Formation Mechanism Of Nail Heads In Panax Notoginseng And Its Influence On Quality

"Evaluating quality by distinguishing appearance" refers to the evaluation of authenticity and quality of medicinal materials based on their appearance.The concepts of "optimal shape","high quality" and "excellent effect" of Daodi herbs were also put forward in the "TCM geo-herbalism",a major project of the National Natural Science Foundation of China.It was also put forward that external shape and internal quality of Daodi herbs could be reflected as clinical effect in the use of medicinal materials.Panax notoginseng is a traditional plant of Panax genus,Araliaceae,and its root is widely used in clinical treatment due to its effects of activating blood flow,stopping bleeding,reducing swelling and relieving pain.Nail head,which is the nodular structure growing on the main root,is usually referred as a characteristic trait in traditional experience identification of P.notoginseng.However,it is still unclear whether there is a correlation between this genuine trait and quality of medicinal materials,as well as the molecular mechanism of nail head formation.On the one hand,clarifying the mechanism of nail head formation in P.notoginseng and its influence on the quality is helpful to uncover the formation mechanism of TCM genuine character.On the other hand,it can also provide basis and guidance for the quality screening,quality evaluation and clinical application of P.notoginseng.In this study,the environmental inducement contributing to formation of nail head was discussed,the molecular mechanism of nail heads formation and its regulatory network were initially revealed,and the influence of nail head on quality was explored.The main results are as follows:Firstly,the morphological characteristics of nail heads in P.notoginseng were summarized,and the characteristics of nail heads were observed in P.notoginseng possessing different sizes,different root shapes,different growing years,different eras,whether transplanted during cultivation or not,and whether processed after harvest or not.It was found that the number of nail heads was affected by the above six factors,among which transplantation might be the main trigger contributing to the increasing number of nail heads.In addition,it was found that the number of nail heads is negatively correlated with the number of lateral roots.Determination of carbohydrate showed that nail heads of P.notoginseng possess a weaker function of nutrients storage,compared with the main root.The microscopic observation showed that the nail head has a wider cortex,while the vascular bundle is connected to the vascular system of the main root.A structure similar to the lateral root primordia was also obtained in the nail head,preliminary concluded that nail heads might be an inflated lateral root,formed by inhibiting the development of lateral roots.In order to explore the correlation between nail heads and quality of P.notoginseng,a method that can simultaneously detect 16 ginsenosides using UPLC-QQQ-MS/MS and a method determing dencichine using UPLC-UV were established.The content of ginsenosides was positively correlated to the number of nail heads,while no significant correlation was obtained between dencichine content and the number of nail heads.Especially,content of ginseoside Rb1,ginsenoside Rf,notoginsenoside R1,notoginseoside R2 and protopanaxatriol-type ginsenoside was significantly higher in P.notoginseng possessing more nail heads.Results of active components determination indicated that P.notoginseng with more nail heads might have better central nervous inhibition effect,vasodilation effect and blood-activating effect,which also provided basis for quality screening of P.notoginseng under different medication needs.In addition,content of ginsenoside Rb1 in nail head were significantly higher than that in the main root and lateral root.The expression levels of key enzyme genes involved in the biosynthesis of ginsenosides were determined by real-time quantitative PCR,and the highly expressed PnFPS,PnDS,PnSS and PnCYP716A47 in nail head were screened,might result in the higher content of ginsenosides in nail heads.Meanwhile,in vitro pharmacological activity of P.notoginseng experiencing different cultivation mode was determined.It was found that transplanted P.notoginseng,possessing higher content of ginsenosides,exhibits stronger in vitro antiplatelet aggregation activity.According to the references,phytohormones,especially auxin and cytokinin,play important roles in root development and root architecture