Study On Mechanism Of Effects Of Arbuscular Mycorrhizal Fungi On Plant Endogenous Hormone Jasmonic Acid

Objective:Traditional Chinese medicine(TCM),the crystallization of tradition experience and wisdom,occupy an important place in Chinese medical system.Chinese materia medica resources are the material basis of TCM,with the quality directly affecting the curative effects and the development of TCM.My supervisor Professor Gou Jiao and her team have been devoting themselves to prophylaxis and treatment of metabolic diseases using the integration of TCM and Western medicine for more than three decades,with principles of“combining TCM and western medicine,combining medicine and drug,and combining scientific and clinic research".She raised the idea of organic bond of "medicine,production,study and research",that“study of prescriptions in prophylaxis and treatment of metabolic diseases with the integration of TCM and Western medicine should run through overall process of origin control,pharmacognostic identification,extract and processing,preparation production and quality inspection”.Chinese materia medica is the source of TCM preparations,and its quality directly determines quality of TCM products,and clinical curative effect.There are 290 million patients with cardiovascular disease now in China,with two people in every five the dead are with cardiovascular disease,and cardiovascular disease has become the biggest health problem.Salvia miltiorrhiza Bunge is a well-recognized medicine for cardiovascular disease,with the compound Danshen dripping pills successfully passed II phase of clinical trial of FDA.Cultivated area of S.miltiorrhiza increased yearly with growing demands;however continuous cropping and extension of cultivation years could cause severe root diseases,with morbidity at 60%to 70%,which adversely affect the quality of S.miltiorrhiza,and restrict its use in treating cardiovascular disease.Abuscular mycorrhizal(AM)fungi are mycorrhizae that evolve obligate symbiosis with about 90 percent of terrestrial plants,and could promote plant growth,increase contents of secondary metabolites,change the equilibrium status of plant endogenous hormones,induce defense reaction on host plant and improve response for stress.Plant hormones,produced within plants,could regulate plant growth,metabolism,response to environment at extremely low concentrations.Jasmonic acid(JA),an important plant hormone,plays crucial role in AMF regulated plant growth and disease resistance,and involves in the colonization of AMF.Transmembrane ionic current,initial signals in stress response signal networking,are the key to reveal mechanism of plant defense.Though molecular mechanism of JA regulating plant defense has been well studied,whether and how AMF is involved in JA regulated plant defense,and how transmembrane ionic current work between AMF and JA remained unclear.Co-cultured hairy root of Salvia miltiorrhiza and Daucus carota were used in this paper to study connections of AMF,JA and ion signal,clarify the effect of AMF on JA and transmembrane ionic current,and reveal the relationship within AMF,JA,and transmembrane ionic current.This research provided guidance for application AMF for disease resistance in the cultivation of medicinal materials,and could promote the application of AMF in Chinese medicinal materials cultivation.Methods:1.Plant hormones were extracted with isopropyl alcohol:water:HCI(2:1:0.002),and methylene chloride,and analyzed by ultra-high performance liquid chromatography-mass spectrometry and multiple reaction monitoring(MRM)quantitative analysis.2.S.miltiorrhiza inoculated and non-inoculated with GI for 45 d were experimented for short-term and long-term.Short-term treatment:SHAM,a JA inhibitor,at high(500 μmol/L),medium(100 μmol/L),and low(10 μmo1/L)concentrations were sprayed to S.miltiorrhiza inoculated with GI for 0.5 h,2 h,6 h,12 h,24 h and 48 h,respectively,while control group was sprayed with distilled water.Long-term treatment:100 μ mol/L MeJA and 100 μ mol/L SHAM were sprayed to S.miltiorrhiza inoculated with GI,respectively,every two days for 2weeks,and analytes were collected after treatment stopped for 1 week,while control group was sprayed with distilled water.Contents of endogenous hormones were determined by UPLC/MS/MS,expression of JA synthesis related genes,AOS、AOC、OPR、JAR1，and signal pathway related genes,JAZ、COI and JMT analyzed by real-time PCR,contents of salvianolic acids,S.miltiorrhiza effective component,were detected using UPLC,and contents of tanshinone were detected by HPLC.3.S.miltiorrhiza inoculated and non-inoculated with GI for 45 d were sprayed with 100 μmol/L MeJA and 100 μmol/L SHAM for 2 weeks respectively,and ion current of Ca2+,K+ H+ were detected by non-invasive micro-test technology(NMT).4.Ingredients of M medium and 6-7-V medium were compared,and sucrose