Both tartaric and pantothenic acids promote Pb-phytoextraction potential of sunflower by regulating calcium and phosphorus uptake

doi:10.21203/rs.3.rs-3983997/v1

Phytoextraction of Pb is a challenging task due to its extremely low mobility within soil and plant systems. In this study, we tested the influence of some novel chelating agents for Pb-phytoextraction using sunflowers. The Pb was applied at control (0.0278 mM) and 4.826 mM Pb as Pb(NO₃)₂ through soil-spiking. After 10 days of Pb addition, four different organic ligands (aspartic, ascorbic, tartaric, and pantothenic acids) were added to the soil at 1 mM concentration respectively. In the absence of any chelate, sunflower plants grown at 4.826 mM Pb level accumulated Pb concentrations up to 104 µg g^-1 DW in roots whereas, 64 µg g^-1 DW in shoot. By contrast, tartaric acid promoted significant Pb accumulation in root (191 µg g^-1 DW; +45.5%) and shoot (131.6 µg g^-1 DW; +51.3%). Pantothenic acid also resulted in significant Pb-uptake in sunflower shoots (123 µg g^-1 DW; +47.9%) and in roots (177.3 µg g^-1 DW; +41.3%). The least effective amongst the chelates tested was ascorbic acid but it still contributed to +39.0 and 45.2% more Pb accumulation in sunflower root and shoots. In addition, plant growth, biochemical, and ionomic parameters were positively regulated by organic chelates. Especially, an increase in the leaf Ca, P, and S was evident in Pb-stressed plants in response to chelates. These results highlight that the use of biocompatible organic chelates positively alters plant physio-biochemical traits contributing to higher Pb-sequestration in sunflower plant parts.

Ascorbic acid

Aspartic acid

Pantothenic acid

Tartaric acid

Lead Phytoextraction

Lead (Pb) is classified 2nd by the USEPA hazardous pollutant among the top 20 pollutants (Lam et al., 2023; Omeka and Egbueri, 2023). The State Environmental Protection Administration of China (SEPA) has established a maximum allowable limit (MAL) of 350 mg kg^− 1 for soils in China (Ernyasih et al., 2023). Whereas the MAL of Pb in fruits and vegetables is 0.3 mg kg^− 1 DW (Kumar et al., 2022; Singh et al., 2023). In Pakistan, the concentration of Pb in fruits and vegetables ranged between 1.8–11 mg kg^− 1, which is above the permissible limits established by FAO (Rehman et al., 2017). Alongside highways, Pb concentration was 0.08–3.98 mg kg^− 1 while, it ranged between 1.95–4.74 mg kg^− 1in soils (Khalid et al., 2018). Additionally, in some parts of Pakistan, concentrations above 30 mg kg^− 1 have been reported (Ahmad et al., 2019). To an estimate, toxic heavy metal pollution affected millions of people worldwide (Peng et al., 2022; Wang et al., 2022). Specifically, exposure to Pb can cause Anemia, cancer, neurological diseases, brain damage, kidney failure, poisoning, and developmental abnormalities (Collin et al., 2022; Cui et al., 2022).

Remediation of Pb-contaminated soils can be achieved by various physical, chemical, and biological methods. The physical removal strategies include excavation, hydraulic flushing, and electrokinetics. A drawback of the physical removal techniques is the disruption of soil structure (Isidro et al., 2022; Kumawat et al., 2022; Zheng et al., 2022). By contrast, Pb-phytoremediation is an in-situ applicable technique that is efficient, eco-friendly, and cheap (Khan et al., 2019; Yadav et al., 2022; Singh and Pant, 2023). During the past few years, we have tested the approach of using various organic ligands to enhance the Pb-removal from the soil through phytoextraction and singled out tartaric acid (TA) as the most significant Pb-solubilizing agent (Khan et al., 2016). Plant roots release TA as a component of root exudates and it can influence the bioavailability of heavy metals in the rhizosphere (Lu et al., 2013). Higher TA levels were found in the root exudates of Sedum alfredii (Cd hyperaccumulator) signifying its role in phytoextraction (Li et al., 2007; Tao et al., 2019). Follow-up studies revealed a significant metal desorption ability of TA contributing to Pb-phytoextraction Chen et al. (2020). As a step further, TA soil spiking enhanced Pb root-to-shoot translocation thereby improving Pb-phytoextraction efficiency in turnip (Khan et al., 2022). To draw parallels with TA, we have compared the effects of some potential biocompatible organic ligands having a role in different plant metabolic pathways.

