The Function Of SmJAZ In Regulating The Biosynthesis Of Salvianolic Acids And Tanshinones In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge(Danshen),as one of the most widely used traditional Chinese medicines,its demands have been growing year by year,and has attracted much attention as a “model medicinal plant” for studying secondary metabolic synthesis and regulation.The active ingredients in S.miltiorrhiza are mainly divided into water-soluble salvianolic acid and lipid-soluble tanshinone.As an important plant hormone,Jasmonic acid(JA)plays an important role in the induction of plant secondary metabolites.Many studies have been reported that exogenous application of methyl jasmonate(MeJA)can promote the accumulation of two active components in S.miltiorrhiza by simultaneously inducing the expression of related genes on the synthetic pathway of salvianolic acid and tanshinone.However,the regulation mechanism has not been studied clearly.The JASMONATE ZIM DOMAIN(JAZ)protein family has been widely reported as an inhibitor of JA signaling pathways involved in the regulation of the accumulation of multiple secondary metabolites in plants,as well as the redundancy and specificity of functions among different members of this family.In this study,we used the hairy roots of S.miltiorrhiza as materials to study the function of the SmJAZ gene family in the MeJA-induced synthesis of salvianolic acid and tanshinone,aiming to explore the redundancy and specificity of the function and to explain the regulation mechanism.The main results of this study are as follows:1.We analyzed the accumulation of secondary metabolites in different tissues from S.miltiorrhiza and its response to MeJA.In the flowering stage of S.miltiorrhiza,Rosmarinic acid(RA)and salvianolic acid B(Sal B)were found to be accumulated in both aboveground and underground tissues,while tanshinone accumulated only in roots and especially in periderm.After treatment with 100 ?M MeJA for 6 d,the contents of salvianolic acid B and total salvianolic acids increased significantly and reached 2.12 and 1.55 times of the control,as well as significantly promoted dihydrotanshinone I(DT-I),cryptotanshinone(CT),tanshinone IIA(TA-IIA),and total tanshinone(TTA),reached 1.13 times,1.45 times,1.55 times and 1.35 times of the control,respectively.Besides,the expression levels of related genes in the biosynthetic pathway of salvianolic acid and tanshinone increased significantly.2.Based on sequence searching the genome of S.miltiorrhiza and gene amplification,we obtained 9 full-length ORFs of SmJAZs,which were named SmJAZ1,-2,-3,-4,-5,-6,-8,-9,and-10,respectively.Phylogenetic tree analysis indicated that the dicotyledonous JAZ can be divided into five subgroups,of which SmJAZ1/2/5/6 belongs to subgroup I(SI),SmJAZ10 belongs to SII,SmJAZ8 belongs to SIV,and SmJAZ3/4/9 belongs to SV.Amino acid sequence alignment revealed that all SmJAZs contain the ZIM and Jas domains.The Jas domain of SmJAZ8 lacks the conserved LPIAR motif,and the N-terminus of SmJAZ1/2/5/6/8 contains the LxLxL-type EAR motif.Promoter analysis revealed that all promoter regions of SmJAZs contain the MYC2 binding element G-box,in addition to functional elements involved in abiotic stress and growth or primary metabolism.3.Tissue-specific expression analysis showed that the expression levels of SmJAZ3/4 in root from seedling or flowering stage of S.miltiorrhiza were significantly higher than that in the aerial part,and SmJAZ4 specifically expressed in periderm.The expression levels of other SmJAZs in the aerial and underground part from seedling showed no significantly,while were significantly higher in stem and leaf from flowering stage plants than those of other parts.The expression of all SmJAZs was significantly increased under MeJA treatment.The expression of SmJAZ8 reached the highest value of 89.6 times after induction for 0.5 h.The expression of SmJAZ1/2/5/6 reached the highest value after induction for 1 h,and was 41.5,11.3,138.4 and 10.6 times of the control,respectively.The expression of SmJAZ10 reached the highest value of 133.8 times after induction for 6 h.The expression of SmJAZ3/4/9 reached the highest value after 12-24 h of induction,and was 3.9,23.0,and 3.2 times of the control,respectively.The expression levels of SmJAZs were affected by different elicitors-induction,indicating that the SmJAZ family may be involved in the crosstalk of multiple hormone signaling pathways.4.We obtained transgenic hairy roots overexpressing SmJAZs by Agrobacterium rhizogene ATCC15834-mediated transformation,respectively.Under MeJA treatment,the contents of Sal B in all positive transgenic lines were significantly decreased.The contents of tanshinones in the hairy roots overexpressing SmJAZ1,SmJAZ2,SmJAZ5,SmJAZ6 and SmJAZ9 were significantly increased,while overexpression SmJAZ3,SmJAZ4 and SmJAZ8 reduced the accumulation of both Sal B and tanshinones.qRT-PCR analysis showed that the expression levels of salvianolic acid synthesis related genes SmRAS1 and SmCYP98A14 in the overexpressing lines were significantly decreased,while the expression levels of tanshinone synthesis related genes SmCPS1 and SmCYP76AH1 showed various changes,indicating that SmJAZs regulate JA-induced tanshinone synthesis by different mechanism.In addition,overexpression of a single SmJAZ significantly changed the expression of other SmJAZs,indicating a co-regulatory effect between these family members.5.Y2 H analysis showed that except for SmJAZ5,most SmJAZs could interact with SmMYC2 a and SmMYB39.SmJAZ1/2/3/4 could interact with SmMYC2 b,and SmJAZ1/2 could interact with SmPAP1.Tissue-specific expression analysis revealed that the transcription factors interacted with SmJAZs showed spatiotemporal expression specificity.Under MeJA treatment,the expression levels of SmMYC2 a,SmMYC2b and SmPAP1 increased significantly,while the expression level of SmMYB39 decreased significantly.In addition to SmJAZ5/6,other SmJAZs can form homodimers,and most SmJAZs can form heterodimers.6.Under MeJA treatment,the contents of salvianolic acid and tanshinone in SmJAZ8-RNAi hairy roots were significantly increased.The expression levels of tanshinone synthesis related genes SmCPS1 and SmCYP76AH1 in RNAi hairy roots increased significantly,while the expression levels of salvianolic acid synthesis related genes SmRAS1 and SmCYP98A14 showed no significant or significant decrease,indicating that SmJAZ8-RNAi may inhibit the synthesis of rosmarinic acid derivatives downstream of SmCYP98A14.The RNA-Seq results indicated that the differential expression genes regulated by JA and SmJAZ8 simultaneously were mainly enriched in primary metabolism,stress response and transcriptional regulation pathway,indicating that SmJAZ8 is involved in transcriptional regulation of JA-induced primary and secondary metabolism in S.miltiorrhiza.