and elements of Na,P,K and Cl were selected as ingredients affecting growth of S.miltiorrhiza hairy root and AMF,then co-cultured system was studied by orthogonal experiment.5.Hairy root of D.carota and co-cultured hairy root of D.carota were treated with 0.1 mmol/L MeJA and 0.1 mmol/L SHAM,respectively for 0.5 h,1 h,2 h,4 h,8 h and 12 h.Contents including JA,OPDA and JA-ile were determined by UPLC/MS/MS;expression of AOC,OPR,JAR1,COI1 and JAZ were detected by real-time PCR.6.Hairy root of D.carota and co-cultured hairy root of D.carota were treated with 0.1 mmol/L MeJA and 0.1 mmol/L SHAM,respectively for 2 h,and ion current of Ca2+,K+ and H+were detected by NMT.Results:1.Study on effects of AMF on contents of plant endogenous hormones in S.miltiorrhiza:Endogenous hormones JA,OPDA,JA-ile,ABA,SA,GA3 and IBA,scanned using anion mode,while MeJA in positive ions mode,were effectively separated using the established of UPLC/MS/MS condition,with the linear correlation coefficient(r)in the range of 0.9914-0.9991;average recovery of JA and ABA in S.miltiorrhiza root and leaf for high,medium and low concentrations were 80.40%-97.00%.Intraday and inter-day precision RSD were below 10.0;Stability and repeatability RSD were below 8.0%.Contents of JA,OPDA,JA-ile,MeJA were significantly higher in S.miltiorrhiza roots and leaves inoculated with GI for 45 d than unvaccinated control group,the content of ABA was significantly lower than that in control group,while GA3,SA,IBA were not detected in S.miltiorrhiza roots nor leaves.2.Study on effect of AMF on endogenous hormones in S.miltiorrhiza treated with MeJA and SHAM:(1)in both short-term and long-term treatment,contents of JA,JA-ile and OPDA in distilled water sprayed S.miltiorrhiza inoculated with GI were significantly higher than that in control group in both over ground and underground part,with expression of JA synthesis related genes,AOC,OPR and JARl,upregulated,while contents of ABA,SA and GA3 were lower than that in control group.(2)Contents of JA,OPDA and JA-ile significantly decreased in over-ground part of short-term SHAM-treated S.miltiorrhiza inoculated with GI,with expression of JA synthesis related genes,AOC,OPR and JARI down-regulated,and expression of JA signal pathway genes COI,JAZ and JMT unchanged.While in the underground part of the same treatment,contents of JA,OPDA and JA-ile increased notably in the first 48 hours,with expression of AOS,AOC,OPR and JAR1 up-regulated,and that of COI,JAZ and JMT changed dramatically,which were different from results of the over-ground part.(3)Contents of JA and its intermediates,OPDA and JA-ile increased notably in both over-and under-ground part of S.miltiorrhiza in control and CI group treated with MeJA for 2 weeks,with expression of AOS,AOC,OPR and JAR1 up-regulated.And the opposite results were observed in SHAM treated groups,with contents decreased drastically and expression down-regulated.(4)Contents of caffeic acid,rosmarinic acid,salvianolic acid b,cryptotanshinone,tanshinone I and Tanshinone IIA in both over-and under-ground part of S.miltiorrhiza significantly increased in the GI group sprayed with distilled water for 2 weeks compared to that in the control group.Their contents also increased notably in MeJA treated control and GI group,while decreased notably in SHAM treated control and GI group.3.Study on mechanism of AMF GI regulating JA transmembrane ionic current in S.miltiorrhiza:(1)Control group treated with distilled water indicated influx of H+,and efflux of Ca2+ and K+,and influx of H+ and K+,and efflux of Ca2+ were detected after treated with SHAM for 2 weeks,while immediate influx of Ca2+ and efflux of H+and K+were observed when treated with MeJA for 2 weeks.(2)When sprayed with distilled water,notable influx of H_,Ca2+,and efflux of K+ were detec’ted in S.miltiorrhiza inoculated with GI for 45 d,and notable efflux of H+ and Ca2+,influx of K+ were observed after 2 weeks treatment of SHAM,while the opposite results were detected after MeJA treatment.4.Study on establishment of co-cultured system of S.miltiorrhiza hairy root:Factors affecting hairy root growth were content of KC1(A),content of Na2HPO4+ and Na2HP04(B)and sucrose(C)in descending order.However,spores in all 9 experiment groups ruptured with infection rate at 0,suggesting the attempts to form co-culture system of S.miltiorrhiza hairy root failed.5.Study on mechanism of actions of MeJA and SHAM on endogenous hormones in co-cultured hairy root of D.carota:(1)contents of JA increased in hairy root of D.carota after inoculated with GI for a month,and a significant difference was observed between samples of 1 h and 8h,while contents of OPDA and JA-ile were lower than that of CK in the first 2 h,and higher after 2 h,which was different from effect of AMF to S.miltiorrhiza.