Antioxidant Systems Response, Mineral Element Uptake and Safe Utilization of Polygonatum Sibiricum in Cadmium-Contaminated Soil

Chinese herbal medicine is widely cultivated in southwest China where the soil cadmium (Cd) contamination of farmland is more serious than that in the whole of China. In this study, Polygonatum sibiricum were exposed to Cd at the concentrations of e − 1 , e 0 , e 2 , and e 4 mg·kg − 1 for up to 30,60, and 90 days and their physiological stress responses, Cd and mineral element uptake, antioxidant enzyme activities, and content changes of pharmaceutical ingredients (polysaccharides) were analyzed to decipher the feasibility of safety use in Cd contaminated soil. Results showed that the activity of antioxidant enzymes (SOD, POD, and CAT) of the aboveground part was enhanced in response to Cd stress after 90 d. Compared with the control, the underground part mobilizes non-enzymatic systems to facilitate the synthesis of polysaccharides (PCP1, PCP2) with antioxidant properties to cope with Cd stress. Mineral elements (P, K, Ca, Mg, Fe, Cu, and Zn) were signicantly changed after 90 d of cultivation. In particular, the changes in iron and zinc contents were signicantly correlated with the activities of SOD and POD. The soil Cd safety thresholds value for Polygonatum sibiricum is e 0 mg·kg − 1 , under which concentration the stimulation of Cd promotes polysaccharides synthesis and biomass growth. amarus under moderate chromium (Cr) stimulation produced more of the therapeutically active secondary metabolites – phyllanthin and hypophyllanthin 16 . A similar phenomenon occurs with another medicinal plant Vaccinium corymbosum and its antioxidant response is activated leading to an increase in phenolic compounds under the Cd stress 17 . However, this “incentive effect” is not costless. Medicinal plants may lose the ability to synthesize active ingredients at high concentrations of heavy metals. Seedlings of St. John's wort completely lose the ability to synthesize or accumulate hyperforin and the concentration of pseudohypericin and hypericin demonstrate a 15 to 20-fold decrease 18 . The mechanism of Cd tolerance in low-accumulating plants is rarely studied, and the response of their antioxidant active components to Cd is not clear yet, which calls into question how to ensure the safe use of low accumulation plants in Cd-contaminated soils. The plant mineral element uptake and tolerance for Cd vary between different plant species, plant growth phases, and soil Cd concentrations, hence it is necessary to systematically study the behavior of low-accumulation plants under Cd stress. This study was specically designed to (1) Determine the physiological stress responses and interaction between enzymatic and non-enzymatic antioxidant systems of Polygonatum sibiricum under Cd stress, (2) Analyse Cd and mineral element uptake situation and polysaccharides content change, (3) Present a soil Cd safety thresholds value for Polygonatum sibiricum growth. part, with a few experiments considering the differences between aboveground and underground antioxidant enzymes. The aboveground SOD activity of Polygonatum sibiricum was similar to that of most plants, but the SOD activity of the underground parts was lower than that of the control group under a higher Cd level (e 0 , e 2 , and e 4 treatment), which showed a difference. The results showed that the response thresholds of SOD, POD, CAT to Cd stimulation are different, and the correlation between the effect of Cd stimulation on the activities of antioxidant enzymes and the concentration of Cd in plants is always variant. Some researchers suggest that Cd inhibits the activity of antioxidant enzymes 27 , and some show that Cd stress could activate antioxidant enzymes 28 , even in some study, the aboveground and underground parts of the same plant have different responses to antioxidant enzyme activity 29 . At the same time, the changes of CAT and POD activity are not uniform, which indicates antioxidant enzyme activities and plant species are also related, exploiting different tolerance behaviours to alleviate Cd-induced oxidative stress.


