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Cadmium (Cd) contamination poses a potential threat to the safety of the cultivation of the medicinal plant Panax notoginseng in Yunnan. Under exogenous Cd stress, field experiments were conducted to understand the effects of lime application (0, 750, 2250 and 3750 kg/h/m2) and foliar spraying with oxalic acid (0, 0.1 and 0.2 mol/L) on accumulation Cd and antioxidant. Systemic and medicinal components of Panax notoginseng. The results showed that under Cd stress, lime and foliar spray with oxalic acid could increase the Ca2+ content of Panax notoginseng and reduce the toxicity of Cd2+. The addition of lime and oxalic acid increased the activity of antioxidant enzymes and changed the metabolism of osmotic regulators. The most significant is the increase in CAT activity by 2.77 times. Under the influence of oxalic acid, the activity of SOD increased to 1.78 times. The MDA content decreased by 58.38%. There is a very significant correlation with soluble sugar, free amino acids, proline and soluble protein. Lime and oxalic acid can increase the calcium ion (Ca2+) content of Panax notoginseng, reduce the Cd content, improve the stress resistance of Panax notoginseng, and increase the production of total saponins and flavonoids. The Cd content is the lowest, 68.57% lower than the control, and corresponds to the standard value (Cd≤0.5 mg kg-1, GB/T 19086-2008). The proportion of SPN was 7.73%, reaching the highest level among all treatments, and the flavonoid content increased significantly by 21.74%, reaching standard medical values ​​and optimal yield.
Cadmium (Cd) is a common contaminant of cultivated soils, migrates easily and has significant biological toxicity. El-Shafei et al2 reported that cadmium toxicity affects the quality and productivity of the plants used. Excessive levels of cadmium in cultivated soil in southwest China have become serious in recent years. Yunnan Province is China’s biodiversity kingdom, with medicinal plant species ranking first in the country. However, Yunnan Province is rich in mineral resources, and the mining process inevitably leads to heavy metal pollution in the soil, which affects the production of local medicinal plants.
Panax notoginseng (Burkill) Chen3) is a very valuable perennial herbaceous medicinal plant belonging to the genus Panax of the Araliaceae family. Panax notoginseng improves blood circulation, eliminates blood stagnation and relieves pain. The main production area is Wenshan Prefecture, Yunnan Province5. More than 75% of the soil in local Panax notoginseng ginseng growing areas is contaminated with cadmium, with levels varying from 81% to over 100% in different areas6. The toxic effect of Cd also significantly reduces the production of medicinal components of Panax notoginseng, especially saponins and flavonoids. Saponins are a type of glycosidic compound whose aglycones are triterpenoids or spirostanes. They are the main active ingredients of many traditional Chinese medicines and contain saponins. Some saponins also have antibacterial activity or valuable biological activities such as antipyretic, sedative and anticancer effects7. Flavonoids generally refer to a series of compounds in which two benzene rings with phenolic hydroxyl groups are connected through three central carbon atoms. The main core is 2-phenylchromanone 8. It is a strong antioxidant that can effectively scavenge oxygen free radicals in plants. It may also inhibit the penetration of inflammatory biological enzymes, promote wound healing and pain relief, and lower cholesterol levels. It is one of the main active ingredients of Panax notoginseng. There is an urgent need to address the problem of cadmium contamination in soils in Panax ginseng production areas and ensure the production of its essential medicinal ingredients.
Lime is one of the widely used passivators for stationary soil purification from cadmium contamination10. It affects the adsorption and deposition of Cd in soil by reducing the bioavailability of Cd in soil by increasing the pH value and changing the soil cation exchange capacity (CEC), soil salt saturation (BS) and soil redox potential (Eh)3, 11. In addition, , lime provides a large amount of Ca2+, forms ionic antagonism with Cd2+, competes for adsorption sites in roots, prevents the transport of Cd into the soil, and has low biological toxicity. When 50 mmol L-1 Ca was added under Cd stress, Cd transport in sesame leaves was inhibited and Cd accumulation was reduced by 80%. A number of similar studies have been reported in rice (Oryza sativa L.) and other crops12,13.