(2)Contents of JA and JA-ile in control and GI group increased after treated with MeJA,while trend of OPDA content differed.Contents of JA and JA-ile in control and GI group decreased after treated with SHAM,with trend of OPDA content different.These results were different that in S.miltiorrhiza.6.Study on mechanism of JA induced transmembrane ionic current in co-cultured hairy root of D.carota:(1)efflux of H+ and K+ and influx of Ca2+were detected in hairy root of D.carota cultured for a month,and immediate efflux of Ca2+ and H+,influx of K+were detected after treated with SHAM for 2 weeks,while immediate influx of Ca H+ and K+were observed when treated with MeJA for 2 weeks.(2)Notable influx of H+,Ca2+,and efflux of K+ were detected in co-cultured hairy root of D.carota for a month,while notable efflux of H+ and Ca2+,influx of K+were observed after 2 weeks of MeJA or SHAM treatment.Conclusion:1.This study developed a new analysis method of endogenous hormones in fresh S.miltiorrhiza using UPLC/MS/MS,and this method was proved with high sensitivity,high specificity,high accuracy,good repeatability,and applicable to the contents determination of endogenous hormones in AMF infected S.miltiorrhiza.2.(1)AMF could notably increase the contents of JA,OPDA and JA-ile in both over-and under-ground part of S.miltiorrhiza,up-regulate expression of JA synthesis related genes,AOS,AOC,OPR and JAR1,and lower the contents of ABA,SA and GA3.(2)Results of changes in underground part short-term treated with SHAM were opposite to that of over-ground part and long-term treatment,which may be contributed to the temporal correlation of SHAM treatment and effects of AMF on JA medicated defense.When treated by SHAM for a short-term,AMF may recognize that as stress and promote JA medicated defense,while treated by SHAM for a long-term,JA pathway adapts and contents of JA,OPDA and JA-ile thus reduced.(3)AMF could notably lower the contents of ABA,SA and GA3 in both over-and under-ground of S.miltiorrhiza,and their contents decreased in both control and GI group after sprayed with MeJA,while increased when sprayed with SHAM,indicating a antagonism of JA to ABA,SA and GA3 in S.miltiorrhiza,which leads to a hormonal equilibrium for plant growth and adapting environmental changes.(4)AMF could significantly increase contents of caffeic acid,rosmarinic acid,salvianolic acid b,cryptotanshinone,tanshinone I and Tanshinone IIA,suggesting AMF could promote the accumulation of secondary metabolites,and/or increase content of JA,and induce the accumulation of secondary metabolites synergically in S.miltiorrhiza for stress defense.3.(1)Transmembrane ionic signals were involved in JA mediated defense.JA was synthesized under the action of JMT when MeJA got into cytoplasm,then JA-ile,synthesized by JAR1,formed compound of SCFCOI1 and activated plasma membrane H+-ATPase to pump out H+ and activate Ca2+ channel though depolarization,followed by the activation of anion channel,leading to efflux of K+.Transmembrane current of Ca2+ and H+ involve in the initial stage of JA activated defense.(2)Ionic signals involve in recognition of S.miltiorrhiza by AMF,and H+,Ca2+ and K+ play crucial roles in symbiosis as initial signals.Notable influx of H+ and Ca2+,efflux of K+ were detected in S.miltiorrhiza inoculated with GI,while after 2 weeks of MeJA treatment,the results turned the opposite,indicating a negative feedback of AMF to JA pathway.4.Growth of S.miltiorrhiza requires medium rich in nutrient,while that of AMF requires medium with low content of nutrient,and AMF may otherwise rupture due to osmotic pressure.Dual-culture systems for AMF and S.miltiorrhiza hairy root are still under investigation,and co-culture system of S.miltiorrhiza hairy root was used in following study on effects of ionic signals in AMF-JA medicated signaling pathway.5.(1)synthesis of OPDA occurs in chloroplast,and the beneficial role of AMF could be affected by the in vitro hairy root instead of the whole plant,which may contribute to the difference of OPDA content in MeJA and SHAM treated co-cultured D.carota hairy root and GI i noculated S.mil tiorrhiza.While M medium,rich in carbohydrate could change the chemical equilibrium of interaction between AMF and plant,which could also lead to the different results in co-cultured D.carota hairy root and potted S.miltiorrhiza.(2)Mechanism of AMF affecting JA in co-cultured D.carota hairy root and potted S.wiltiorrhiza were different from that in S.miltiorrhiza,indicating hairy root could not replace the function of the whole plant,and hairy root is not an ideal material for the mechanism study of AMF effects to JA.6.AMF could regulate ion channels positively and negatively when enhance defense through JA pathway.When MeJA increases dramatically,AMF could pump out Ca2+ by negative regulation of JA to prevent excess Ca2+ in cytoplasm,with influx of K+to prevent charge imbalance.