One such potential biocompatible organic ligand is vitamin C which has not been tested as a chelate for phytoextraction of metals. Ascorbic acid is an integral component of plant development and defense pathways (Reviewed by Akram et al., 2017). It has been reported that exogenous vitamin C reduced Pb-induced oxidative damage prevented the degradation of photosynthetic pigments and improved plant physio-biochemical status (Alamri et al., 2018). Another organic ligand we tested is pantothenic acid (vitamin B₅) involved in the synthesis of various metabolic precursors including the 4´-phosphopantetheine moiety of CoA and acyl-carrier protein (Alberts and Vagelos, 1966; Coxon et al., 2005). In plants, both are vital for various metabolic and energy-yielding including Kreb’s cycle, β-oxidation, isoprenoid synthesis, fatty acids synthesis, and biosynthesis of lignin (Kleinkauf, 2000; Miller and Rucker, 2020). Taken together, pantothenate is a metabolically active organic ligand with a metal-binding carboxylate moiety. Lastly, we also tested the chelating ability of aspartic acid for Pb phytoextraction, since amino acids naturally possess metal binding and detoxification abilities (Kocaman, 2023). In plants, aspartic acid can alleviate oxidative stress and detoxify various HMs (Sun et al., 2020). In this context, the combined application of EDTA and aspartic acid in a 1:1 ratio significantly enhanced the phytoextraction of Cd than EDTA-citric acid (Kou et al., 2020). Interestingly, an aspartic acid-derived polymer, polyaspartic acid (PASP) is a prominent metal complexing agent indicated by various studies (He et al., 2019; Ji et al., 2021; Li et al., 2022). Due to its organic nature, aspartic acid and its derived polyaspartic acid are non-toxic and highly biodegradable chelates (Mu’azu et al., 2018). The PASP is a water-soluble anionic polypeptide and is biocompatible (Tabata et al., 2000; Juriga et al., 2016). Nonetheless, aspartate application promoted phytoextraction of Cd in Bidens pilosa (Li et al., 2022), in Pennisetum sp. (Li et al., 2020), Pb and Cd accumulation in Solanum nigrum (He et al., 2019) and multiple other plant species (Zhang et al., 2013, 2019; He et al., 2019; Xie et al., 2021). Its phytoextraction ability is particularly due to hydroxyl and carboxylic functional groups (Tarai et al., 2021). Besides soil amendment with aspartates can improve soil microbial health and can improve soil nutrient availability (Liu et al., 2019; Cao et al., 2021; Ji et al., 2021).

Carefully summarizing this information, all the studied organic compounds have diverse cellular roles in plants and their exogenous supplementation as chelates could benefit plants in dual ways. Since all the molecules of the soil-applied chelates would not bind with Pb due to complex soil interactions, this means that these unbound/ freely available chelating molecules could be taken up by plants. Therefore, we hypothesized that biocompatible chelates could enhance Pb-phytoextraction as well as benefit plants under a Pb-contaminated environment. The primary objectives of this study were to identify biodegradable and eco-friendly chelates that could pave the way for low-cost, eco-friendly cleanup of Pb-contaminated soils.

2.1. Experiment details

The relative effectiveness of four organic chelating agents for Pbphytoextractionby sunflower was investigated in a pot experiment. Plastic pots (30 × 45 cm) were filled with 10 kg soil and various physio-chemical characteristics of the soil were determined (pH 8.1; organic matter 0.65%; total N 0.08%; available P10 ppm; available K207 ppm; saturation 35%; Pb 0.0278 mM and electrical conductivity 2.1 dsm⁻¹). Ten sunflower seeds (variety FH-331) were sown in each pot and after germination, three seedlings per pot were retained. The Pb treatments (added as Pb(NO₃)₂) were applied at control (already detected in the soil as 0.0278 mM) and 4.826 mM Pb concentrations after 20 d of germination. After 10 days of Pb application (30 d old plants), different chelates viz. Control, aspartic acid (D), ascorbic acid (AsA), tartaric acid (TA), and pantothenic acid (PA) were applied to sunflower plants via soil-spiking at 1 mM concentration each. Later, different growth and biochemical parameters were recorded from 60-day-old plants (after 30-d of chelate application).

2.2. Morpho-biochemical analyses from plant samples

a. Plant growth traits

After 30-d of chelate application, the plants were carefully removed from pots. The roots were washed with distilled water and dried by using blotting paper. The analysis of various plant growth parameters was performed by determining the root and shoot length and their fresh and dry biomasses. Additionally, the number of leaves was counted, and the leaf area was calculated.

b. Photosynthetic pigments

Photosynthetic pigments can be determined by the light absorption of chlorophyll solutions (Figure S1). For chlorophyll determination, 100 mg of fresh leaf tissue was first extracted with 80% acetone after overnight incubation. For chlorophylls, the λ_max was recorded at 663 and 645 (Mackinney, 1941; Arnon, 1949), whereas for carotenoids, the OD was recorded at 480 nm (Kirk and Allen, 1965). The following equations were used to calculate leaf pigment concentrations.