Introduction
Southwest China is one of the main production areas of Chinese herbal medicine 1 , but the region has a long history of discharge of Cd-containing wastewater from zinc smelting, waste dumping, and the application of phosphate fertilizers with high Cd content, resulting in compound soil pollution of heavy metals is particularly prominent 2 . Among the various methods for remediation of contaminated soil, phytoremediation uses hyperaccumulation to extract heavy metals from the soil. Although this method is eco-friendly to eliminate heavy metals or other hazardous chemicals over other physical and chemical soil remediation techniques and does not cause secondary soil contamination 3 , it is di cult to promote phytoremediation techniques due to its long restoration cycle and the fact that the economic bene ts of cultivated land cannot be guaranteed during the restoration cycle 4 . The purpose of agricultural land soil remediation is to ensure the safety of production and utilization of agricultural products 5 . Using the plants with high economic value and low heavy metal accumulation could break through the current dilemma of plant remediation and has great application potential.
In preliminary studies, we found that the Chinese herbal medicine Polygonatum sibiricum has the characteristic of low accumulation of Cd, and because it is rich in polysaccharides and other medicinal active components, it has a high added value and is an advantageous plant for the safe use of Cd-contaminated soil. For "low accumulation" cash crops, the antioxidant system is the main mechanism of resistance to heavy metal stress 6 . Heavy metals induce oxidative stress by generating free radicals and reactive oxygen species (ROS), which can interact with lipids, proteins, pigments, and nucleic acids, leading to lipid peroxidation and damage to cell membranes, impairing cellular physiology and the ability to adapt to the environment 7 . Harmful effects of the oxidative state of cells can be mitigated by enzymatic and non-enzymatic antioxidant effects in plants (Singh et al. 2006 10 . Numerous studies showing that polysaccharides especially heteropolysaccharides containing proteins and phenols also have antioxidant properties [11][12][13] . So, this research put polysaccharides, the main material to evaluate the economic value of Chinese herbal medicine 14 , into the non-enzymatic antioxidant system to conduct research and analysis. Several studies have found that Cd has the potential to induce the synthesis of plant metabolites 15 , including certain pharmacologically active substances with antioxidant properties. Phyllanthus amarus under moderate chromium (Cr) stimulation produced more of the therapeutically active secondary metabolites -phyllanthin and hypophyllanthin 16 . A similar phenomenon occurs with another medicinal plant Vaccinium corymbosum and its antioxidant response is activated leading to an increase in phenolic compounds under the Cd stress 17 . However, this "incentive effect" is not costless. Medicinal plants may lose the ability to synthesize active ingredients at high concentrations of heavy metals. Seedlings of St. John's wort completely lose the ability to synthesize or accumulate hyperforin and the concentration of pseudohypericin and hypericin demonstrate a 15 to 20-fold decrease 18 .
The mechanism of Cd tolerance in low-accumulating plants is rarely studied, and the response of their antioxidant active components to Cd is not clear yet, which calls into question how to ensure the safe use of low accumulation plants in Cd-contaminated soils. The plant mineral element uptake and tolerance for Cd vary between different plant species, plant growth phases, and soil Cd concentrations, hence it is necessary to systematically study the behavior of lowaccumulation plants under Cd stress. This study was speci cally designed to (1) Determine the physiological stress responses and interaction between enzymatic and non-enzymatic antioxidant systems of Polygonatum sibiricum under Cd stress, (2) Analyse Cd and mineral element uptake situation and polysaccharides content change, (3) Present a soil Cd safety thresholds value for Polygonatum sibiricum growth.

