| Tanshinones are an important part of the active compounds of Salvia miltiorrhiza Bge.with important medical and commercial value.Biogenesis pathway analysis is hot in the research on tanshinones and has made important progress in recent years.According to the apparent characteristics of medicinal materials and previous studies,tanshinones mainly accumulate in the periderm of roots.Besides,we found that the root transitioned from white to red in the Salvia miltiorrhiza seedling period,indicating tanshinones were likely temporally developmentally specificity.The above shows that the tanshinones have a typical "spatial-temporal" distribution phenomenon.Based on this phenomenon,the following questions are raised.1.What is the mechanism and regulation mechanism of the "spatialtemporal" distribution of tanshinones? 2.Is there a special transport mechanism for tanshinones in Salvia miltiorrhiza? 3.What is the biological significance of the gradual accumulation of tanshinones in young roots and directed accumulation in the periderm? With these problems,firstly,we revealed the "spatial-temporal" distribution of tanshinones,and combined transcriptome information to explain the mechanism of the "spatial-temporal" distribution.Then,mine the transcriptional regulators and transporters of tanshinones by integrating the two dimensions of information.Finally,combined with the discovery of allelopathic effects of Tanshinone IIA,explored the physiological significance of the "spatial-temporal" distribution characteristics of tanshinones.The main research content includes five parts:1.A "spatial-temporal" metabolic map of tanshinones was constructed.Based on the UPLC-Q/TOF-MS metabolic analysis platform,the roots of different developmental stages of Salvia miltiorrhiza seedling and the different root tissues of mature Salvia miltiorrhiza were analyzed.It was found that there were almost no tanshinones in the roots for a period after seed germination.Tanshinones gradually began to accumulate at20-25 DAG(Days After Germination)with red dots distributed in the roots,and the content increased sharply at 35 DAG with the roots turning red.So,it is proposed that the tanshinones have an accumulation characteristic in the seedling stage.The results of different tissue analyses showed that the tanshinones were concentrated in the periderm,with a small amount in the phloem and almost no in other tissues.Here,we systematically characterized the "spatial-temporal" distribution of tanshinones,laying a foundation for further research on combining with molecular data.2.A "spatial-temporal" transcriptional map of Salvia miltiorrhiza root was constructed.A spatiotemporal transcriptome landscape of Salvia miltiorrhiza root was depicted based on the same batch of samples as the metabolome.It was found that the transcript profile of tanshinones pathway genes was consistent with the distribution of tanshinones,which is highly expressed in the periderm,and significantly high transcription at the 35 DAG.It was found that the specific expression of downstream pathway genes is a central factor for the specific distribution of tanshinones.According to the transcriptome characteristics,the root development process of Salvia miltiorrhiza seedlings could be divided into two stages,0-20 DAG was the first stage,and 25-40 DAG was the second stage.The functional enrichment results showed that genes in the first stage mainly carried out the primary metabolic process,and the second stage started the synthesis and metabolism of secondary metabolites,such as terpenes and phenylpropanoids.3.Discovery of potential key transcriptional regulators specific to "spatial-temporal" feature of tanshinones.It was found that the transcription levels of Sm ERF98 and Sm ERF72 were consistent with the "spatial-temporal" distribution of tanshinones.Both of them were nuclear-expressed genes with typical transcription factor expression characteristics.Further combined with Y1 H,EMSA,and DLR experiments proved that Sm ERF72 could bind to GCC-box motifs in promoter of KSL1,CYP76AH3,IDI1,and HMGR to activate their transcription,and Sm ERF98 could bind to the GCC-box in the promoter of KSL1,CYP76AH3,IDI1 to activate their transcription.By constructing overexpressing transgenic Salvia miltiorrhiza,it was found that the transcription levels of multiple tanshinones pathway genes were significantly increased and the contents of Tanshinone IIA and Cryptotanshinone were significantly improved.Sm ERF98 and Sm ERF72 are potential regulators of the specific distribution of tanshinones.This discovery will provide target genes for quality regulation and breeding of Salvia miltiorrhiza.4.Research on the transport mechanism of Tanshinone ⅡA.Microscopic observation found that the red dots represented tanshinones could transport to the intercellular space from cellular.The ABC transporter family was reported to participate in the transport of secondary metabolites.A total of 127 ABC family transporter genes were obtained through genome-wide annotation,which were distributed in 8 subfamilies,of which the ABCG family had the most members(46%).It was found that Sm ABCG1 was localized on the cell membrane,with the highest expression in roots compared with stems,leaves,flowers,and other organs.Besides,its transcription level was induced by methyl jasmonate.Yeast transport assay showed that Sm ABCG1 can transport Tanshinone IIA,and 100 μM Tanshinone IIA can promote the expression of Sm ABCG1 protein.After overexpression of Sm ABCG1,the contents of Tanshinone IIA and Cryptotanshinone in roots were significantly increased.Collectively,the results show that Sm ABCG1 has the function of transporting Tanshinone IIA.This discovery will provide a strong basis for revealing the transport process and mechanism of tanshinones in Salvia miltiorrhiza root.5.Research on the allelopathic effect of Tanshinone ⅡA.Tanshinones are specifically synthesized in the periderm and transported to the outside of the cells,implying their potential physiological interaction with the environment.Further,it was found that Tanshinone IIA could be diffused into the root-soil environment.Arabidopsis thaliana roots were severely inhibited during co-cultured with Salvia miltiorrhiza.Besides,it was found that 100 μM Tanshinone IIA could seriously inhibit the root development of Arabidopsis thaliana under sterile conditions.Both the metabolic and transcriptional characteristics showed the toxic effect of Tanshinone IIA on Arabidopsis thaliana roots.The significant reduction in root IAA content may be one of the reasons for hindering root development.Consequently,the above results indicated that Tanshinone IIA can be secreted into the root-soil environment,and it is very likely to inhibit the growth and development of other plants to preempt the ecological niche.In summary,this project revealed the "spatial-temporal" distribution of tanshinones and explained the mechanism of their distribution initially.It was found that the transcript regulators Sm ERF98 and Sm ERF72,and the transporter Sm ABCG1 are very likely to mediate the biosynthesis and specific accumulation of tanshinones,respectively.In addition,Tanshinone IIA was found to inhibit Arabidopsis root development.The toxic effects indicated Tanshinone IIA may be new allelochemical.Therefore,we propose the hypothesis that tanshinones are gradually synthesized in the seedling stage,accumulated in the periderm directly,then transported from the intracellular to the extracellular,and finally secreted to the soil environments to preempt niche.Conclusively,this project will help to reveal the tissue-specific,time-specific characteristics of plant secondary metabolites. |
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