C_a= 0.0127D₆₆₃- 0.00269 D₆₄₅ × DF

C_b = 0.0229 D₆₄₅ - 0.00468D₆₆₃× DF

C = C_a + C_b = 0.0202D₆₄₅ + 0.00802 D₆₆₃ × DF

Car = D₄₈₀ + 0.114 D₆₆₃ – 0.638 D₆₄₅ × DF

Where “C_a” and “C_b” represent chlorophyll a and b, carotenoids are denoted by “Car” and the “DF” represents the dilution factor.

c. Cytosolutes

The analysis of different organic solutes was performed. Leaf proline was determined after the extraction of fresh plant material with 5-sulfosalicylic acid (3%). After centrifugation, 2 mL of supernatant was mixed with 2 mL acid-ninhydrin reagent and 2 mL glacial acetic acid followed by mixing and incubation in a boiling water bath for 1 h. After terminating the reaction on ice, 4 mL toluene was added and stirred vigorously to extract free proline. Finally, the OD of the upper pink layer was recorded at 520 nm. Free proline was then calculated.

Proline = [(µg proline/mL × mL toluene) / (115.5 µg/µmole]/ [(g sample/5]

The determination of soluble sugars and total free amino acids was carried out by using plant-EtOH extracts (Shafiq et al., 2019). Soluble sugars were determined by mixing 1 mL of EtOH extracts with 1 mL cold anthrone reagent (0.2% anthrone in 65% H₂SO₄). The OD of the reaction mixture was recorded at 560 nm (Yemm and Willis (1954) and sugars were calculated using a standard curve prepared from D-glucose. The total free amino acids were determined by the method of Moore and Stein (1948). For this, 1 mL of plant-EtOH extracts were mixed with 1 mL of ninhydrin (0.2%) and 1 mL of pyridine solution (10%) and incubated in a boiling water bath for 30 minutes. The OD of the blue solution was recorded at 570 nm and FAA concentration was calculated using a standard curve prepared from L-glycine. Additionally, the total soluble proteins were estimated by using the Bradford (1976) assay.

d. Ionomics

The analysis of various inorganic ions was performed after wet-acid digestion (Netondo et al., 2004). Briefly, a 500 mg dry plant sample was digested at 300 ^oC using a mixture of (HNO₃and H₂O₂) until a clear solution was obtained, which was then filtered and diluted to 25 mL. The concentration of Na⁺, K^+, and Ca²⁺ was performed using a flame photometer (Model Jenway, PFP, UK). The concentration of phosphorus was spectrophotometrically determined from the digested sample by Barton reagent and the sulfur determination was carried out by using barium chloride by turbidity method (AOAC, 1984). For the determination of nitrates, dry leaf material was extracted with de-ionized water after incubation at 45 ^oC for 1 h. The solution was then centrifuged at 5000 rpm for 15 minutes and the supernatant was mixed with salicylic acid under highly acidic conditions. This was followed by the addition of NaOH, and the OD was recorded at 410 nm. The NO₃^- concentration was calculated from a standard curve prepared by using KNO₃ (Cataldo et al. 1975).

2.3. Pb-phytoextraction: Bioaccumulation, translocation, and removal percentage

The Pb²⁺ ions in plant root and shoot samples were determined by Atomic Absorption Spectrophotometer (AAS; Hitachi Polarized Zeeman) and the Pb²⁺ concentrations in sunflower plant parts were calculated.

Pb concentration (μg g⁻¹) = Reading × Dilution factor

Moreover, the bioaccumulation factor (BAF) was calculated.

Different translocation factors were also calculated. The TF_rootrepresents the ratio of metal concentration in the plant root to metal concentration in the soil. Whereas the TF_shootprovides the ratio of metal concentration in the plant shoot to metal concentration in the soil. Finally, the TF_rsgive us the ratio of metal concentration in the plant shoot to metal concentration in the root.

The following formulas were used to calculate the TFs as;

Lastly, the ability of sunflowers to remove Pb from the contaminated soil was estimated as phytoextraction efficiency (PE).

Phytoextraction efficiency percentage (PE %) = (C_i- C_t/ C_i) x 100

Where C_i represents the initial Pb concentration in soil and C_t represents the Pb on t^th-day.

d. Statistical analysis

The pot experiment was conducted in a completely randomized layout (CRD) with three replications per treatment. Data from all the parameters was analyzed by Two-Way ANOVA (CoStat, version 6.2; CoHort Software, Monterey, CA, USA). Mean values were compared using the LSD test at P<0.05. The percentage changes for all the recorded parameters were computed to the respective controls. Whereas the clustered heatmap was generated for both control and Pb-stressed plants separately by using R-studio. All the remaining figures were prepared in Microsoft Excel 365.

