Study On The Mechanism And Relative Alleviation Measures Of Ammonium Toxicity To Panax Notoginseng

Panax notoginseng（Burk.）F.H.Chen,or Sanqi,belongs to the Araliaceae,which is a famous traditional Chinese medicine containing various active ingredients such as saponins,dencichine,etc.It plays an important role in the prevention and treatment of cardiovascular and cerebrovascular diseases.Yunnan province in China is the geo-authentic area of Sanqi,where has the largest planting area as well as raw materials production,accounting for more than 95%nationwide.At present,annual Sanqi planting area in Yunnan has been exceeding 53,000 hectares,from which around 50,000 tons of medicinal materials have been harvested,with an agricultural value of up to 10 billion Yuan.The income from cultivation and processing,as well as from other relevant industries,has become one of the most important economic pillars of Yunnan province.With the increasing of planting scale and market demand,partial and over fertilization has become a severe problem in Sanqi cultivation.Since shading and covering are necessary in Sanqi cultivation,soil temperature is usually low and soil water is usually high,soil under this condition may accumulate NH₄⁺for a long time.Even worse,over fertilization may aggravate the toxicity of ammonium on Sanqi.Therefore,based on the problem of ammonium toxicity in Sanqi cultivation,following studies were conducted.Nitrogen（N）species,rates,ways of application in Sanqi cultivation,as well as soil N conteng,were investigated.Moreover,soil nitrification rate of Sanqi planting mode was simulated in lab and field.Furthermore,effects of N application forms and rates on the growth of Sanqi were carried out by a field experiment.In addition,the mechanism of ammonium and nitrate combined application on the ammonium toxicity alleviation of Sanqi was studied by hydroponics and sand culture experiments.Then,effects of balanced N and potassium（K）application on the yield and quality of Sanqi was carried out with a pot culture and a field experiment.The main results of this study were following:（1）N fertilizers in Sanqi cultivation are mainly ammonium or amine containing synthetic fertilizers.Annual N application rates of 2-yr and 3-yr old Sanqi are 270⁴00 kg·hm^-2,450⁵60 kg·hm^-2,respectively.Topdressing is the main way of N application,for 3⁶times a year.Average total N content of Sanqi planting soil is 1.98 g·kg^-1,and average available N content is 147 mg·kg^-1 which is above the rich grade.Simulation results showed Sanqi planting had little effects on the nitrification rate of soil.Soil with urea application had a higher nitrification rate than that with ammonium sulfate.With the increasing of ammonium sulfate application rate and soil water content,the rate of soil nitrification firstly increased and then decreased,was the highest when soil water was70%of field water holding capacity and ammonium sulfate application rate was 200mg·kg^-1.Under the planting mode with no shading and covering,soil cost 15 d to convert300 kg·hm^-2 ammonium sulfate,but cost 30 d under Sanqi planting mode.Thus,Sanqi planting mode makes soil to accumulate NH₄⁺easier than regular planting mode.（2）Ammonium-and amide-N application had a better effects on promoting Sanqi growth than nitrate-N application when N rate was 150 kg·hm^-2.However,nitrate-N was the better one when N application rate was 300 kg·hm^-2.When N application rate was 150kg·hm^-2,the three N forms had little effects on the content of carbon and nitrogen metabolites in Sanqi.With the increasing of N rate,ammonium-N had a more beneficial effect on the uptake of N but less beneficial for P,K and other microelements for Sanqi,than that with amide-and nitrate-N.Generally,150 kg·hm^-22 of amide-N application had a better effect on P,N and other microelements uptake.Moreover,with the increasing of N rate,Sanqi with ammonium-N was easier to generate reactive oxygen than that with amide-and nitrate-N.Altogether,Sanqi had the highest reactive oxygen content but the lowest antioxidant enzymes activity,when ammonium-N application rate was 300kg·hm^-2.