Foliar spraying of crops to control the accumulation of heavy metals is a new method for controlling heavy metals in recent years. Its principle is mainly related to the chelation reaction in plant cells, which results in the deposition of heavy metals on the cell wall and inhibits the uptake of heavy metals by plants14,15. As a stable diacid chelating agent, oxalic acid can directly chelate heavy metal ions in plants, thereby reducing toxicity. Research has shown that oxalic acid in soybeans can chelate Cd2+ and release Cd-containing crystals through the upper trichome cells, reducing Cd2+ levels in the body16. Oxalic acid can regulate soil pH, enhance the activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and regulate the penetration of soluble sugar, soluble protein, free amino acids and proline. Metabolic regulators17,18. Acid and excess Ca2+ in the plant form a calcium oxalate precipitate under the action of nucleating proteins. Regulating the Ca2+ concentration in plants can effectively achieve the regulation of dissolved oxalic acid and Ca2+ in plants and avoid the excessive accumulation of oxalic acid and Ca2+19,20.
The amount of lime applied is one of the key factors influencing the repair effect. It was found that the dosage of lime ranged from 750 to 6000 kg/m2. For acidic soil with a pH of 5.0~5.5, the effect of applying lime at a dose of 3000~6000 kg/h/m is significantly higher than at a dose of 750 kg/h/m221. However, overapplication of lime will result in some negative effects on the soil, such as significant changes in soil pH and soil compaction22. Therefore, we defined the CaO treatment levels as 0, 750, 2250 and 3750 kg hm-2. When oxalic acid was applied to Arabidopsis thaliana, it was found that Ca2+ was significantly reduced at a concentration of 10 mmol L-1, and the CRT gene family, which affects Ca2+ signaling, responded strongly20. The accumulation of some previous studies allowed us to determine the concentration of this test and further study the effect of the interaction of exogenous supplements on Ca2+ and Cd2+23,24,25. Therefore, this study aims to explore the regulatory mechanism of exogenous lime and oxalic acid leaf spray on Cd content and stress tolerance of Panax notoginseng in Cd-contaminated soil and further explore ways to better ensure medicinal quality and efficacy. Panax notoginseng production. He provides valuable guidance on increasing the scale of herbaceous plant cultivation in cadmium-contaminated soils and achieving the high-quality, sustainable production required by the pharmaceutical market.
Using the local ginseng variety Wenshan Panax notoginseng as the material, a field experiment was conducted in Lannizhai, Qiubei County, Wenshan Prefecture, Yunnan Province (24°11′N, 104°3′E, altitude 1446 m). The average annual temperature is 17°C and the average annual precipitation is 1250 mm. The background values ​​of the studied soil were TN 0.57 g kg-1, TP 1.64 g kg-1, TC 16.31 g kg-1, OM 31.86 g kg-1, alkali hydrolyzed N 88.82 mg kg-1 , phosphorus free. 18.55 mg kg-1, free potassium 100.37 mg kg-1, total cadmium 0.3 mg kg-1, pH 5.4.
On December 10, 2017, 6 mg/kg Cd2+ (CdCl2·2.5H2O) and lime treatment (0, 750, 2250 and 3750 kg/h/m2) were mixed and applied to the soil surface in a layer of 0~10 cm of each plot. . Each treatment was repeated 3 times. Test plots are randomly located, each plot covering an area of ​​3 m2. One-year-old Panax notoginseng seedlings were transplanted after 15 days of tillage. When using a sunshade net, the light intensity of Panax notoginseng inside the sunshade net is about 18% of normal natural light intensity. Cultivation is carried out according to local traditional cultivation methods. Before the ripening stage of Panax notoginseng in 2019, spray oxalic acid in the form of sodium oxalate. Oxalic acid concentrations were 0, 0.1 and 0.2 mol L-1, respectively, and NaOH was used to adjust the pH to 5.16 to simulate the average pH of the litter leach solution. Spray the upper and lower surfaces of the leaves once a week at 8:00 am. After spraying 4 times in the 5th week, 3-year-old Panax notoginseng plants were harvested.
In November 2019, three-year-old Panax notoginseng plants were collected from the field and sprayed with oxalic acid. Some samples of three-year-old Panax notoginseng plants that needed to be measured for physiological metabolism and enzyme activity were placed in tubes for freezing. , quickly frozen with liquid nitrogen and then transferred to a refrigerator at -80°C. Some root samples to be measured for Cd and active ingredient content at maturity stage were washed with tap water, dried at 105°C for 30 minutes, at constant weight at 75°C, and ground in a mortar for storage.