3.1. Sunflower biomass and leaf photosynthetic pigments as influenced by chelating agents under Pb-contamination

Soil contamination with Pb did not influence shoot length, root length, root fresh weight, shoot dry weight, and root dry weight (P>0.05), however an increase in the number of leaves and shoot fresh weight was recorded (P<0.05; Table1). Application of all the chelates improved shoot growth characteristics (Table1) and maximum improvements were recorded in response to pantothenic acid under Pb-stress (P<0.001). Improvements in root growth traits under Pb-stress were also recorded in response to pantothenic acid (P<0.001); whereas other chelates also improved root growth traits (P<0.05). Overall, the growth attributes of sunflower followed this trend under the influence of chelates as pantothenic acid > tartaric acid > aspartic acid > ascorbic acid > control. Apart from growth, changes in various photosynthetic pigments under Pb-stress were non-significant (Figure S1). However, both pantothenic and tartaric acid resulted in improvements in total chlorophyll contents under Pb-stress (P<0.05; Figure S1).

3.3. Cytosolutes in sunflower leaf tissues as influenced by chelating agents under Pb-contamination

Total soluble sugars, free amino acids, free proline, and protein content in sunflower leaves in response to soil spiking of Pb remain unaffected (P>0.05). An increase in the leaf-free proline concentration was recorded under Pb-stress in response to all the chelates (Table 2). In addition, the application of both tartaric acid and pantothenic acid improved the concentration of soluble sugars, free amino acids, and soluble proteins (P<0.01). Under control conditions, the pantothenic acid application resulted in slight improvements in the concentration of cytosolutes (Table 2).

3.4. Ionomic profile of sunflower as influenced by chelating agents under Pb-contamination The treatments with no chelates, demonstrated non-significant (P<0.05; Table 3) statistical differences in terms of different ions i.e. nitrates (NO^3-), phosphates (PO₄^3-), sulfates (SO₄^2-), potassium (K⁺), calcium (Ca²⁺) and sodium (Na⁺) in sunflower leaves in response to soil spiking of Pb as well as in control (P<0.05; Table 3). Ascorbic acid and aspartic acid treatments demonstrated higher ionic concentrations of PO₄^3- and K⁺ while non-significant difference in concentration of NO^3-, SO₄^2-, Ca^2+, and Na⁺in response to soil spiking of Pb in comparison to control (P<0.05; Table 3). Tartaric acid and pantothenic acid treatments demonstrated the highest ionic concentration of PO₄^3-, SO₄^2-and K⁺ while non-significant difference in concentration of NO^3-, Ca^2+, and Na⁺in response to soil spiking of Pb in comparison to control (P<0.05; Table 3).

3.5. Uptake of Pb in sunflower plant parts as influenced by chelating agents

The exposure of Pb-contamination resulted in marked Pb-uptake in sunflower plant parts (Figure 1A and B). The concentration of Pb in roots was higher (ranging up to 104 µg g^-1 DW) compared with the shoot Pb fraction (64 µg g^-1 DW). Soil spiking with chelates substantially enhanced root Pb-uptake among sunflower plants (P<0.001). Under Pb-stress, the maximum concentration of Pb in roots was promoted by tartaric acid soil amendment (up to 191µg g^-1 DW; +45.5%) followed by pantothenic acid (177.3 µg g^-1 DW; +41.3%). Not only this, the ascorbic acid and aspartic acids also enhanced Pb-uptake in sunflower roots by over 38% respectively (P<0.001; Figure 1A). In terms of shoot, maximum Pb-uptake was promoted by tartaric acid (131.6 µg g^-1 DW; +51.3%) followed by pantothenic acid (123.6 µg g^-1 DW; +47.9%), ascorbic acid (117 µg g^-1 DW; +45.2%) and aspartic acid (112.6 µg g^-1 DW; +45.2%) respectively (P<0.001; Figure 1B). Comparative analysis of different physio-biochemical attributes revealed that Pb stress caused prominent negative effects on Ca²⁺, P, S, and cytosolutes. However, an increase in plant biomass and free proline was also evident (Figure 1C).

3.6. Pb-phytoextraction and relative effectiveness of different organic ligands

Maximum Pb-phytoextraction efficiency (%) and bioaccumulation factor were caused by tartaric acid followed by pantothenic, ascorbic, and aspartic acids respectively (P<0.001). All four chelates promoted Pb-uptake in root and shoot from soil (Table-4; P<0.05); whereas the root/shoot transfer of Pb was insignificant among chelates (P>0.05; Table-4). The relative effectiveness of different chelates in terms of their effects on all the morpho-biochemical and ionomic traits (Figure 2A-D). Both tartaric and pantothenic acid promoted maximum recovery in Ca, S, and P uptake (Figure 2C and D). Moreover, a prominent recovery in some key biochemical traits promoted Pb-uptake. A clustered heatmap of all traits after the application of different chelates under control and Pb-stress is presented in Figure 3.

