Regulation Mechanism Of Tanshinone Biosynthesis And CDNA-AFLP Analysis Of Salvia Miltiorrhiza And Salvia Castanea Diels F. Tomentosa Stib

Salvia miltiorrhiza (Danshen in Chinese) is one of the most important and populartraditional Chinese medicinal plants and has been widely used for prevention and treatment ofcoronary heart disease, chronic renal failure, atherosclerosis, myocardial infarction, anginapectoris, myocardial ischemia, liver fibrosis and cirrhosis. There are two major groups of activeconstituents in S. miltiorrhiza, polyphenolics and tanshinones. The former, mainly includingsalvianolic acid B, rosmarinic acid, caffeic acid, danshensu, and protocatechualdehyde arewater-soluble. The latter, mainly including tanshinone ⅡA, tanshinone Ⅰanddihydrotanshinone Ⅰ are lipophilic.Tanshinones are synthesized via at least two pathways in plant, the mevalonate (MVA)pathway in the cytosol and the2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in theplastids. Hydroxymethyl glutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzymein the MVA pathway, and mevinolin (Ormeno et al.) is a highly specific inhibitor of HMGR.1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) is the second enzyme in the MEPpathway, and fosmidomycin (Foster et al.) is a specific inhibitor of DXR. By blocking theMVA and MEP pathway with mevinolin and fosmidomycin and inhibiting IPP transport withDLG and NAPP in S. miltiorrhiza hair roots, roots growth, tanshinones profiles and key genesexpression patterns were comprehensively analyzed to elucidate regulatory mechanism oftanshinones biosynthesis and the crosstalk between the MVA and MEP pathways. Our previouswork has revealed that tanshinones accumulation was induced significantly by drought stressin pot experiment. In this study using S. miltiorrhiza hair root culture system, the regulationmechanism of tanshinones biosynthesis by polyethylene glycol (PEG) and absicsci acid (ABA)were investigated and the interaction of ABA, reactive oxygen species, methyl jasmonate (MJ),nitric oxide (NO) were revealed.Salvia castanea Diels f. tomentosa Stib is a forma of S. castanea Diels, only produced inLinzhi of Tibet and grows at the altitude between1950and3750m. Our previous work hasrevealed that The contents of most of tanshinone components were higher in S. castanea Diels f.tomentosa Stib than that in S. miltiorrhiza. However, content of salvianolic acid B in S.castanea Diels f. tomentosa Stib was only11.36%of that in S. miltiorrhiza. The normal rootcolor of S. miltiorrhiza was brick red. However, roots with other colors were usually found.Luckily, a white root of S. miltiorrhiza was obtained in our field experiments. In this study, themetabolic and transcriptomic profiles of normal root of the two species, the white root and thehydroponic culture root of S. miltiorrhiza were analyzed by HPLC and cDNA-AFLP method to discover novel genes involved in tanshinone or polyphenolic biosynthesis. The mainconclusions were the following.1.The root growth was inhibited more efficiently by MEV than by FOS. NAPP increasedthe growth. DLG had no effect on the growth but could strengthen the inhibitory effects oftanshinone by MEV and FOS. The areas of peak1-2and4-8were inhibited by MEV, and thoseof peak1-3and5-8were reduced by FOS. For peak1-3,5,6and8, FOS inhibited theiraccumulations more effectively than MEV. Contents of most tanshinones could be inhibited byDLG and NAPP. However, every inhibitor had its preferred components to inhibit. Theinhibitory effects of tanshinones contents by MEV and FOS were significantly strengthened byDLG. Expression of HMGR in the MVA pathway in S. miltiorrhiza hairy roots weredramatically stimulated by MEV at6h and followed by a sharp decrease at24h, while DXRexpression in the MEP pathway were up-regulated by FOS at6h and followed by a sharpdecrease at24h. In summary, our results suggested that the MVA pathway played a major rolein cell growth of S. miltiorrhiza hairy roots. Although the MVA pathway played an importantrole in the biosynthesis of eight tanshinone compounds, the MEP pathway was the main sourceof precursors in the biosynthesis of most of the tanshinones. However, both of cell growth andtanshinone production could partially depend on the crosstalk between the two pathways. Theinhibitor-mediated changes were also reflected in transcript of the genes in the two pathways.2.Both PEG and ABA could inhibite S. miltiorrhiza hairy root growth and increasetanshinones accumulation. Tanshinones production induced by PEG and ABA were mainlydepended on MEP