Plant material and growth conditions
Experiments were conducted in soil-cultivated pots, and Polygonatum sibiricum was cultivated in wooden pots of 35 cm × 35 cm × 20 cm, each with a soil mass of 15 kg. The experimental soil for potted plants was collected from the Soil Contamination Remediation Project site in Mianzhu, Sichuan Province, with 20% (volumetric ratio) of humus and 5% (mass ratio) of sulfuric acid-type NPK fertilizer added to the soil (total Cd 0.12 mg·kg -1 , available Cd 0.029 mg·kg -1 , pH 7.4). The Cd in the experimental soil was added in the form of CdCl 2 -2.5H 2 O, and the designed soil Cd concentration gradients were CK-0 mg·kg -1 , e -1 -0.37 mg·kg -1 , e 0 -1 mg·kg -1 , e 2 -7.39 mg·kg -1 , and e 4 -54.60 mg·kg -1 . The 2-year-old seedlings of Polygonatum sibiricum were harvested from a traditional Chinese medicine cultivation base in Neijiang City, Sichuan Province. The collection of Polygonatum sibiricum complies with guidelines in Sichuan province and regulations in China. Plants were transplanted to pots after two weeks of soil equilibration then exposed to Cd stress. From each pot, three plants were randomly collected after 30, 60, and 90 d of cultivation for the measurement of plant biomass, Cd content and mineral element uptake, and polysaccharide content, and the other three plants were collected for the measurement of the antioxidant system parameters. The roots of all plants were soaked in 0.01 mol·L -1 EDTA-2Na solution for 10 minutes to remove heavy metal ions and precipitates adsorbed on the surface.

Cd and mineral element contents measurement
Weigh the dried root, rhizome, stem, and leaf samples by 0.1 g using an analytical balance. Samples were placed in a crucible with 10 ml of HNO 3 and 2 ml of HClO 4 overnight, then digested on an electric plate until nearly dry and transferred to a 15 ml centrifuge tube, which was xed with 1% HNO 3 to 15 ml. Samples were analysed by inductively coupled plasma mass spectrometer (SHIMADZU ICPE-9000, JPN).

Enzyme and polysaccharides antioxidant activity analysis
The activity of SOD was determined according to the method of Jia, et al. 19 . The activity of CAT and POD was evaluated using the improved methods by Azevedo, et al. 20 . Using the pyrogallol autoxidation method according to Zhang, et al. 21 to determine the antioxidant activity of polysaccharides.

Extraction of Polysaccharides
Using the method of graded extraction, 0.1g of dried avin was taken, and the residue was degreased by re uxing at 80℃ for 24 h. The residue was dried to obtain the defatted avin sample. The sample was decocted in 10 mL of distilled water for 2 h each time, sonicated for 1 h. The ltrate was ltered, combined, and transferred to a 50 ml ask, where the liquid polysaccharide sample was PCP1. Taking the rst stage of ltration and adding 0.1% NaOH solution to extract, did the same steps as above and acquired the polysaccharide sample called PCP2. The second ltrate was extracted by adding 0.5% NaOH solution, repeated the same steps as step 2 then obtained the polysaccharide sample named PCP3.

Determination of polysaccharide content and molecular weight
The glucose solution was dried to constant weight (105°C), 33 mg was taken and transferred to a 100 ml ask. Add 0.2% anthrone -sulfuric acid solution in an ice water bath and slowly add the solution to the scale, mix well and let it cool for 10 min in a 100℃-water bath, then immediately put it in an ice-water bath. The absorbance at 582 nm was measured by UV-Vis spectrophotometer (MAPADA UV-6100S, CHN) for 10 min. The polysaccharide liquid sample was treated as above, the absorbance was measured and the polysaccharide content was calculated against the standard curve. Polysaccharide molecular weight was determined by high-performance gel permeation chromatography according to Peng, et al. 22 .

Statistical analysis
Collection and aggregation of raw data using Excel and mapping using Origin 9.0 software. The biomass, Cd, and mineral element content, enzyme activities, and contents of polysaccharide data were subjected to correlation analysis, one-factor ANOVA, and Duncan multiple using GraphPad Prism 8.0 software.