Removal of Pb from the soil is a challenging task due to its lower mobility in the soil-plant system compared with other toxic metals. From soil, Pb is mainly accumulated by plant roots, and its translocation to above-ground plant parts is generally low. About 90% of the Pb taken up by the plants is primarily retained inside plant roots (Pourrut et al., 2011) which makes it challenging for Pb-phytoextraction without using suitable plant species and complexing agents. In the present study, we tested the changes in the phytoextraction potential of sunflowers in response to some biocompatible organic complexing agents under Pb stress.

a). Effect of Pb-toxicity and chelates on growth, pigments, and cytosolutes

The sunflower plants exposed to Pb at 4.826 mM did not experience a reduction in the root growth traits whereas an increase in the number of leaves and shoot fresh biomass was recorded. The applied chelates also promoted growth traits differentially and maximum improvements were recorded in response to pantothenic acid application followed by tartaric acid. Moreover, an increase in the chlorophyll contents under Pb stress due to tartaric acid and pantothenic acid application. It has been reported earlier that exposure to Pb does not always reduce plant growth (Ejaz et al., 2022; Ngugi et al., 2022). Findings of our previous experiments also depicted that Pb contamination at levels above 4 mM stimulated the growth of spinach (Khan et al., 2016) and turnip (Khan et al., 2022) potentially due to hormesis. Such findings of increased growth in response to toxic metal contaminants have been reported earlier in many plants (Hajiboland et al., 2013; Durenne et al., 2018; Salinitro et al., 2021). Hormetic mechanisms usually contribute to accelerated growth rates after initial impairment of cellular homeostasis followed by defensive and adaptive responses leading to growth overcompensation (Li et al., 2022; Qin et al., 2022). Chelating agents can also enhance plant growth under Pb stress (Harmon, 2022; Kalyvas et al., 2022). Furthermore, the tartaric and pantothenic acid-mediated improvements in the total chlorophyll contents could be due to hormetic mechanisms necessary to cope with limited photochemical efficiency under Pb stress (Agathokleous et al., 2020; Khan et al., 2022). Apart from this, an increase in the leaf-free proline concentration was recorded under the Pb-stress in response to all the chelates. Maximum concentration in soluble sugars, free amino acids, and proteins was recorded in response to tartaric acid and pantothenic acid. Likewise, the concentration of different cytosolutes was increased by chelates application (Samy, 2022).

b). Ionomics: Chelate-assisted Pb-phytoextraction in sunflower

The Pb contamination at 4.826 mM did not affect the concentration of macronutrients including NO₃^--N, P, K, Ca, and S in sunflower leaves. By contrast, the application of all four organic acids (ascorbic, aspartic, tartaric, and pantothenic acids) improved the uptake of macronutrients in the leaf especially Ca, nitrate-N, P, and S. The beneficial role of different chelates in the uptake of micronutrients is well reported (Welch and Shuman, 1995; López-Rayo et al., 2016). However, how chelating agents alter macronutrient uptake is poorly understood. Based on our findings, we proposed that the two organic chelates aspartic and pantothenic acids would directly serve as N sources. Whereas the uptake of P, K, Ca, and S could be due to chelate-mediated improvements in plant root system architecture. It is pertinent to mention both tartaric and pantothenic acids used in this study improved sunflower root growth characteristics. Positive regulation of root system architecture is directly linked to the uptake of N, P, K, Ca, and S and nutrient use efficiency (Jose, 2023).

In the absence of any chelate, the sunflower roots accumulated Pb up to 104 µg g^− 1 DW whereas the Pb fraction in the shoot was 64 µg g^− 1 DW. So, shoot Pb concentration was almost half compared with Pb in root. As reported earlier, Pb in plants is pre-dominantly retained by roots and for some plants, root-Pb accumulation can be up to 90% of the total Pb-uptake (Pourrut et al., 2011). Besides, it is not a very mobile element within soils which limits its uptake in plants as well (Chen et al., 2019). Earlier we found that turnip plants when exposed to 4.83 mM Pb accumulated 132.4 µg g^− 1 DW Pb in the root while 273.1 µg g^− 1 DW Pb in the shoot (Khan et al., 2019). Whereas spinach plants grown at 4.83 mM Pb level had 237.6 µg g^− 1 DW Pb in root while 113.5 µg g^− 1 DW Pb in shoot (Khan et al., 2016). On a comparative basis, three different plant species that exhibit a tap root system but with various modifications might influence Pb-uptake capacities. Spinach develops extensive branching into vertical and seminal roots extending from the main tap root. Whereas the fibrous root system of the sunflower mainly originating from the embryonic taproot limits Pb-uptake in roots (Strubińska and Hanaka, 2011). In the case of turnip, the higher potential to accumulate Pb in the shoot can be related to the storage tap root modification. The increased lateral growth of turnip might damage casparian strips leading to relatively higher Pb contaminant transfer in shoot.

