Response Of Paeonia Ostii On Cu Stress And The Application Of Paeonia Ostii On Utilization Of Cu Contaiminated Soil

China is rich of copper resources, but the copper mining, smelting and tailings resulted in serious soil contamination around the mines. In recent years, the enhancement of utilization of Cu contaminated soil is one of the hot spots. This study explored the applied value of Fengdan (Paeonia ostii) in remediation of Cu contaminated soil by field investigation and field experiments. In addition, the response of Fengdan seedlings on Cu stress by hydroponics in the laboratory was studied. The mainly results as follows:This study optimized the chromatographic conditions of HPLC and fumbled out the optimal method to in prepare paeonol sample. The results showed that the column temperature controlled at 25 ℃ and methanol-water ratio adjusted to 70:30 (v:v) would preferably separated paeonol with other components; Compared with the traditional method of drying, it could obtain the most paeonol extracted directly from the fresh moutan cortex, followed by freeze-drying mothod. Compared with the direct extraction of fresh sample, dried in the shade、in the sum in the oven at 30℃,40℃,60℃,80℃ reduced the content of paeonol by 47%72%,40%,85%,92%,98%, respectively. Therefore, the moutan cortex should be stored at low temperature to reduce the loss of paeonol. The distribution of paeonol content in Fengdan plants was moutan cortex>duramen> stem bark> stem duramen> leaves> petiole.The alleviation effect of paeonol on oxidation stress caused by excess Cu was investigated in this study.14-day-old seedlings of soybean (Glycine max L. cv. Zhonghuang 50) were treated by 0.32 and 10 μmol·L-1 Cu. The results showed that 1 foliar applications of 10 μmol·L-1 paeonol pure solution or paeonol crude extracts could significantly reduce the MDA and H2O2 contents caused by Cu treatment. In Cu treated soybean leaves sprayed in vivo or soaked in vitro by paeonol, the G-POD activity improved significantly compared to the leaves treated with H2O, but NADH-POD activity significantly reduced; In the leaves without Cu treated, the G-POD and NADH-POD activity did not change significantly compared to the leaves treated with H2O when sprayed by paeonol, but the NADH-POD activity decreased significantly after soaked in paeonol in vitro. The role of paeonol on H2O2 accumulation significantly weakened when the activity of POD was inhibited by NaN3 and aminotriazole which were POD inhibotors. The results showed that paeonol reduce the accumulation of H2O2 is mainly achieved by affecting the activity of POD.Attempting to discover whether paeonol accumulation in moutan cortex was related to soil factors (especially copper), the moutan cortex and the corresponding soil samples from Fenghuangshan copper mine and circumjacent regions were collected. The paeonol and Cu contents in moutan cortex, soil Cu content were determined. The results showed that paeonol content increased with the increase of soil total Cu content at lower soil Cu background levels (≤300 mg·kg-1); Paeonol content of moutan cortex collected from the sampling points with 100 mg·kg-1 of total Cu was generally high; but higher soil total Cu (>400 mg·kg-1) content inhibited the accumulation of paeonol; The paeonol and soil available Cu content showed the similar trends; Paeonol content and Cu of moutan cortex showed significant positive linear relationship (r=0.3742*, n=48). No significant relationships were found between the paeonol content in moutan cortex and soil pH, and contents of organic matter, available nitrogen, available potassium and available phosphorus in soils.Despite the background levels of Cu in the soil were significantly different (ranged from 30 mg·kg-1 to 1000 mg·kg-1), but Cu content of moutan cortex did not exceed<Green Trade Standards of Importing & Exporting Medicinal Plants & Preparations> (WM2-2001) (Cu≤ 20 mg·kg-1), and fulfiled the quality requirements.Forty-four kinds of microbes were identified in soil of three regions by PCR-DGGE method. The analysis of correlation between paeonol and soil bacterial diversity showed that paeonol content and soil bacterial diversity in 20 samples were significantly positive linear associated at p<0.01 level. We investigated the number of strains appeared in different soil samples. Five bacterium species including Geobacte sp. strain TL18, Oceanicaulis sp. TL22, Desulfovibrio sp. TL28, Desulfovermiculus sp. TL29 and Simplicispira sp. TL31 were not detected in soils in which paeonol concentrations in moutan cortex of Fengdan were< 6.5 g·kg-1. It could be speculated that the five bacteria may stimulate paeonol accumulation, but it also required further experimental verification.Using pot experiment, the growth, quality, paeonol and Cu contents of peony seedlings were studied after different levels of Cu applied into soils. The results show that different concentrations of Cu (50,100 and 200 mg·kg-1) had no significant impact on the growth of Fengdan plants and the appearance of the moutan cortex; Application of Cu at 50 mg·kg-1 has a promoted trend on plant growth. Appropriate Cu content (≤100 mg·kg-1) played a role in promoting paeonol accumulation, but when the Cu concentration is too high (200 mg·kg-1), Dan accumulation; the wooden hearts paeonol content of Cu concentration increases significantly increased.Under hydroponic conditions, the nutrient solution supplemented with copper inhibited root elongation of Fengdan, and with the copper concentration increased, the inhibition effect was more pronounced, and the copper treatment also significantly inhibited the total biomass and root biomass, but no significant difference between treatments groups and control group. After copper treatment, Cu content of roots was significantly higher than the control group, but paeonol content was not significantly different from control group. The inhibition of root net elongation rate under 5 μmol·L-1 Cu treatment at different times was about 50%, and with the treatment time extend, the inhibition rate gradually reduced; Seedlings total and root biomass were decreased 29.2% and 29.5% compared with control group after 14 days treatment, paeonol content increased but had no significant difference compared with control.Histochemical staining results showed large amounts of reactive oxygen species (ROS) accumulated in Fengdan seedling roots after treated by 5 μmol·L-1 Cu for 24 h. Antioxidant enzymatic system such as CAT and POD activities increased significantly compared with the control; GR activity gradually decreased with the extension of the Cu treatment. AsA, DHA and total AsA contents of the root increased significantly when treated for 72 h under 5 μmol·L-1 Cu, while AsA/DHA values and soluble protein content had no significant changes in the entire process of Cu treatment. PAL activity was higher than that of the control at 72 h under Cu treatment; Lignin content and POD activity increased at first and then decreased to the same level with the control. It indicated POD played a key role in lignin synthesis.The soil background of copper contaminated farmlands, growth situation of peony, copper and paeonol contents of moutan cortex were investigated, and the income of peony was compared to other crops. The results showed that, growth status of the medicinal peony variety Fengdan was significantly better than ornamental variety Huhong. Multivariate analysis of variance results showed that the investigation phases and planting ground significantly impact paeonol content. The results of paeonol content in different farmlands showed that the paeonol content was higher in moutan cortex of mediun-pollution farmland than the low and high-pollution farmlands; Paeonol content of moutan cortex investigated in flowering phase was higher than the germination and defoliating phase. The assay of copper content showed that the moutan cortex grown in different test farmlands in defoliating phase were lower than the green industry standards of the national medicinal plants import and the export. The income of planting Fengdan was 4.31 times,12.58 times and 14.09 times of sweet potato, rapeseed and corn, respectively. Therefore, it has good value of using Fengdan for repairing moderate copper contamination soil.