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 influence of biomass occurred at e2 and e4 treatment after 90 d of cultivation. In comparison to CK treatment, the biomass of e2 and e4 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 e0 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 e0 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.
Cd content in different parts of plants
Cd levels in plants increased in a dose-dependent manner (Fig.2). The highest Cd content in roots, rhizomes, stems, and leaves occurring in the e4 treatment after 90 d cultivation under which condition the plant Cd content was 239.04, 16.38, 12.84, and 16.41 mg/kg, respectively. The roots Cd content higher than other parts in all treatments and significantly increased with cultivation time (Fig.2A). The Cd content in the medicinal site rhizomes of 0.36 (30 d, e-1),0.43 (30 d, e0), 0.33 (60 d, e-1),0.64 (60 d, e0), 0.20 (90 d, e-1), 0.69 (90 d, e0) mg•kg-1, respectively (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 e2 and e4 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 beneficial 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 e4 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, e0, e2 treatment, respectively. However, for the underground parts, the high Cd treatment shows lower SOD activity through the full cultivation time. Furthermore, the correlation coefficients 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 e4 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 e2 and e4 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 (e0, e2, and e4 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 e0 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 (e2 and e4) 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 find a facilitative effect of Cd on K uptake except for the e4 treatment. The phenomenon might be related to the ability of Cd can increase the influx 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 significantly promoted by Cd stress and increased by 140.03%, 101.25%, 27.11%, and 38.35%, respectively, in e-1, e0, e2, and e4 treatment after 60 d of cultivation. However, it was altered after 90 d of cultivation that Ca uptake was inhibited except e2 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 Cd2+ 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, e0, e2, and e4 treatment. Pearson correlation coefficients 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, significant 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 deficient, 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 deficiencies, 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 e4 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 e0 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 first 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 influence on their biological activities 39. PCP1 has the lowest molecular weight with the best performance in scavenging superoxide radicals which could find 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 significantly 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 significant 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 43-45.