Weigh 0.2 g of dry plant sample, place it in an Erlenmeyer flask, add 8 ml HNO3 and 2 ml HClO4 and cover overnight. The next day, use a curved funnel placed in an Erlenmeyer flask for electrothermal digestion until white smoke appears and the digestive juices run clear. After cooling to room temperature, the mixture was transferred to a 10 ml volumetric flask. Cd content was determined using an atomic absorption spectrometer (Thermo ICE™ 3300 AAS, USA). (GB/T 23739-2009).
Weigh 0.2 g of dry plant sample, place it in a 50 ml plastic bottle, add 1 mol L-1 HCL in 10 ml, cap and shake well for 15 hours and filter. Using a pipette, pipet the required amount of filtrate, dilute it accordingly and add SrCl2 solution to bring the Sr2+ concentration to 1g L-1. Ca content was measured using an atomic absorption spectrometer (Thermo ICE™ 3300 AAS, USA).
Malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) reference kit method (DNM-9602, Beijing Prong New Technology Co., Ltd., product registration), use the corresponding measurement kit. No.: Beijing Pharmacopoeia (accurate) 2013 No. 2400147).
Weigh about 0.05 g of Panax notoginseng sample and add anthrone-sulfuric acid reagent along the sides of the tube. Shake the tube for 2-3 seconds to thoroughly mix the liquid. Place the tube on a tube rack to develop color for 15 minutes. Soluble sugar content was determined by ultraviolet–visible spectrophotometry (UV-5800, Shanghai Yuanxi Instrument Co., Ltd., China) at a wavelength of 620 nm.
Weigh 0.5 g of a fresh sample of Panax notoginseng, grind it into a homogenate with 5 ml distilled water, and then centrifuge at 10,000 g for 10 minutes. The supernatant was diluted to a fixed volume. The Coomassie Brilliant Blue method was used. Soluble protein content was measured using ultraviolet–visible spectrophotometry (UV-5800, Shanghai Yuanxi Instrument Co., Ltd., China) at a wavelength of 595 nm and calculated based on the standard curve of bovine serum albumin.
Weigh 0.5 g of fresh sample, add 5 ml of 10% acetic acid, grind to a homogenate, filter and dilute to constant volume. The color development method was used with a ninhydrin solution. Free amino acid content was determined by UV–visible spectrophotometry (UV-5800, Shanghai Yuanxi Instrument Co., Ltd., China) at 570 nm and calculated based on the leucine standard curve28.
Weigh 0.5 g of a fresh sample, add 5 ml of a 3% solution of sulfosalicylic acid, heat in a water bath and shake for 10 minutes. After cooling, the solution was filtered and brought to a constant volume. The colorimetric method with acid ninhydrin was used. Proline content was determined by ultraviolet–visible spectrophotometry (UV-5800, Shanghai Yuanxi Instrument Co., Ltd., China) at a wavelength of 520 nm and calculated based on the proline standard curve29.
Saponin content was determined by high-performance liquid chromatography with reference to the Pharmacopoeia of the People’s Republic of China (2015 edition). The basic principle of high-performance liquid chromatography is to use high-pressure liquid as the mobile phase and apply ultrafine particle separation technology of high-performance column chromatography to the stationary phase. The operating technique is as follows:
HPLC Conditions and System Suitability Test (Table 1): Use octadecylsilane bound silica gel as the filler, acetonitrile as the mobile phase A and water as the mobile phase B. Perform gradient elution as shown in the table below. The detection wavelength is 203 nm. According to the R1 peak of the total saponins of Panax notoginseng, the number of theoretical plates should be at least 4000.
Preparation of standard solution: Accurately weigh ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 and add methanol to prepare a mixture containing 0.4 mg ginsenoside Rg1, 0.4 mg ginsenoside Rb1 and 0.1 mg notoginsenoside R1 per 1 ml of solution.
Preparation of test solution: Weigh 0.6 g Panax ginseng powder and add 50 ml methanol. The mixed solution was weighed (W1) and left overnight. The mixed solution was then gently boiled in a water bath at 80°C for 2 hours. After cooling, weigh the mixed solution and add the prepared methanol to the first mass W1. Then shake well and filter. The filtrate is left for analysis.
Accurately collect 10 μL of the standard solution and 10 μL of the filtrate and inject them into a high performance liquid chromatograph (Thermo HPLC-ultimate 3000, Seymour Fisher Technology Co., Ltd.) to determine the saponin 24 content.