Biomass and plants height
The aboveground (stems and leaves) biomass and underground (roots and rhizomes) biomass showed opposite changes after 30 d cultivation (Fig.1A -B): the biomass of underground parts was smaller than that of the control group, while the biomass of aboveground showed a growth trend as the Cd concentration increases. But the negative in uence of biomass occurred at e 2 and e 4 treatment after 90 d of cultivation. In comparison to CK treatment, the biomass of e 2 and e 4 groups were decreased by 40.22% and 63.90% (underground biomass), and by 33.27% and 53.85% (total biomass), respectively.
However, compared with CK treatment, the biomass of underground part and total plants exposed to e -1 and e 0 treatment was increased by 24.03% and 25.41% (underground biomass) and by 18.66% and 22.23% (total biomass), respectively, after 90 d of cultivation (Fig. 1B -C). The aboveground biomass (Fig.1A) and plant height (Fig.1D) showed a stable growth state at all Cd treatments after 90 days of cultivation. This phenomenon indicates that soil Cd at the concentration of e -1 and e 0 mg/kg has positive effects on the growth of the whole plant and Polygonatum sibiricum exhibited good tolerance to Cd during persistent interaction with Cd in the soil.  (Fig.2B), was lower than the limit value of Cd content in Pharmacopoeia of the People's Republic of China but it fails to meet the requirements in e 2 and e 4 treatment due to an excessive soil Cd concentration. After 90 d of cultivation, the Cd content in stems and leaves was higher than 30 d of cultivation and increased as Cd levels increased (Fig.2C -D). Previous studies have also shown that the roots could be the highest Cd content parts of plants 23,24 because roots are the primary organs in the response to Cd stress in soil and Cd can complex with proteins, cellulose or pectates, or insoluble Cd-phosphate in the root cell wall 25 . This characteristic of Cd uptake of roots is consistent with the accumulation of heavy metals in root-hoarding plants. Root-hoarding plants store heavy metals mainly in the roots, and only a small amount of heavy metal is transferred to the ground that reduces damage to the photosynthetic, respiratory, and reproductive systems 26 . This "root-retention" characteristic of Polygonatum sibiricum is bene cial to improve survivability in Cd-contaminated soil and ensure the safety of medicinal parts.

Antioxidant enzyme system
Aboveground and underground parts showed different patterns of SOD and POD activity (Fig.3A -B). For the aboveground part after 30 d of cultivation, SOD activity increased with Cd levels raise, and it was maximum high at e 4 treatment, which was 52.17% higher than that of the control group. After 90 d of cultivation, the activity of SOD could attach to 1.47, 1.45, and1.27 times higher than CK in the e -1 , e 0 , e 2 treatment, respectively. However, for the underground parts, the high Cd treatment shows lower SOD activity through the full cultivation time. Furthermore, the correlation coe cients revealed that there was a negative correlation between the underground parts Cd content and SOD activity (r = -0.5538, p < 0.05) ( Table 1), indicating that the response of SOD to Cd is suppressed slightly. The POD activity of aboveground/underground parts increased/decreased as the Cd levels increased and the maximum and minimum activity both occurred in e 4 treatment after 30 d of cultivation. After 90 d of cultivation, the POD activity was 6.41 and 6.47 times higher than CK in e 2 and e 4 treatment. As shown in Fig. 3C, the CAT activities increased as the cultivation times increased, and the aboveground enzyme activity was higher than that of the underground part. Only after 60 d of cultivation, Cd demonstrates a stimulating effect on enzyme activity.
Typically, studies of plant antioxidant enzyme activity focused on the aboveground part, with a few experiments considering the differences between aboveground and underground antioxidant enzymes. The aboveground SOD activity of Polygonatum sibiricum was similar to that of most plants, but the SOD activity of the underground parts was lower than that of the control group under a higher Cd level (e 0 , e 2 , and e 4 treatment), which showed a difference. The results showed that the response thresholds of SOD, POD, CAT to Cd stimulation are different, and the correlation between the effect of Cd stimulation on the activities of antioxidant enzymes and the concentration of Cd in plants is always variant. Some researchers suggest that Cd inhibits the activity of antioxidant enzymes 27 , and some show that Cd stress could activate antioxidant enzymes 28 , even in some study, the aboveground and underground parts of the same plant have different responses to antioxidant enzyme activity 29 . At the same time, the changes of CAT and POD activity are not uniform, which indicates antioxidant enzyme activities and plant species are also related, exploiting different tolerance behaviours to alleviate Cd-induced oxidative stress. mineral element uptake The changing macronutrients (P, K, Ca, and Mg) in Polygonatum sibiricum are shown in Fig. 4A-D in response to the Cd stress. Phosphorus (P) is an essential macronutrient that not only supports plant growth but also reduces the toxicity of cadmium by chelating or forming complexes with cadmium in plants, thereby reducing the damage to cell function caused by Cd 30 . In e -1 and e 0 treatment, the P content was increased by 27.6%, 17.7%, respectively, and 27.9%, 39.32% respectively, after 30 and 60 d of cultivation compared to CK treatment. But under higher Cd stress (e 2 and e 4 ) and long-term Cd stress of 90 d, the P content was declined. This indicates that Cd can affect the uptake and accumulation of elemental P in Polygonatum sibiricum while P was described as having no effect on Cd uptake 31 .

