| Root of the perennial herb Salvia miltiorrhiza Bunge, a species of Salvia belong to familyLabiatae, is well-known as a Chinese traditional medicine named Danshen, and is widely usedto treat cardiovascular diseases, blood circulation disorders and inflammation. The importantmedicinal constituents in the Danshen are generally known as secondary metabolites includinglipid-soluble terpenes and water-soluble phenylpropanoids. Where the phenylpropanoids weremainly ascribed to salvianolic acids such as danshensu, protocatechuic acid, cinnamic acid,caffeic acid, p-coumaric acid, rosmarinic acid, salvianolic acid A, B, C and D etc., and theterpenes were named tanshinones including dihydrotanshinone â… , cryptotanshinone,tanshinone â… a nd tanshinone â…¡A, etc. Of these, content of the salvianolic acid B andTanshinone â…¡ Awere preferred as important criterias in determining quality of the Danshen inChina. As important signaling compounds, generally salicyclic acid (SA) and methyljasmonate, gibberellic acid (GA3) were applied to plant in a series of eliciting production ofterpenoids and phenolic acids, elevating stress resistance of plant and regulating plant growth.This study was carried out to demonstrate that the H2O2molecule involved in theJA-signal-transduction using S. miltiorrhiza seedlings as material. For better understandingmechanism of secondary metabolites production underlying sugar-metabolism and hormoneslevels, contents of salvianolic acids in roots, tanshinones contents in roots, leavesphotosynthetic ability, sugars metabolism, C/N rations as well as endogenous hormones levelsof seedlings and fruiting plants of S. miltiorrhiza were investigated under treatments of SA,MeJA and GA3foliar applications. As a part of the study, the effects of different sugars ascarbon nutrients in mediums on contents of salvianolic acids in cultures of S. miltiorrhiza hairyroots were investigated to prove the conclusion obtained in the study. The object of the studywas focused on the relationship between secondary metabolites production andphotosynthesis-sugars metabolism, hormones level in plants. The main results of the study areas follows:(1) The application of MeJA to the S. miltiorrhiza seedling activitated the antioxidantenzyme system in leaves during waterloss process after detached, that activities of polyamineoxidase (PAO), peroxidase (POD) and superoxide dismutase (SOD) was promoted and accumulation of H2O2was increased, meantime the activities of ascorbate peroxidase (APX)and glutathione reductase (GR) as well as H2O2content in chloroplast were increasedcorrespondingly. Further the experiment on cell-free system showed that the raising of H2O2concentrations (in a certain range) in media correspondly increased activities of APX, GR andSOD and contents of H2O2in the suspended-chloroplast in vitro. These results suggested thatantioxidant enzymes system and H2O2molecule were involved in JA signal transduction toincur stress responses in plant.(2) The results showed that the SA treatment positively developed leaf microstructure byincreasing leaf stomata density, reducing interspace in mesophyll tissue, decreasing both sizeof mesophyll cell and number of chloroplast in mesophyll cell, but size of the chloroplast andnumber of grana-structures in the chloroplast were markedly increased; meantime contents ofchlorophyll a and b in the leaves, net photosynthetic rate of leaves, contents of sucrose andsoluble sugar in shoots of the seedlings, were together significantly increased but activities ofacid invertase in both shoots and roots of the seedlings were significantly decreased. Similarlywith this, the activities of acid invertase in both roots and leaves as well as neutrial invertaseactivity in leaves of the S. miltiorrhiza fruiting plant were enhanced by the SA application,while contents of soluble sugar and sucrose of the fruiting plants were increased.While the MeJA application resulted in a series contrary effects for decreasing density ofleaf stomata, inducing a number of abnormal stomata, increasing interspace in mesophylltissue, notably reducing number and size of chloroplast in mesophyll cells and destructinggrana structures in the choloroplast; meantime contents of chlorophyll a and carotenoid of theleaves, leaf net photosynthetic rate as well as contents of sucrose and total amount of solublesugar in roots of the seedlings were together decreased, but