Nonetheless, to enhance the phytoavailable fraction of Pb, the use of different complexing agents has been recommended (Evangelou et al., 2007; Khan et al., 2019; Zulkernain et al., 2023; Chen et al., 2024). The use of all four chelates, aspartic, ascorbic, pantothenic, and tartaric acids promoted Pb-uptake in sunflower roots by 38, 39, 41.3 and 45.5% whereas shoot Pb-fraction was promoted by 43.1, 45.2, 47.9 and 51.3% respectively. Additionally, maximum Pb in root (191 µg g^− 1 DW) and shoot (131.6 µg g^− 1 DW) was recorded due to tartaric acid. Tartaricis naturally synthesized by certain plant species through the catabolism of ascorbic acid and is secreted in the rhizosphere by plant roots through root exudates (Melino et al., 2009; Lu et al., 2013). Its exudation and Cd binding affinity have been well-reported in Sedum alfredii (a hyperaccumulator) by Tao et al. (2019). Compared with citric, malic, oxalic acids, and EDTA, we also found that tartaric acid enhanced Pb phytoextraction in spinach and turnip (Khan et al., 2016; 2022). Interestingly, ascorbic acid-mediated Pb-phytoextraction was less compared to tartaric acid application indicating that exogenous vitamin C to sunflower might not contribute to endogenous tartaric acid synthesis, but this remained untested in this study. Also, the synthesis of tartaric acid occurs only in members of some families including Vitaceae and Geraniaceae (Narnoliya et al., 2018).

Apart from this, the use of pantothenic acid was quite effective in enhancing Pb-uptake in sunflower roots and shoots. In plants, pantothenic acid is involved in various metabolic and energy-yielding including Kreb’s cycle, β-oxidation, isoprenoid synthesis, fatty acids synthesis, and biosynthesis of lignin (Kleinkauf, 2000; Miller and Rucker, 2020). Our results indicated that pantothenic acid soil spiking enhanced Ca, P, and S uptake in sunflowers under Pb stress. Calcium acts as a secondary messenger in plants and pantothenic acid-mediated enhancements in Ca might have contributed to regulation of Pb-detoxification pathways. In agreement with this, it has been reported that Ca²⁺-dependent plant responses to Pb²⁺ are regulated by Low-Affinity Cation Transporter 1 leading to Pb stress tolerance (Wojas et al., 2007). In our experiments, the concentration of Ca²⁺ was also enhanced due to tartaric acid application which also contributed to the highest Pb-phytoextraction among four chelates. Also, both tartaric and pantothenic acids contributed to higher P contents and Pb-tolerance which can be explained by phytochelatins by binding with PO₄³⁻ ions. The phosphate-induced reduction in free Pb²⁺ through P-conjugation and phytochelatin synthesis has been reported in Vetiveria zizanioides L. (Andra et al., 2010).

Lastly, aspartic acid in our study promoted 70% Pb-uptake in shoot and ranked in 3rd place (tartaric acid > 95%; pantothenic acid > 90%) but it was better than ascorbic acid (> 40%). As mentioned earlier, the carboxylic moiety of aspartic acid bonds with Pb and forms stable complexes that can be transported through xylem vessels. Besides, it can also improve soil health and nutrient availability (Liu et al., 2019; Cao et al., 2021). Nonetheless, aspartic acid promoted Pb phytoextraction in different plant species (Zhang et al., 2013; He et al., 2019; Xie et al., 2021). Also, its polymer, polyaspartic acid (PASP) is a biocompatible and biodegradable chelate with significant metal binding ability (Tabata et al., 2000; Mu’azu et al., 2018; He et al., 2019; Ji et al., 2021; Li et al., 2022). Above all, our results rank tartaric acid as the most suitable organic ligand for Pb-phytoextraction, followed by pantothenic, aspartic, and ascorbic acids.

Upon testing the influence of some novel chelating agents for Pb-phytoextraction using sunflower and concluded that tartaric acid promoted a 47.9% Pb accumulation in sunflower plants followed by pantothenic acid (44.%). Least effective among the chelates was aspartic acid which contributed to a + 40% increase in Pb uptake. Above all, we also concluded that these enhancements in the total Pb uptake by chelates are primarily due to improvements in plant ionomics (especially Ca, P, and S) which potentially contributed to the regulation of Pb-toxicity through signaling and redox mechanisms. Our results therefore highlight the potential of using biocompatible organic chelates to clean Pb-contaminated sites using sunflower plants.