Standard curve: measurement of a mixed standard solution of Rg1, Rb1 and R1. Chromatography conditions are the same as above. Calculate the standard curve by plotting the measured peak area on the y-axis and the concentration of saponin in the standard solution on the x-axis. The saponin concentration can be calculated by substituting the measured peak area of ​​the sample into the standard curve.
Weigh 0.1 g of P. notogensings sample and add 50 ml of 70% CH3OH solution. Ultrasonic extraction was carried out for 2 hours, followed by centrifugation at 4000 rpm for 10 minutes. Take 1 ml of supernatant and dilute it 12 times. The flavonoid content was determined using ultraviolet-visible spectrophotometry (UV-5800, Shanghai Yuanxi Instrument Co., Ltd., China) at a wavelength of 249 nm. Quercetin is one of the standard common substances8.
Data were organized using Excel 2010 software. SPSS 20 statistical software was used to conduct analysis of variance on the data. Pictures were drawn using Origin Pro 9.1. Calculated statistical values ​​include mean ± SD. Statements of statistical significance are based on P < 0.05.
At the same concentration of oxalic acid sprayed on the leaves, the Ca content in the roots of Panax notoginseng increased significantly as the amount of lime applied increased (Table 2). Compared to the absence of lime, the Ca content increased by 212% when adding 3750 kg/h/m2 of lime without spraying oxalic acid. For the same amount of lime applied, the Ca content increased slightly as the concentration of oxalic acid spray increased.
The Cd content in roots ranges from 0.22 to 0.70 mg kg-1. At the same spray concentration of oxalic acid, as the amount of lime added increases, the Cd content of 2250 kg/h decreases significantly. Compared to the control, the Cd content in the roots decreased by 68.57% after spraying with 2250 kg hm-2 lime and 0.1 mol l-1 oxalic acid. When limeless and 750 kg/h of lime were applied, the Cd content in the roots of Panax notoginseng decreased significantly with increasing concentration of oxalic acid spray. When 2250 kg/m2 lime and 3750 kg/m2 lime were applied, the root Cd content first decreased and then increased with increasing oxalic acid concentration. In addition, bivariate analysis showed that lime had a significant effect on the Ca content of Panax notoginseng roots (F = 82.84**), lime had a significant effect on the Cd content in Panax notoginseng roots (F = 74.99**), and oxalic acid. acid (F=7.72*).
As the amount of lime added and the concentration of sprayed oxalic acid increased, the MDA content decreased significantly. There was no significant difference in the MDA content in the roots of Panax notoginseng without the addition of lime and with the addition of 3750 kg/m2 of lime. At application rates of 750 kg/h/m2 and 2250 kg/h/m2, the lime content of 0.2 mol/L oxalic acid spray treatment decreased by 58.38% and 40.21%, respectively, compared with no oxalic acid spray treatment. The lowest MDA content (7.57 nmol g-1) was observed when spraying 750 kg hm-2 lime and 0.2 mol l-1 oxalic acid (Fig. 1).
Effect of foliar spraying with oxalic acid on malondialdehyde content in Panax notoginseng roots under cadmium stress. Note: The legend in the figure indicates the concentration of oxalic acid at the spray (mol L-1), different lowercase letters indicate significant differences between treatments of the same lime application. number (P < 0.05). Same below.
Except for the application of 3750 kg/h lime, there was no significant difference in SOD activity in Panax notoginseng roots. When adding 0, 750 and 2250 kg/h/m2 of lime, SOD activity when treated by spraying with oxalic acid at a concentration of 0.2 mol/l was significantly higher than without the use of oxalic acid, increasing by 177.89%, 61.62% and 45.08% respectively. SOD activity in the roots (598.18 U g-1) was the highest in the absence of lime application and when treated by spraying with oxalic acid at a concentration of 0.2 mol/l. When oxalic acid was sprayed at the same concentration or 0.1 mol L-1, SOD activity increased with increasing amount of lime added. After spraying with 0.2 mol/L oxalic acid, SOD activity decreased significantly (Fig. 2).
Effect of spraying leaves with oxalic acid on the activity of superoxide dismutase, peroxidase and catalase in the roots of Panax notoginseng under cadmium stress
Like SOD activity in roots, POD activity in roots treated without lime and sprayed with 0.2 mol L-1 oxalic acid was the highest (63.33 µmol g-1), which is 148.35% higher than control ( 25.50 µmol g-1). With increasing oxalic acid spray concentration and 3750 kg/m2 lime treatment, POD activity first increased and then decreased. Compared with the treatment with 0.1 mol L-1 oxalic acid, the POD activity when treated with 0.2 mol L-1 oxalic acid decreased by 36.31% (Fig. 2).
