Study On The Regulatory Of Nitrogen Forms On Growth And Alkaloid Metabolism Of Atropa Belladonna L.

Atropa belladonna L.(A.belladonna)is a perennial herb in the Atropa of the Solanaceae,and the only medicinal plant source of tropane alkaloids(TAs)in China(authorized by pharmacopoeia of The People's Republic of China 2010),with its main medicinal ingredients hyoscyamine and scopolamine.However,the alkaloids content in A.belladonna is usually very low,and the content of scopolamine is far less than hyoscyamine.This led people to pay attention to the relationship between the synthesis and accumulation of hyoscyamine and scopolamine and the environmental conditions,as well as to find suitable incubation conditions to increase the yield of hyoscyamine and scopolamine.Nitrogen is one of the important factors affecting plant growth and metabolism.Different forms of nitrogen have different effects on plants,and nitrogen forms are also related to the synthesis and accumulation of alkaloids.In this paper,we took A.belladonna as experimental material,combined with sand culture and nutrient solution culture,setting different ratios of ammonium to nitrate(0/100,25/75,50/50,75/25,100/0),to investigate the changes of growth and nitrogen metabolism related physiological indexes and alkaloid contents under different treatments of nitrogen forms at different treating time.Besides,we studied the effects of nitrogen forms on the upstream products,signal molecule and key enzyme genes in the secondary metabolism of A.belladonna,which helps us explore the mechanism of nitrogen forms regulating the growth,nitrogen utilization and the alkaloids synthesis of A.belladonna from cell and gene levels.We hope the work may contribute some information for the research of the relationship between secondary metabolism of A.belladonna and environmental factors,and provide theoretical basis for the rational allocation and efficient utilization of nitrogen fertilizer in the process of cultivation of A.belladonna.The main results areas follows.1.During the whole treating period,both the shoot dry mass and root dry mass of A.belladonna increased gradually with the rise of NO3-content,in which the shoot dry mass reached the max value under treatment of 100%NO3--N,whereas the root dry mass was highest at NH4+/NO3-of 25/75.The chlorophyll content of A.belladonna was determined.It was found that when NH4+ proportion was bigger,the chlorophyll content of the plant was higher.The above results indicate that A.belladonna has the characteristics of nitrate plant,with 100%NO3-nutrient solution it can accumulate more dry matter,the presence of a small amount of NH4+(NH4+?25%)does not affect its growth and can increase the synthesis of chlorophyll.Therefore,combining ammonium with nitrate and at the ratio of 25/75 to supply nitrogen is more conducive to the growth of A.belladonna.2.Investigating the variation of the contents of main nitrogen compounds and key enzymes activities of nitrogen metabolism in treatments of different nitrogen forms with treating time,we found at the beginning of treatment,total ammonium nutrition increased the content of soluble protein,but with the extension of treating time,50/50(NH4+/NO3-)was more conducive to the accumulation of soluble protein.The content of free amino acids(FAA)overall was elevated with the increase of ammonium ratio in the treated solution,which reached the highest at NH4+/NO3-of 75/25.The nitrate content rose with the increase of nitrate in the treated solution.When the nitrate was more than50% in the treated solution,the nitrate content in plant decreased during the whole treating period.NR activity went up with the increase of the proportion of NO3-,but it went down with treating time prolonging.With the increasing proportion of NH4+,GS activity rose first and then decreased,at the same time totally nitrate treatment reduced GS activity over treating time when totally ammonium treatment increased GS activity.That is to say,with the increase of nitrate nitrogen in the nutrient solution,A.belladonna can accumulate more nitrate,which promotes NR activity,While addition of ammonium can increase the GS activity,facilitating the GS/GOGAT cycle,thereby increasing the leaf soluble protein and free amino acids contents,so the combination of ammonium and nitrate with ratio of 25/75 is more conducive to the nitrogen metabolism of A.belladonna.3.Studing the changes of leaf scopolamine and scopolamine contents in different forms of nitrogen at different treating time,we found although the initial amount of hyoscyamine and scopolamine under mixed nitrogen of 50/50(NH4+/NO3-)were not sohigh,there was a continuous promotion of TAs contents during the whole treating period,especially scopolamine increased significantly.Within the whole treatment period,the content of scopolamine under 100%NO3-treatment was less than other treatments.The results indicate that the nitrogen forms significantly affect the accumulation of TAs,which also affect the conversion of hyoscyamine to scopolamine.The 50/50(NH4+/NO3-)treatment is more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.4.In order to clarify the mechanism of nitrogen forms regulating alkaloids synthesis of A.Belladonna,we took A.Belladonna seedlings treated 28 d as material to study the effects of different nitrogen forms on the precursor putrescine(Put),signal molecule nitric oxide(NO)and key enzyme genes PMT,TR? and H6 H expression levels in secondary metabolism.The results showed that the content of Putrescine(Put)was almost consistent with the change of key enzymes activities in the synthesis of Put,they both increased with the rise of ammonium ratio,reaching the highest at 75/25(NH4+/NO3-).The detection of signaling molecule nitric oxide(NO)showed that the NO concentration decreased with the decrescent nitrate proportion.Further detection of gene expression levels of PMT,TR? and H6 H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6 H in roots.When the ratio of ammonium to nitrate was 50/50,PMT,TR? and H6 H in leaves and roots had higher expression levels.It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes.50/50(NH4+/NO3-)treatment increased the gene expression of TR ? in both leaves and roots as well as PMT and H6 H in roots,promoting the synthesis of Put to hyoscyamine and the conversion of hyoscyamine to scopolamine.In summary,nitrate is conducive to the growth of A.belladonna,a certain proportion of ammonium can promote the secondary metabolism of the plant.Changes of nitrogen forms has direct effects on the nitrogen metabolism and chlorophyll content,leading to differences in the primary growth of A.belladonna.The regulation of secondary metabolism by nitrogen forms plays a role by altering the expression of key enzymes in the secondary metabolism.