The Mechanisms Of NO In Cd Accumulation And The Saponins Synthesis In Panax Notoginseng Roots

Cadmium (Cd) is a non-essential element with no known biological function in flora and fauna. Cd pollutant is a limiting factor for plant growth and crop production. Due to its high solubility, Cd can easily and rapidly be taken up by plant roots and translocated to above-ground parts. Therefore, Cd contamination in crops poses a potential risk to human health and compromises the commercial value of the products, especially for plants with edible roots. Cd mines and smelters is a main source of Cd contamination. Because Cd is usually in combination with Pb and Zn, the processes of Pb and Zn smelting can also increase Cd entry to environment.Panax notoginseng (Burk.) F.H. Chen is one of the precious traditional herbs in China. The roots are regarded as main medicinal part and are used for the treatment of cardiovascular disease, inflammation, trauma and hemorrhagen. Due to its high medicinal value, this plant is widely cultivated in Wenshan autonomous prefecture, Yunnan Province, China. However, this area contains several Pb and Zn mineral deposits and background value of the amount of heavy metals in soil of the very area is high. Moreover, in order to pursue more economic benefits, excessive application of pesticides and fertilizers lead to increasing exacerbation of heavy metal contamination in growing zone of Panax notoginseng. Cd contamination of Panax notoginseng roots is directly correlated with its medicinal safety, making this a primary obstacle for drug safety and its international trade. Thereby, minimizing or preventing Cd contamination of Panax notoginseng roots is an urgent problem that remains to be solved.To solve this problem, we examined Cd toxicity on Panax notoginseng and its response to Cd stress under hydroponic conditions in this study. We also analyzed the mechanisms of Cd-induced NO production and its regulatory roles in Cd accumulation and saponin synthesis. These results could provide new research insights and theoretical bases for controlling Cd contamination by biological methods in future. The main findings of this study are as follows:(1)The annual Panax notoginseng seedlings were subjected to 0,25,50 and 100 Cd treatment for 24h, the Cd toxicity to Panax notoginseng roots and its response to Cd stress were analyzed. The results showed that Cd stress has an effect on absorption of mineral elements. The concentrations of Ca and K in Panax notoginseng roots decreased significantly; whereas, the contents of Mg in Panax notoginseng roots showed a trend of first increase and then decrease with the increase of Cd treatment concentration. The results from membrane integrity revealed that the staining degree of Evans blue and OD600 per fresh weight enlarge gradually with the rise of Cd concentration. In addition, ROS and H2O2 contents in root cells also increase significantly with the rise of Cd cocentration. These results indicated that Cd toxicity causes oxidant stress in root cells. By detection the antioxidant enzymes activity, we found that the CAT and POD activities were enhanced with a dose-dependent manner after Cd treatment for 24 h. After 25,50 and 100μmol/L Cd treatment, the SOD activities were also increased by 1.34,2.35 and 2.06 times higher than that of control, respectively. These results made it clear that Cd has a toxicity effect on Panax notoginseng roots and give rise to oxidative sress and activation of antioxidant enzymes may be involved in the alleviation of Cd toxicity by elimination Cd-induced ROS production.(2) We then analyzed the effects of endogenous NO on Cd accumulation in Panax notoginseng roots by adding different additives into Cd treatment solution. The results showed that the content of endogenous NO appeared a trend of first increase and then decrease with the rise of Cd concentration, the top point reached at 50μM. Adding tungstate or L-NAME, a nitrate reductase inhibitor or NO synthase inhibitor, the Cd-induced NO production was reduced by 72 and 4%, respectively, indicating that Cd-induced NO production in Panax notoginseng roots primarily through the nitrate reductase pathway. Cd accumulation in Panax notoginseng roots was analyzed by adding NO donator (SNP), tungstate and cPTIO into Cd treatment solution. The results showed that SNP promoted Cd accumulation in root cell wall but decreased by the addition of tungstate and cPTIO into Cd treatment solutions. In addition, SNP could also promote the increase in contents of hemi cellular1 and pectin and activities of pectin methylesterase activity (PME); whereas, this promotion could be inhibited by tungstate or cPTIO. These results suggested that nitrate reductase-dependent NO production enhances Cd accumulation in Panax notoginseng through affecting root cell wall properties.(3) Panax notoginseng is one of the most important Chinese medicines. Therefore, we further analysis the roles of nitrate reductase-mediated NO production in the regulation of Panax notoginseng saponin (PNS) synthesis in Cd-stressed Panax notoginseng roots. The results showed that endogenous NO promoted the Rb1 synethsis but reduced the Re and Rgl accumulation. However, the concentrations in the total saponin did not show significant changes after the plants were treated by Cd, Cd+SNP and Cd+tungstate. To further analysis the mechanisms of the effect of NO on saponin systhesis, we analysis the expression profiles of the saponin key genes under Cd stress conditions. The results showed that endogenous NO promoted the transcription of β-AS, CAS ans SE, but deceased the transcription of DS. and had a suppression effect on information flow from 2,3-osidosqualene to dammarane saponin. So endogenous NO restained the synthesis of tatal saponin.