formation.Therefore,we studied the role of phytohormones in the formation of nail heads in P.notoginseng.Firstly,we developed a method that can simultaneously determine 8 endogenous phytohormones using UPLC-QQQ-MS/MS,and the contents of phytohormones in the main root,lateral root and nial head of P.notoginseng were determined.The results showed that nail head of P.notoginseng had significantly higher content of trans-zeatin riboside and jasmonic acid related phytohormones,which might be the key phytohormone inhibiting development of lateral root and promoting the accumulation of ginsenosides.We also studied the spatial distribution of plant hormones by imprint mass spectrometry imaging,and found that in early developing lateral root and nail head,auxin showed a higher expression in primordium of both structure,while trans-zeatin riboside specifically accumulated in the cortex of nail head.Based on the transcriptomic sequencing data and real-time quantitative PCR of main root,lateral root and tuberculate of P.notoginseng,PnCYP735A1 involved in the cytokinin biosynthesis pathway,as well as PnIAA14 involved in the auxin signal transduction pathway were screened.Function of PnCYP735A1 and PnIAA14 was heterologously verified in Arabidopsis thaliana by overexpressing in deletion mutant of homogous genes.It was found that atcyp735a1 and atiaal4 deletion mutants all showed similar phenotypes towards lateral root development,and the number of lateral root was reduced after overexpression of PnCYP735A1 and PnIAA14.These results suggested that PnCYP735A1 and PnIAA14 might inhibit lateral root development and thus lead to the formation of nail heads.In order to further explore its upstream regulation mechanism,2 MYB family transcription factors,PnMYB31 and PnMYB78,and 1 bHLH family transcription factor,PnbHLH31 highly expressed in the nail head were screened out by transcriptome and real-time quantitative PCR.Transient expression of PnMYB31,PnMYB78 and PnbHLH31 in Nicotiana tabacum confirmed their localization in the nucleus.Based on existing genomic data,promoter sequences of PnFPS,PnDS,PnSS,PnCYP716A47,PnCYP735A1 and PnIAA14 were amplified,and the binding sites on promoter sequences were analyzed.MYB family binding sites MBSIIG motif and bHLH family binding sites G-box were found in all of them.By constructing the 3 transcription factors and 6 promoters into the Dual-LUC experimental system vector and analyzing the changes in fluorescence intensity,it was found that PnMYB31 and PnMYB78 could transcriptively activate the expression of PnFPS,PnSS,PnIAA14 and PnCYP735A1,while PnbHLH31 inhibited the expression of PnIAA14.PnMYB31,PnMYB78 and PnbHLH31 were fused with MBP tag proteins and purified.It was confirmed by EMSA that PnMYB31 specifically binds to MBSIIG motif of proPnFPS and proPnSS.PnMYB78 can specifically bind to MBSIIG sequence of proPnIAA14,proPnCYP735A1 and proPnFPS,while MBP-PnbHLH31 can directly bind to G-box in proPnIAA14.The combination of PnMYB31/PnMYB78/PnbHLH31 with proPnFPS,proPnSS,proPnIAA14 and proPnCYP735A1 was further confirmed by Y1H.It was found that PnMYB31 and PnMYB78 could interact with PnbHLH31 in yeast respectively by BiFC experiment.The interaction between these 3 transcription factors was further proved by BiFC.Based on the results above,we established a possible internal mechanism for nail head formation,that is,auxin promotes lateral root initiation,while PnIAA14,and PnCYP735A1 influence lateral root meristem from breaking through the epidermis and promoting cortical cell division by regulating auxin signal transduction and cytokinin biosynthesis,thus leading to formation of nail head.PnMYB31-PnbHLH31 and PnMYB78-PnbHLH31 complexes regulates the formation of nail heads by influencing the expression of PnIAA14 and PnCYP735A1,thus intervening auxin signal transduction and cytokinin biosynthesis.On the other hand,the two complexes also regulates the accumulation of ginsenosides by binding with promotors of PnFPS and PnSS,leading to the increasing content of ginsenosides.The injury stress during transplantation is the environmental inducements contributing to the increasing number of nail heads.As the characteristic trait of P.notoginseng,nail head formation mechanism was illustrated in this research,and its influence on quality was verified,thus provides basis for quality screening of P.notoginseng under different medication requirements.The correlation between apparent characters and quality of Daodi herbs was proved,furthermore providing guidance for realizing "excellent effect" of Daodi herbs in clinical use.