With the exception of spraying 0.2 mol/l oxalic acid and adding 2250 kg/h/m2 or 3750 kg/h/m2 lime, CAT activity was significantly higher than the control. When spraying 0.1 mol/l oxalic acid and adding 0.2250 kg/m2 or 3750 kg/h/m2 lime, CAT activity increased by 276.08%, 276.69% and 33.05%, respectively, compared to treatment without spraying oxalic acid. CAT activity in roots was highest (803.52 μmol/g) in the no-lime treatment and in the 0.2 mol/L oxalic acid treatment. CAT activity was the lowest (172.88 μmol/g) when treated with 3750 kg/h/m of lime and 0.2 mol/L oxalic acid (Fig. 2).
Bivariate analysis showed that CAT activity and MDA activity of Panax notoginseng roots were significantly associated with the amount of oxalic acid or lime sprayed and the two treatments (Table 3). SOD activity in roots was significantly related to lime and oxalic acid treatment or oxalic acid spray concentration. Root POD activity was significantly dependent on the amount of lime applied or the lime and oxalic acid treatment.
The content of soluble sugars in the roots decreased with increasing amount of lime application and concentration of oxalic acid spray. There was no significant difference in the content of soluble sugars in Panax notoginseng roots without lime application and when 750 kg/h/m of lime was applied. When 2250 kg/m2 of lime was applied, the soluble sugar content when treated with 0.2 mol/L oxalic acid was significantly higher than that when treated without spraying oxalic acid, increasing by 22.81%. When 3750 kg h/m2 of lime was applied, the soluble sugar content decreased significantly as the concentration of sprayed oxalic acid increased. The soluble sugar content when treated with 0.2 mol L-1 oxalic acid decreased by 38.77% compared to that without spraying oxalic acid. In addition, the 0.2 mol·L-1 oxalic acid spray treatment had the lowest soluble sugar content, which was 205.80 mg·g-1 (Fig. 3).
Effect of foliar spraying with oxalic acid on the content of soluble total sugar and soluble protein in Panax notoginseng roots under cadmium stress
Soluble protein content in roots decreased with increasing amounts of lime application and oxalic acid spray treatment. Without the addition of lime, the soluble protein content when treated with oxalic acid spray at a concentration of 0.2 mol L-1 was significantly reduced by 16.20% compared to the control. There were no significant differences in the soluble protein content of Panax notoginseng roots when 750 kg/h of lime was applied. Under the application conditions of 2250 kg/h/m of lime, the soluble protein content of 0.2 mol/L oxalic acid spray treatment was significantly higher than that of non-oxalic acid spray treatment (35.11%). When 3750 kg·h/m2 of lime was applied, the soluble protein content decreased significantly as the oxalic acid spray concentration increased, with the lowest soluble protein content (269.84 μg·g-1) when the oxalic acid spray was 0.2 mol·L-1 . treatment (Fig. 3).
There were no significant differences in the content of free amino acids in the root of Panax notoginseng in the absence of lime application. As the spray concentration of oxalic acid increased and the addition of 750 kg/h/m2 of lime, the content of free amino acids first decreased and then increased. Compared with the treatment without spraying oxalic acid, the content of free amino acids increased significantly by 33.58% when spraying 2250 kg hm-2 lime and 0.2 mol l-1 oxalic acid. The content of free amino acids decreased significantly with increasing spray concentration of oxalic acid and the addition of 3750 kg/m2 of lime. The free amino acid content of 0.2 mol L-1 oxalic acid spray treatment was reduced by 49.76% compared with non-oxalic acid spray treatment. The free amino acid content was highest without oxalic acid spray and was 2.09 mg g-1. The 0.2 mol/L oxalic acid spray treatment had the lowest free amino acid content (1.05 mg/g) (Fig. 4).