activities of acid invertase in bothshoots and roots of the seedlings were significantly increased. Similarly with this, activities ofacid invertase in both roots and leaves as well as neutrial invertase activity in leaves of the S.miltiorrhiza fruiting plant were decreased by the MeJA application, while contents of solublesugar and sucrose in both roots and shoots of the fruiting plants were markedly decreased.When the GA3were applicated to the fruiting plants of S. miltiorrhiza, contents of solublesugar and sucrose in roots were significantly decreased.In a conclusion, the SA application to S. miltiorrhiza seedling was in favor of leafmicrostructure to accelerate leaves photosynthetic capacity and accelerated sucroseaccumulation in S. miltiorrhiza plants; whereas the MeJA application damaged leafmicrostructure to impair leaves photosynthetic capacity and led decrease-trend in sucroseaccumulation of the plants; the GA3application decreased sucrose accumulation in roots the S.miltiorrhiza fruiting plants. (3) Corresponding with sucrose accumulation, contents of tanshinone â…¡A,tanshinoneâ… in roots of the S. miltiorrhiza seedlings and contents of tanshinone â…¡A,cryptotanshinone, tanshinoneâ… as well as total amount of tanshinones in roots of the S.miltiorrhiza fruiting plants were together increased following to the foliar application ofsalicyclic acid (SA); while the MeJA application to S. miltiorrhiza seedlings increased contentsof tanshinones in the roots, and contents of sucrose and soluble sugar were decreased in theroots but increased in the shoots. Whereas the tanshinone contents were significantly decreasedin roots of S. miltiorrhiza fruiting plants along with decreasing in contents of sucrose andsoluble sugar in both shoots and roots of the plants under the MeJA and GA3treatments.Furthermore, treatment of higher concentration sucrose as carbon sucrose in media positivelyaccelerates production and accumulation of tanshinones in cultures of S. miltiorrhiza hairyroots comparing with treatments of glucose and fructose. This demonstrated that sucroseaccumulation in plant accelerated terpenes production in plant.Amounts of the tanshinones accumulation in roots of S. miltiorrhiza fruiting plants weremuch higher than which in roots of the seedlings, and contents of tanshinones in S. miltiorrhizahairy roots marked enhanced following to increase of survival time after treatment, thisindicated that terpenes production in plant was correlated to growth stage.On the other hand, comparing changes of endogenous hormones, such as ABA, IAA andZr in seedlings and fruiting plants of S. miltiorrhiza induced by the applications of SA,MeJAor GA3, with tanshinones production, we found that the different hormones and activesubstances variously modulated tanshinons production, namely that regulation effects weredifferent for different hormones.(4) The SA application resulted in overall increase whereas the MeJA and GA3applications caused decrease of contents of sucrose in seedlings and fruiting plants of S.miltiorrhiza, meanwhile, excepting reducing sugar content in shoots of the GA3-treated fruitingplants, contents of reducing sugar in the different-aged plants under the SA, MeJA or GA3treatments, as well as contents of salvianolic acids in roots of the different-aged plants weretogether increased. While higher concentration fructose as carbon source in media causedhigher contents of rosmarinic acid and more total amount of salvianolic acids in cultures of S.miltiorrhiza hairy roots than treatments of same media concentrations of sucrose and glucose.This suggested that high concentration fructose but not glucose and sucrose acceleratedproduction of salvianolic acids in plants.Comparing the changes in levels of endogenous hormones in different-aged S.miltiorrhiza plants with contents of salvianolic acids in their root under treatments of SA,MeJA or GA3applications, we found that production of salvianolic acids was correlated with hormones balance in plants, so it is concluded that the regulation effects of SA and hormoneson phenolic acids production in plant are similar and target to multiple points.(5) In this study, treatment of the fructose significantly promoted growth of the S.miltiorrhiza hairy roots compared with treatments of the sucrose and glucose. Mechanisms ofthis should be carefully carried out in the future. |
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