Funding support and acknowledgments

The study was conducted in the absence of any financial support and was part of the Ph.D. dissertation of Mr. Asif Ghafoor. All the chemicals and resources were provided by the University of Lahore, Pakistan.

Ethical Approval

Not applicable

Consent to Participate

Not applicable

Consent to Publish

All the authors agree to its submission and publication in Environmental Science and Pollution Research.

Competing Interests

The authors declare that they have no competing financial interests of any sort.

Authors Contributions

AG performed the experiments, analyses and collected the data. FS planned the experiment and wrote the initial draft of this manuscript. SA contributed to data analysis and visualization. Both MA and LZ supervised the project and updated the manuscript.

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Table 1: Relative changes in some growth traits of sunflower in response to soil spiking of Pb and to four different organic ligands. Morphological parameters were recorded after 30-days of chelate application (Mean, n = 3; ± SE).

Source of variation		Shoot length		Root length	Shoot FW	Root FW	Shoot DW	Root DW	Leaves	Leaf area
Source of variation		(cm)	(cm)		(g)	(g)	(g)	(g)		(cm²)
0.0278 mM Pb	No chelate	64.0^f+0.5	11.3^c+0.7		29.2^h+0.5	12.4^ef+0.6	15.5^g+0.7	6.19^ef+0.3	21^c	42.1^ef+2.1
	Ascorbic acid	74.6^de+1.1	12.7^c+0.9		42.6^fg+1.1	14.3^de+0.6	22.2^fg+1.1	7.14^de+0.3	22^c	48.5^de+1.9
	Aspartic acid	78.3^d+1.0	15.3^bc+1.2		46.1^ef+2.6	15.5^cd+0.6	25.7^ef+2.6	7.70^cd+0.3	30^a	52.4^cd+2.0
	Tartaric acid	83.2^bc+1.3	17.0^b+0.7		53.3^cd+1.8	16.4^c+0.6	32.9^cd+1.8	8.16^c+0.3	23^bc	55.5^c+2.0
	Pantothenic acid	85.3^b+1.8	17.5^b+1.5		55.6^bc+1.0	18.5^b+0.5	35.2^bc+1.0	9.18^b+0.3	24^bc	62.4^b+1.7
4.826 mM Pb	No chelate	69.5^f+1.5	13.2^c+1.0		41.4^g+1.2	12.3^f+0.4	21.0^g+1.2	6.14^f+0.2	25^b	41.8^f+1.5
	Ascorbic acid	73.8^e+1.5	14.1^c+0.5		49.4^de+1.9	15.2^cd+0.6	29.0^de+1.9	7.58^cd+0.3	22^c	51.5^cd+2.2
	Aspartic acid	82.1^c+1.4	15.3^bc+1.5		56.4^bc+1.9	16.4^c+0.6	35.9^bc+1.9	8.13^c+0.3	23^c	55.3^c+2.1
	Tartaric acid	86.1^b+1.3	17.0^b+1.6		57.8^b+2.5	18.2^b+1.0	37.4^b+2.5	9.40^b+0.5	23^c	61.5^b+3.5
	Pantothenic acid	94.6^a+2.4	20.4^a+1.4		61.5^a+1.8	20.1^a+0.7	41.1^a+1.8	10.0^a+0.3	24^bc	69.0^a+2.2

Different lowercase letter(s) within a column differ significantly at 95% confidence index (LSD_0.05). FW, Fresh weight; DW, Dry weight

Table 2: Relative concentration of some cell cytosolutes in sunflower leaves in response to soil spiking of Pb and to four different organic ligands. Analysis of biochemical traits were performed from leaves collected after 30-d of chelate application (Mean, n = 3; ± SE).

Source of variation		TSS	TFA	Proline	Soluble proteins
Source of variation		(mg g^-1 FW)	(mg g^-1 FW)	(µg g^-1 FW)	(mg g^-1 FW)
0.0278 mM Pb	No chelate	1.358^ab+0.35	20.16^cd+0.32	12.95^c+0.23	62.23^d+1.67
	Ascorbic acid	1.505^ab+0.42	22.33^c+0.93	14.20^c+0.10	74.41^cd+10.1
	Aspartic acid	1.559^ab+0.32	22.59^c+1.26	13.84^c+0.44	74.10^d+5.00
	Tartaric acid	1.870^a+0.35	25.49^abc+1.59	13.76^c+0.68	83.55^cd+7.71
	Pantothenic acid	1.666^ab+0.24	24.29^bc+2.47	13.54^c+0.85	79.30^cd+3.87
4.826 mM Pb	No chelate	1.206^b+0.20	17.77^d+0.78	15.55^c+1.57	68.17^d+4.26
	Ascorbic acid	1.617^ab+0.10	24.21^bc+0.98	18.30^b+0.51	86.11^cd+9.49
	Aspartic acid	1.675^ab+0.11	24.39^bc+1.41	18.88^ab+0.58	90.32^bc+7.17
	Tartaric acid	2.107^a+0.22	28.16^a+1.16	20.13^a+0.34	102.06^a+3.93
	Pantothenic acid	1.913^a+0.10	26.37^ab+1.97	19.08^ab+0.67	97.50^ab+2.36