Potassium(K) is the most abundant inorganic cation in plant cells (Benito et al. 2014
). The K content in all treatments reached maximum value after 90 d of cultivation and, to varying degrees, could nd a facilitative effect of Cd on K uptake except for the e 4 treatment. The phenomenon might be related to the ability of Cd can increase the in ux of K + ions by binding to K channels and opening them permanently 32,33 while the complexation of ATP with CD proved that the absorption of K decreased and the available energy of membrane transport system decreased lead to disruption in the plasma membrane and caused the decline of K under Cd concentrations as a result of K leakage 34 .
Calcium (Ca) content was signi cantly promoted by Cd stress and increased by 140.03%, 101.25%, 27.11%, and 38.35%, respectively, in e -1 , e 0 , e 2, and e 4 treatment after 60 d of cultivation. However, it was altered after 90 d of cultivation that Ca uptake was inhibited except e 2 treatment. It has been reported that the Ca content in plants growing in Cd-contaminated solutions is reduced in different plants, possibly due to competition between Cd 2+ and divalent cations during the absorption process 35,36 . But researches also showed that the action of Cd on Ca channels and transporter proteins lead to an increase in their transcription and translation, thus allowing greater Ca uptake and compensating for the blocking effect of Ca channels 32 . Thus, it can be seen that the interaction between Ca and Cd is adjusted according to the concentration of Cd and the duration of stress.
Magnesium (Mg) content decreased progressively with increasing plant cultivation time. In particular, compared with CK treatment after 90 d of cultivation, the Mg content declined by 71.46%, 45.05%, 66.26%, and 38.99%, respectively, in e -1 , e 0 , e 2 , and e 4 treatment. Pearson correlation coe cients between Mg contents and POD activity (r = −0.5664, p < 0.05) (Table 1) indicated that the toxicity of Cd can promote the reduction of Mg which in turn affected the enzyme activity that Mg was a master activator of more than 300 enzymes 37 .
In this study, signi cant positive relationships were found between Cd content and Iron (Fe), Copper (Cu) and Zinc (Zn) content (r = 0.7613, 0.6337 and 0.6320, p < 0.05). Moreover, Fe, Cu and Zn content also correlated strongly with each other (r = 0.6654, 0.8199 and 0.5671, p < 0.05). After 90 d of cultivation, Fe, Cu, and Zn content increased under high Cd treatment compared with CK treatment (Fig 4E -G). It had also been shown that Fe and Zn content were strongly negatively correlated with SOD and POD activity (r = -0.7291 and -0.5768, -0.6349 and -0.7501, p < 0.05) and PCP1 and TPCP content (r = -0.6956 and -0.6445, -0.7306 and -0.6420, p < 0.05). Fe, Cu, and Zn have in the formation of enzymes that are crucial in the plant antioxidative mechanisms and Cd have replacement/displacement of Fe, Cu, and Zn in enzymes or other molecules by different macromolecules. Thus, this effect may plunge regulatory mechanisms into a state of Fe/Cu/Zn de cient, leading to an increment in their uptake as an over compensatory mechanism 38 . The toxicity of Cd to plants disrupts the uptake and distribution of mineral elements in tissues, leading to mineral de ciencies, overcompensation, or imbalance, which in turn affects the activity of related enzymes and causes damage to the plant's antioxidant system.