pathway. However, MVA pathway also made some contribution to theincrease of tanshinones production, especially to ABA-induced dihydrotanshinonⅠandtanshinon ⅡA accumulation. Four tanshinones components shared the similar regulationmechanism by PEG and ABA, but it was different in detail.3.The decreases of root growth and tanshinone production induced by PEG and ABAtreatment were reversed by ROS scavengers, catalase (CAT) and superoxide dismutase (SOD).Both PEG and ABA trigger ROS burst in S. miltiorrhiza hairy roots. These results indicatedthat the PEG and ABA-induced increase of tanshinone production was probably via ROSsignaling.4.Both MJ and SNP were effective elicitors to stimulate tanshinone production.Cryptotanshinone accumulation was the most sensitive to MJ elicitation among four tanshinonecompounds, while the biosynthesis of cryptotanshinone and tanshinone ⅡA was more sensitiveto SNP elicitation than that of the other compounds. The MVA and the MEP pathways wereboth activated by MJ and SNP. ROS mediated MJ-induced tanshinone production, butSNP-induced tanshinone production was ROS-independent. 5.Both PEG and ABA triggered MJ accumulation in S. miltiorrhiza hairy root. Thetanshinone production induced by PEG and ABA were completely reversed by inhibitors ofABA and JA biosyntheisis, tungstate and ibuprofen (IBU), but that by MJ was only reversedIBU and MEP pathway inhibitor. Simultaneously, the expression of two key genes encodingHydroxymethyl glutaryl-coenzyme A reductase (HMGR) and1-deoxy-D-xylulose5-phosphatereductoisomerase (DXR) in MVA and MEP pathways were up-regulated by MJ.6.The enhancement of tanshinone production in S. miltiorrhiza hairy root by PEG andABA were reversed by L-NAME and c-PTIO. The increases of tanshinone contents by SNPtreatment were also observed in our experiment and they were largely arrested by the MEPpathway blocker. Simultaneously, PEG and ABA triggered the burst of NO. In conclusion, werevealed that the increase of tanshinone production induced by PEG and ABA was probablyNO-dependent and NO mainly induced the MEP pathway to increase tanshinone production7.The contents of tanshinone components and rosmarinic acid were higher in S. castaneaDiels f. tomentosa Stib root (S1) than those in S. miltiorrhiza root (S4). However, salvianolicacid B content in S1was much lower than that in S4. The contents of tanshinone componentsin the white root (S3) and the hydroponic culture root (S2) of S. miltiorrhiza were not detected.However, the polyphenolic components contents were in S2and S3not less than those in S4.8.928differentialy expressed transcript derived fragments (TDFs) were observed betweenS2and S4, and391of them were highly expressed in S4and537of them were in S2.596TDFswere observed between S3and S4,217of them were in S3and379of them were in S4.776TDFs were obtained between sample1and4,385of them were in S3and291of them were inS4.975of the total TDFs were isolated from the gel and re-amplified,573of them wereselected to sequence. Finally,323TDFs were successfully sequenced. The majority of323TDFs belonged to no hits (66%), and only109TDFs showed significant sequence similaritiesto protein in the databases by BLASTX analysis (eValue≤10-5). Only86TDFs weresuccessfully annotated in terms of their associated molecular functions, biological processesand cellular components by Blast2GO tool for Gene Ontology (GO) assignments. About60%of the TDFs were involved in biological processes and they encoded a broad set of transcriptsrepresented within cellar metabolic, primary metabolic and biosynthetic processs. There were67TDFs annotated within molecular function strategy. Most of them were about nucleotidebinding, oxidoreductase activity, protein binding and transferase activity. About60%of thesequences represented the strategy of cellar component. Within the sequences of the strategy,plastid, chloroplast and cytoplasmic membrane-bounding vesicle were well represented. Ofthese sequences,27were highly homologous to the enzyme in KEGG database, and22wereassigned to the metabolic pathways. Excitingly,14TDFs were assigned to secondary metabolic pathways. The contents of tanshinone and phenolic components and the key genes involved intheir biosynthesis in S. miltiorrhiza hairy root were induced by yeast extraction (YE). Theexpression of9TDFs were coup-regulated with the key genes involved in tanshinone andphenolic biosynthesis, including genes encoding lipoxygenase, catalase, jasmonate zim-domainprotein, hd domain class transcription factor, pyruvate decarboxylase and three unknowngenes.