Relationship Of Salicylic Acid With Baicalein And Baicalein In Scutellaria Baicalensis Georgi

The medicinal substance content is one of the key points in deciding the quality of the Chinese herbal plants.Most of these substances are the secondary metabolites in herbal plants,and the secondary metabolites in plants play a primary role in response to environment stresses.Therefore,it is very important to elucidate the key factors in regulating the secondary metabolites in herbal plants.Salicylic acid(SA)as one of the signal molecules can regulate the secondary metabolites accumulation,and it was synthesized via phenylalanine lyase(PAL)and isochorismate synthase(ICS)pathways in plants.But the effect of the two pathways on the SA synthesis and the effects of SA on the secondary metabolis accumulation in herbal plants are far from clear.For this purpose,we studied the relationship between the enzymes and SA,and the relationship between the SA and main medicinal substances in Scutellaria baicalensis Georgi.We cloned the ICS gene in Scutellaria baicalensis Georgi with the method of rapid amplification of c DNA ends(RACE),amplificed the sics and spal sequence,and constructed the virus induced gene silencesilence(VIGS)vector pTRV2-sics and pTRV2-spal.Then we silenced the PAL and ICS gene with the method of VIGS,treated the seedlings with 10,20,40 mg/L exogenous SA and with drought,high salt and high temperature stresses.The results indicated(1)VIGS treatment resulted in significant silence of PAL and ICS expression.PAL silence resulted in the significant decrease of free salicylica acid(FSA)content,but a slight decrease of total salicylic acid(TSA)content.ICS silence resulted in a significant decrease of FSA and TSA content.The baicalin content decreased significantly when the FSA or FSA/TSA decreased,but the baicalein did not.PAL silence significantly affected the FSA,ICS affected TSA content.FSA significantly affected the baicalin,rather than baicalein content.We analyzed the correlation of PAL,ICS with SA,and SA with metabolites.Both FSA and TSA exhibited a strong positive correlation with PAL and ICS,respectively.FSA and TSA positively correlated with baicalin and negatively correlated with baicalein(2)The effects of exogenous SA treatment on the PAL and ICS expression varied.The 20 mg/L treatment resulted in the increase of the genes expression and enzyme activities,and the 10 and 40 mg/L treatments resulted in the decrease of the expression and enzyme acitivities at most sampling time.The perturbation of enzymes activity had significant effects on the endogenous SA accumulation.The results of spearman analysis show that PAL correlated more strongly with the FSA(coefficient 0.883,p<0.01)than did ICS(coefficient 0.586,nonstatistically),while,ICS correlated more strongly with the TSA(coefficient 0.510,nonstatistically)than PAL(coefficient 0.517,nonsignificant).FSA correlated more strongly positively with baicalin(coefficient 0.667,p<0.05)than did TSA(coefficient 0.267,nonsignificant),while TSA correlated more strongly negatively with baicalein(coefficient-0.65,p=0.058)than did FSA(coefficient-0.133,nonsignificant).(3)Drought stress treatment inhibited the PAL and ICS expression,while high salt and high temperature facilitated the expression.The results of spearman analysis show that PAL correlated more strongly with the FSA(coefficient 0.651,p<0.05)than did ICS(coefficient 0.429,nonstatistically),and ICS correlated strongly with the FSA and than that of TSA(coefficient 0.643,nonsignificant).FSA correlated more strongly positively with baicalin(coefficient 0.717,p<0.05)than did TSA(coefficient 0.683,nonsignificant),while TSA correlated more strongly negatively with baicalein(coefficient-0.59,p<0.05)than did FSA(coefficient-0.383,nonsignificant).According to these results,we drawn conclusions as below.(1)Both the PAL and ICS pathways are involved in SA synthesis in S.baicalensis.The effects of the two pathways on SA synthesis were different.PAL mainly affects FSA ranther than TSA synthesis,and ICS reversely.(2)FSA was more strongly correlated with PAL than ICS,and TSA was more strongly with ICS than PAL in S.baicalensis.(3)The PAL and ICS pathways of SA synthesis were negatively regulated,and SA might be involved the regulation.The negative regulation mechanism was necessary for plants to avoid injury by the ecessive accumulation of SA.(4)SA can significantly affect the baicalin and baicalein acculumation in S.baicalensis.FSA mainly affects baicalin accumulation,and ICS mainly affects baicalein accumulation.(5)There was correlation between SA and baicalin or baicalein accumulation.Both the FSA and TSA positively correlated with baicalin,but negatively with baicalein.(6)The correlation between SA and baicalin or baicalein consistent with a coordinated utilizing both phenylpropanoid skeletons and other intermediates,by which synthesized flavonoids to regulate the physiological state of plants.(7)There exists complex retulatory network in plant secondary metabolism.Although SA can regulate the secondary metabolites,the genetic mechanism are lacking.The regulatory mechanism of SA on the particular secondary metabolites in S.baicalensis needs further to be elucidated.