Polysaccharide content and its antioxidant properties
Compared to the control group, Polygonatum sibiricum was appropriately stimulated to increase polysaccharide content in all treatments through 30 d of cultivation, while at a higher Cd level, this stimulatory effect was reduced as evidenced by the inhibition of polysaccharide synthesis at e 4 treatment instead of after 60 and 90 d of cultivation (Fig.5). However, total polysaccharides after 90 d of cultivation decreased by 8.45%, 20.25%, 46.12%, and 50.77%, respectively, compared with 30 d of cultivation. But it's worth noting that the control group decreased by 16.31% as well. The depletion of polysaccharides in rhizomes is presumed to be due, on the one hand, to the growing period and, on the other hand, to excessive Cd stress. Among them, the Cd stress showed the best promotion effect on polysaccharides synthesis at e 0 treatment.
The antioxidant activity of the three polysaccharides in the rhizome of Polygonatum sibiricum was in the order of PCP1> PCP2> PCP3 ( Table 2). The polysaccharide from the rst step and second step showed the scavenging rate of superoxide anions at 5.61% and 3.06%, respectively. The polysaccharides from the last step did not show antioxidant activity. Evidence had proved that the molecular weight distributions of polysaccharides had a great in uence on their biological activities 39 . PCP1 has the lowest molecular weight with the best performance in scavenging superoxide radicals which could nd a similar result that high molecular weight polysaccharides are less active than low molecular weight 40 .
Not only are sugars a source of nutrients and a component of the structural parts of plants, but more and more researches are now showing that sugars play an important role in plant stress tolerance 41,42 . Most of the polysaccharides in plants are heteropolysaccharides, which not only consist of various kinds of monosaccharides but also proteins, phenols, etc. The antioxidant functional groups of these substances can signi cantly enhance the antioxidant properties of plant polysaccharides. Therefore, the role of polysaccharides as non-enzymatic antioxidants in plant stress tolerance cannot be ignored. From correlation analysis (Table.2), there was a signi cant positive correlation (r =0.8394, p < 0.01) between polysaccharides and POD activity. Fewer studies have investigated the role of polysaccharides as part of a non-enzymatic antioxidant system in plant resilience, but several studies have shown that plant polysaccharides have antioxidant effects and mitigate heavy metal toxicity [43][44][45] .

Conclusion
This study found that the soil Cd safety threshold concentration is e 0 mg•kg -1 . In e -1 and e 0 treatment, the Cd content of Polygonatum sibiricum rhizome meets the consumption standard of heavy metals in Chinese herbal medicine stipulated in the Pharmacopoeia of the People's Republic of China, and the biomass of Polygonatum sibiricum increased showing a Cd tolerance and utilization safety. For the aboveground part of Polygonatum sibiricum, SOD and CAT activity are increased to cope with ROS generated by oxidative stress at higher Cd concentrations. For the underground part, enzymatic and non-enzymatic systems act synergistically which embodies an enhancement in antioxidant enzyme activity and an increase in polysaccharide synthesis at lower Cd treatment. Both enzymatic and non-enzymatic systems are partially inhibited at higher Cd treatment. The stimulatory effect of Cd changes the mineral element uptake of Polygonatum sibiricum especially in high Cd treatment and caused an in uence on the enzyme system of plants. In conclusion, the safe utilization of Polygonatum sibiricum can be guaranteed, and it has high application potential in soil remediation areas with lower Cd contamination.

Declarations Declaration of interest statement
The authors declare that they have no competing interests.
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Competing interests
The authors declare that they have no competing interests.