（3）Under hydroponics,with ammonium-N concentration increased from 5 mM to 30 mM,the intergrity of plasma membrane of Sanqi root tip was gradually destroyed.Sanqi had obvious ammonium toxicity effects,with the significantly reduction of root activity and relative elongation,as well as heavily chlorosis leaves and markedly biomass decreasing,when ammonium-N concentration was 15 mM.But the ammonium toxicity effects of Sanqi could be visibly alleviated by 15 mM nitrate-N combined with 15 mM ammonium-N.On the one hand,combined application significantly reduced the uptake of NH₄⁺,meanwhile increased the activity of GOGAT,GDH and NR,thereby improved the assimilation of NH₄⁺to increase the ratio of organic N/total N.On the other hand,combined application improved the contents of sucrose,glucose,total soluble sugar as well as the activity of SS,SPS,MDH and ICDH,resulting the content of organic carbon increased.Therefore,Sanqi with ammonium-and nitrate-N combined application promoted the balance of C/N,compared to that only with ammonium-N application.Moreover,the C/N ratio of Sanqi under ammonium stress had no significant difference when nitrate-N addition rate increased from 0.15 mM to 15 mM.It demonstrated that the nitrate signal function might regulate the carbon and nitrogen metabolism in Sanqi under ammonium stress.（4）RNA-Seq analysis showed that Sanqi treated with15 mM nitrate-N（15N）and 15 mM nitrate-N combined with 15 mM ammonium-N（15AN）had 117 up-regulated and 268down-regulated DEGs compared to that treated with 15 mM ammonium-N（15A）.The DEGs were significantly enriched in 8 KEGG pathways,including the TCA cycle,photosynthesis,starch and sucrose metabolism,etc..Selected 5 up-regulated and 3down-regulated genes under 15N and 15AN but not under 15A for RT-qPCR,which involved in citrate synthase（ACLA-3）,dormancy-associated gene-1/auxin-repressed protein（DRM1/ARP）,ABC transpoter,etc.,the expression patterns of all these genes were similar to RNA-Seq.It further confirmed the reliability of RNA-Seq results.In addition,enzymes activities and metabolites contents of the TCA cycle that ACLA-3participates in were analysized,results indicated that 15AN could reduce the NH₄⁺content and increase metabolites contents of the TCA cycle in Sanqi.Thus,ACLA-3 plays an important role in the alleviation of ammonium toxicity by ammonium-and nitrate-N combination.（5）Under hydroponics,15 mM ammonium-N significantly inhibited root elongation,increased reactive oxygen content but reduced antioxidant enzymes activity of Sanqi,while released by SNP.NO content of Sanqi was significantly positive correlated with its NR activity under ammonium stress,which could promoted by exogenous nitrate.Meanwhile,both nitrate and SNP addition significantly accelerated the assimilation of NH₄⁺to organic N and increased the content of organic carbon under ammonium stress,thereby improved the C/N of Sanqi.Nevertheless,these effects of could inhibited by tungstate（NR inhibitor）or cPTIO（NO scavenger）.（6）Under field condition,compared to conventional N and K application rate(N?K₂O=1?1,300 kg·hm^-2 for 2-yr old,450 kg·hm^-2 for 3-yr old),reducing N(150kg·hm^-2 for 2-yr old,225 kg·hm^-2 for 3-yr old)but keeping K application rate（N?K₂O=1:2）could reduce root rot incidence of Sanqi by 19%,increase survival rate by11%,improve the yield of shoot and root yield by 3%and 8%,respectively,and raise total saponins content by 3%.So N?K₂O=1:2 is a more balancing way of N and K for Sanqi cultivation.Hydropoincs experiment showed the expression of 9 genes were inhibited by high N,15 genes were improved by high K in the saponins biosynthesis pathway of Sanqi.Total saponins content was negatively correlated with N/K ratio,but positively correlated with chlorophyll content,net photosynthetic rate and total soluble sugar content.Pot culture showed that compared to high N and high K,low N but high K significantly reduced N/K ratio,improved chlorophyll content,net photosynthetic rate and total soluble sugar content,then accelerated saponin accumulation of Sanqi.In summary,application 150 kg·hm^-22 ammonium-or amide-N has a better improving effect on plant growth of Sanqi.However,application ammonium-N 300 kg·hm^-2 costs more than 30 d for soil to convert ammonium,increases the content of reactive oxygen but decreases the activity of antioxidant enzyme and the uptake of mineral elements,then inhibites the growth of Sanqi.So,ammonium-N over application causes ammonium toxicity,the mechanism is carbon and nitrogen metabolism imbalanced by excessive ammonium accumulation in the tissue of Sanqi.Ammonium and nitrate combined application could prevent Sanqi suffer ammonium toxicity.Combined application can induce NR to improve NO synthesis,which might effectively scavenge reactive oxygen.Meanwhile,combined application can up-regulate ACLA-3 expression to improve the TCA cycle metabolism,which could maintain C/N balance.Moreover,reducing the application rate of conventional N but keeping K can reduce the incidence of root rot,improve survival rate,yield and quality of Sanqi.Therefore,in Sanqi cultivation,the recommendation way of N and K application are as following:ammonium or amide combined with nitrate,N?K₂O=1?2,N and K rates for 2-yr old are 150 kg·hm^-22 and 300kg·hm^-2,respectively,for 3-yr old are 225 kg·hm^-22 and 450 kg·hm^-2,respectively.