Effect of spraying leaves with oxalic acid on the content of free amino acids and proline in the roots of Panax notoginseng under cadmium stress conditions
The proline content in the roots decreased with an increase in the amount of lime applied and the amount of spraying with oxalic acid. There were no significant differences in the proline content of Panax ginseng root when lime was not applied. As the spray concentration of oxalic acid increased and the application of 750 or 2250 kg/m2 of lime increased, the proline content first decreased and then increased. The proline content of 0.2 mol L-1 oxalic acid spray treatment was significantly higher than that of 0.1 mol L-1 oxalic acid spray treatment, increasing by 19.52% and 44.33%, respectively. When 3750 kg/m2 of lime was added, the proline content decreased significantly as the concentration of sprayed oxalic acid increased. After spraying 0.2 mol L-1 oxalic acid, the proline content decreased by 54.68% compared with that without spraying oxalic acid. The lowest proline content was when treated with 0.2 mol/l oxalic acid and amounted to 11.37 μg/g (Fig. 4).
The total saponin content in Panax notoginseng is Rg1>Rb1>R1. There was no significant difference in the content of the three saponins with increasing concentration of oxalic acid spray and concentration without lime application (Table 4).
The R1 content after spraying 0.2 mol L-1 oxalic acid was significantly lower than without spraying oxalic acid and applying a lime dose of 750 or 3750 kg/m2. At a sprayed oxalic acid concentration of 0 or 0.1 mol/L, there was no significant difference in R1 content with increasing amount of lime added. At a spray concentration of 0.2 mol/L oxalic acid, the R1 content in 3750 kg/h/m2 of lime was significantly lower than 43.84% without adding lime (Table 4).
As the spray concentration of oxalic acid increased and 750 kg/m2 of lime was added, the Rg1 content first increased and then decreased. At lime application rates of 2250 and 3750 kg/h, the Rg1 content decreased with increasing oxalic acid spray concentration. At the same concentration of sprayed oxalic acid, as the amount of lime increases, the Rg1 content first increases and then decreases. Compared with the control, except for the Rg1 content in three concentrations of oxalic acid and 750 kg/m2 lime treatments, which was higher than the control, the Rg1 content in Panax notoginseng roots in other treatments was lower than the control. control. The maximum content of Rg1 was when spraying 750 kg/h/m2 of lime and 0.1 mol/l oxalic acid, which was 11.54% higher than the control (Table 4).
As the spray concentration of oxalic acid and the amount of lime applied increased at a flow rate of 2250 kg/h, the Rb1 content first increased and then decreased. After spraying 0.1 mol L-1 oxalic acid, the Rb1 content reached a maximum value of 3.46%, which was 74.75% higher than without spraying oxalic acid. For other lime treatments, there were no significant differences between different concentrations of oxalic acid spray. After spraying with 0.1 and 0.2 mol L-1 oxalic acid, as the amount of lime increased, the Rb1 content first decreased and then decreased (Table 4).
At the same spray concentration with oxalic acid, as the amount of lime added increased, the content of flavonoids first increased and then decreased. No significant difference in the content of flavonoids was detected when spraying different concentrations of oxalic acid without lime and 3750 kg/m2 of lime. When adding 750 and 2250 kg/m2 of lime, as the concentration of sprayed oxalic acid increased, the content of flavonoids first increased and then decreased. When applying 750 kg/m2 and spraying oxalic acid at a concentration of 0.1 mol/l, the content of flavonoids was maximum – 4.38 mg/g, which is 18.38% higher than when adding the same amount of lime, and there was no need spray oxalic acid. The content of flavonoids when treated with 0.1 mol L-1 oxalic acid spray increased by 21.74% compared to the treatment without oxalic acid and the treatment with lime at a dose of 2250 kg/m2 (Fig. 5).
Effect of spraying leaves with oxalate on the content of flavonoids in the root of Panax notoginseng under cadmium stress
Bivariate analysis showed that the soluble sugar content of Panax notoginseng roots was significantly dependent on the amount of lime applied and the concentration of oxalic acid sprayed. The content of soluble protein in the roots was significantly correlated with the dosage of lime and oxalic acid. The content of free amino acids and proline in the roots was significantly correlated with the amount of lime applied, the concentration of spraying oxalic acid, lime and oxalic acid (Table 5).
The R1 content in Panax notoginseng roots was significantly dependent on the concentration of sprayed oxalic acid, the amount of lime, lime and oxalic acid applied. The content of flavonoids depended significantly on the concentration of oxalic acid spray and the amount of lime added.
