control group, E2. Cd stress group, E3. Choline supp. After Cd stress, E4. Choline supp., E5. Choline supp. Before Cd stress Fig. 10  A Pearson correlation coefficient of relationship between parameters analyzed in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplementations before and after 50 mg/L Cd stress. The correlation indicated by the colored boxes is significant at the p < 0.05 level. Abbrevia- tions used in the figure are as follows: FW, fresh weight; DW, dry weight; Chl, Chlorophyll content; TPC, total phenolic content; GSH, glutathione content; AsA, Ascorbic acid; H2O2, hydrogen peroxide content; MDA, malondialdehyde content; SOD, superoxide dismutase activity; Cd Acc.-L, Cd accumulation in leaves; Cd Acc.-R, Cd accumulation in roots Page 12 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 Conclusion Exogenous application of various osmoprotectants, phy- tohormones, and organic acids is an environmentally appropriate way to eliminate heavy metal toxicity as well as heavy metal accumulation in plant tissues [90, 91]. Ma et al. [92] indicated the effect of exogenous application of syringic acid in S. lycopersicum varieties under the toxic concentrations of Pb. And, they detected that syringic acid has protected the plant from Pb toxicity and reduced Pb uptake to tissues. Ahmad et al. [93] also presented nitric oxide (NO) application restricted the Cd uptake and enhanced growth and development in the shoots and roots of S. lycopersicum. Shen et al. [35] indicated the effects of exogenous abscisic acid (ABA) on Cd toxicity in purple flowering stalk (Brassica campestris L. ssp. chinen- sis). And, exogenous ABA alleviated Cd toxicity mainly by reducing the reactive oxygen species (ROS) through activating the antioxidant enzyme system and accumu- lating more Cd in roots. Our results indicate that cho- line, an osmoprotectant, prevents the inhibition of plant growth by reducing Cd uptake. To our knowledge, this is the first demonstration of how foliar choline supplemen- tations may influence the phenotypic, biochemical, and physiological changes in S. lycopersicum under Cd stress. These findings provide evidence that choline affects the localization of plasma membrane transporters and con- sequently inhibits Cd uptake and long-distance transport. As highlighted in this study, choline supplementations also enhanced plant growth, decreased ROS production, maintained the antioxidative system, and decreased the Cd accumulations of plant organs. It is worth mention- ing that this study reveals a sustainable, low-cost, high- efficiency, and environmentally friendly method for reducing heavy metal accumulation in plants and miti- gating heavy metal toxicity. In field conditions, although the methods used to remove heavy metals from agricul- tural products or soil are realized mainly by chemicals such as copperoxyethylidene diphosphonic complexes, metasilicate pentahydrate, sodium tripolyphosphate, or mercaptoacetic acid, these heavy metal removers in the current situation bring about a secondary chemical waste problem and also pose a great risk to employee health in addition to the secondary waste problem. However, it can achieve a reduction in the Cd accumulation and elimina- tion of Cd toxicity by applying only low doses of choline compound in the form of spraying instead of applications Fig. 11  Heatmap of the relationship between parameters analyzed in Solanum lycopersicum seedlings exposed to choline chloride (10 mM) supplemen- tations before and after 50 mg/L Cd stress. The correlation illustrated with colored boxes is significant at the p < 0.05 Page 13 of 15Akpinar and Cansev BMC Plant Biology (2024) 24:977 that require extra energy and costs such as chemical precipitation, filtration, electrochemical processes, ion exchange methods, and evaporation method. Our study is the first to reveal physiological responses, and the evaluation of agronomic responses, including fruit yield and fruit quality in agricultural production, is among our next goals. Consequently, it is suggested that studies on choline-induced membrane lipid remodeling under heavy metal stress should be conducted in the future, as this research holds significant promise for the industries dealing with crop production in soils contaminated with heavy metals. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12870-024-05653-w. Supplementary Material 1 Acknowledgements Thanks to the Scientific Research Projects Council of Bilecik Seyh Edebali University, Turkey for supporting (Research Project No. 2021-01.BŞEÜ.11-02) and COST Action CA22102 E-NICHE for funding. Author contributions Aysegul AKPINAR and Asuman CANSEV have contributed equally to this work. Funding This work was supported by the Scientific Research Projects Council of Bilecik Seyh Edebali University, Turkey (Research Project No. 2021-01.BŞEÜ.11 − 02). Data availability No datasets were generated or analysed during the current study. 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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1093/jexbot/52 https://doi.org/10.1093/jxb/erh113 https://doi.org/10.1093/jxb/erh113 https://doi.org/10.1111/j.1399-3054.2006.00620.x https://doi.org/10.1104/pp.103.033548 https://doi.org/10.1104/pp.103.033548 https://doi.org/10.1007/s10534-021-00315-y Choline supplementation reduces cadmium uptake and alleviates cadmium toxicity in Solanum lycopersicum seedlings Abstract Introduction Materials and methods Plant material Experimental design and treatments Determination of cd content in tissues Chlorophyll content, dry and fresh weight Hydrogen peroxide (H2O2) content Malondialdehyde (MDA) content Superoxide dismutase (SOD) activity Glutathione (GSH) assay Ascorbic acid (AsA) content Total polyphenol content (TPC) Statistical analysis Results and discussion Conclusion References