Different lowercase letter(s) within a column differ significantly at 95% confidence index (LSD_0.05). TSS, Total soluble sugars; TFA, Total free amino acids; FW, Fresh weight

Table 3: Ionomic profile of sunflower leaf tissue in response to soil spiking of Pb and four different organic ligands. Analysis of biochemical traits were performed from leaves collected after 30 days of chelate application (Mean, n = 3; ± SE).

Source of variation		Na⁺		K⁺	Ca²⁺	NO₃^-	PO₄^3-	SO₄^2-
Source of variation		(mg g^-1 DW)	(mg g^-1 DW)		(mg g^-1 DW)	(mg g^-1 DW)	(mg g^-1 DW)	(mg g^-1 DW)
0.0278 mM Pb	No chelate	7.4^abc+0.2	26.2^ab+0.3		9.6^de+0.1	15.0^ab+1.1	8.2^f+0.5	8.5^cd+0.3
	Ascorbic acid	6.6^abc+0.6	25.9^b+0.3		11.1^de+1.1	15.8^ab+0.4	10.5^e+0.2	10.2^bc+0.6
	Aspartic acid	6.5^bc+0.7	25.9^b+0.3		11.4^cd+0.9	15.8^ab+0.3	10.6^e+0.2	10.4^bc+0.5
	Tartaric acid	6.7^abc+0.6	25.0^b+0.3		13.4^ab+0.5	16.4^ab+0.8	13.1^c+0.1	11.4^b+0.5
	Pantothenic acid	6.5^bc+0.8	25.3^b+0.3		12.6^bc+0.6	16.1^ab+0.2	12.9^cd+0.3	10.7^bc+0.6
4.826 mM Pb	No chelate	8.2^a+0.6	27.1^a+0.3		7.4^e+0.8	14.9^b+1.3	7.3^f+0.4	7.2^d+0.4
	Ascorbic acid	7.5^ab+0.7	26.8^a+0.3		12.7^bc+0.6	16.6^a+0.5	12.2^d+0.5	11.2^b+0.5
	Aspartic acid	7.5^ab+0.4	26.8^a+0.3		12.6^bc+0.4	16.8^a+0.5	12.3^d+0.5	11.3^b+0.6
	Tartaric acid	6.4^c+0.5	25.9^b+0.3		14.5^a+0.5	17.0^a+0.7	14.9^a+0.4	13.4^a+0.4
	Pantothenic acid	7.0^abc+0.4	26.2^b+0.3		13.6^ab+0.3	17.0^a+0.5	14.2^b+0.3	12.9^a+0.9

Different lowercase letter(s) within a column differ significantly at 95% confidence index (LSD_0.05). DW, Dry weight; Na⁺, Sodium; K⁺, Potassium; Ca²⁺, Calcium; NO₃^-, Nitrates; PO₄^3-, Phosphate; SO₄^2-, Sulfates

Table 4: Relative effectiveness of various organic ligands on various key phytoextraction indicators with reference to the ability of sunflower to remediate Pb-contaminated soils.

Source of variation		PE (%)		BAF	TF (Root/Soil)	TF (Shoot/Soil)	TF (Root /Shoot)
Source of variation
4.826 mM Pb	No chelate	42.0^h+0.6	0.72^e+0.0		0.45^a+0.0	0.28^g+0.0	0.62^a+0.1
	Ascorbic acid	71.9^c+0.4	2.56^c+0.0		1.52^a+0.0	1.04^c+0.0	0.69^a+0.0
	Aspartic acid	70.2^d+0.3	2.35^c+0.0		1.41^a+0.0	0.94^d+0.0	0.67^a+0.0
	Tartaric acid	80.7^a+0.2	4.17^a+0.1		2.47^a+0.1	1.70^a+0.0	0.69^a+0.0
	Pantothenic acid	75.1^b+1.5	3.05^b+0.3		1.80^a+0.2	1.25^b+0.1	0.70^a+0.0

Different lowercase letter(s) within a column differ significantly at 95% confidence index (LSD_0.05). PE, Phytoextraction efficiency; BAF, Bioaccumulation factor; TF, Translocation factor.

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Graphical Abstract

Both tartaric and pantothenic acids promote Pb-phytoextraction potential of sunflower by regulating calcium and phosphorus uptake

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Abstract

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1. Introduction

2. Materials and Methods

3. Results

4. Discussion

Conclusions

Declarations

References

Tables

Supplementary Files

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