Many amendments have been used to reduce cadmium levels in plants by fixing cadmium in the soil, such as lime and oxalic acid30. Lime is widely used as a soil amendment to reduce cadmium levels in crops31. Liang et al. 32 reported that oxalic acid can also be used to remediate soil contaminated with heavy metals. After varying concentrations of oxalic acid were added to contaminated soil, soil organic matter content increased, cation exchange capacity decreased, and pH increased33. Oxalic acid can also react with metal ions in the soil. Under Cd stress conditions, the Cd content in Panax notoginseng increased significantly compared to the control. However, if lime is used, it is significantly reduced. When 750 kg/h/m of lime was applied in this study, the Cd content of roots reached the national standard (Cd limit is Cd≤0.5 mg/kg, AQSIQ, GB/T 19086-200834), and the effect was good. . The best effect is achieved by adding 2250 kg/m2 of lime. The addition of lime creates a large number of competition sites for Ca2+ and Cd2+ in the soil, and the addition of oxalic acid reduces the Cd content in the roots of Panax notoginseng. After mixing lime and oxalic acid, the Cd content of Panax ginseng root decreased significantly and reached the national standard. Ca2+ in soil is adsorbed to the root surface through a mass flow process and can be absorbed into root cells through calcium channels (Ca2+ channels), calcium pumps (Ca2+-AT-Pase) and Ca2+/H+ antiporters, and then transported horizontally. to the roots. Xylem23. There was a significant negative correlation between Ca and Cd content in roots (P < 0.05). The Cd content decreased with increasing Ca content, which is consistent with the idea of ​​antagonism between Ca and Cd. ANOVA showed that the amount of lime had a significant effect on the Ca content in the root of Panax notoginseng. Pongrack et al. 35 reported that Cd binds to oxalate in calcium oxalate crystals and competes with Ca. However, the regulatory effect of oxalic acid on Ca was insignificant. This shows that the precipitation of calcium oxalate from oxalic acid and Ca2+ is not simple precipitation, and the coprecipitation process may be controlled by several metabolic pathways.
Under cadmium stress, a large amount of reactive oxygen species (ROS) is formed in plants, damaging the structure of cell membranes36. Malondialdehyde (MDA) content can be used as an indicator to judge the level of ROS and the degree of damage to the plasma membrane of plants37. The antioxidant system is an important protective mechanism for scavenging reactive oxygen species38. The activities of antioxidant enzymes (including POD, SOD, and CAT) are typically altered by cadmium stress. The results showed that MDA content was positively correlated with Cd concentration, indicating that the extent of plant membrane lipid peroxidation deepened with increasing Cd concentration37. This is consistent with the results of the study by Ouyang et al.39. This study shows that MDA content is significantly influenced by lime, oxalic acid, lime and oxalic acid. After nebulization of 0.1 mol L-1 oxalic acid, the MDA content of Panax notoginseng decreased, indicating that oxalic acid could reduce the bioavailability of Cd and ROS levels in Panax notoginseng. The antioxidant enzyme system is where the plant’s detoxification function takes place. SOD removes O2- contained in plant cells and produces non-toxic O2 and low-toxic H2O2. POD and CAT remove H2O2 from plant tissues and catalyze the decomposition of H2O2 into H2O. Based on iTRAQ proteome analysis, it was found that the protein expression levels of SOD and PAL were decreased and the expression level of POD was increased after lime application under Cd40 stress. The activities of CAT, SOD and POD in the root of Panax notoginseng were significantly affected by the dosage of oxalic acid and lime. Spray treatment with 0.1 mol L-1 oxalic acid significantly increased the activity of SOD and CAT, but the regulatory effect on POD activity was not obvious. This shows that oxalic acid accelerates the decomposition of ROS under Cd stress and mainly completes the removal of H2O2 by regulating the activity of CAT, which is similar to the research results of Guo et al.41 on the antioxidant enzymes of Pseudospermum sibiricum. Kos. ). The effect of adding 750 kg/h/m2 of lime on the activity of enzymes of the antioxidant system and the content of malondialdehyde is similar to the effect of spraying with oxalic acid. The results showed that oxalic acid spray treatment could more effectively enhance the activities of SOD and CAT in Panax notoginseng and enhance the stress resistance of Panax notoginseng. The activities of SOD and POD were decreased by treatment with 0.2 mol L-1 oxalic acid and 3750 kg hm-2 lime, indicating that excessive spraying of high concentrations of oxalic acid and Ca2+ may cause plant stress, which is consistent with the study of Luo and etc. al. Wait 42.