Study On Programmed Cell Death During The Formation Of "Withered Skin" And "Hollow Heart" And Distribution Of Secondary Metabolites Of Astragalus Membranceus

[Objective] Huangqi is a famous traditional Chinese medicine.Hunyuan,Shanxi Province,is a traditional authentic production areas of Huangqi.It has a long growth period,imitates wild cultivation and good quality.The structure of “withered skin” and “hollow heart” are important characteristics of Astragalus membranaceus with high age genuine medicinal materials,which are different from fast-growing.There are three questions about the development of “withered skin” and “hollow heart” structure and its influence on the distribution of main secondary metabolites in high-age genuine Chinese medicinal materials:(1)whether the development of “withered skin” in A.membranaceus is a PCD event;(2)whether the development of “hollow heart” in A.membranaceus is a PCD event;(3)whether the distribution of secondary metabolites in different tissues and formation of “withered skin” and “hollow heart” structure in the root of A.membranaceus affects the distribution of secondary metabolites in different tissues.Above these problems are helpful to reveal the scientific connotation of “distinguishing the features” of Huangqi.[Method] In this study,paraffin sections,semi thin sections,ultrathin sections,light microscopy and transmission electron microscopy were used to observe the developmental process and the cytological characteristics of the “withered skin” and “hollow heart” in the root of A.membranaceus.DAPI staining and TUENL in situ end labeling technique were used to observe the DNA fragmentation of the cells,combined with DNA extraction and agarose gel electrophoresis was determined to determine the programmed cell death(PCD)of “withered skin” and “hollow heart”.On the basis of histological observation,the effects of secondary structure and the formation of “withered skin” and “hollow heart” on the distribution of secondary metabolites in different tissues in the root of were studied by using frozen section,laser microdissection and UPLC-Q/TOF-MS.[Results](1)The results of cytological and molecular biological studies on “withered skin” showed that the development of “withered skin” in the roots of A.membranaceus originated from the parenchyma cells near the primary periderm of the secondary phloem,and then a new periderm was produced,which is the structure of the rhytidome,it's a dynamic process.According to the development characteristics,the development process of “withered skin” is divided into four stages: initial stage,pre-rhytidome stage,rhytidome formation stage and mature stage.In the process of development,the rhytidome shows the characteristics of PCD,which is a PCD event in the process of plant growth.It is characterized by the degradation of mitochondrion and endoplasmic reticulum,the fusion of small vacuoles into large vacuoles,the rupture of vacuole membrane,the existence of phagocytic organelles and flocculent structures in vacuoles,the degradation of cytoplasm,the thinning and distortion of cell walls,the deformity of nucleus,and the degradation of nucleolus.The results of DAPI staining and TUNEL detection showed that in the early stage of rhytidome formation and rhytidome formation,the nucleus was TUNEL positive,while in the mature stage,DAPI and TUNEL were both negative.DNA results showed that DNA degradation was tail like.(2)The “hollow heart” is an abnormal structure in the roots of A.membranaceus.The development of the abnormal structure results in cork cell ring in the secondary xylem.Macroscopical observation: there were black spots on the surface of the cross section of the root.It was found that the black spots gradually expanded on the vertical section,and the decayed extended to the root head.The results of paraffin and semi thin sections showed that the black spots were caused by the filling of the vessels with some substance.“hollow heart” originated from the parenchyma cells around the abnormal vessels that restore the ability of meristem.Then,the parenchyma cells divide radially and form cork cambium.Cork cambium continues to differentiated into cork,produced a ring structure,that is,the interxylary cork.The development of interxylary cork is also a PCD event.The ultrastructural features of the cell wall were the generation of marginal vesicles,the degradation of mitochondrion and Golgi bodies,the disordered of membrane system,the density of cytoplasm and the deformity of nucleus.The degradation of nucleus usually occurs in the early stage and the formation stage of interxylary cork,and TUNEL-positive.While in the mature stage,DAPI staining and TUNEL assays are negative.Gel electrophoresis showed that DNA was diffused.(3)The results showed that there were 15 compounds in periderm,9 saponins and 4 flavonoids,17 compounds in secondary phloem,11 flavonoids and 3 saponins,and 18 compounds in secondary xylem,12 flavonoids,Three saponins.It can be seen that there are more saponins in periderm than in secondary phloem and secondary xylem,and flavonoids are abundant and even in secondary phloem and secondary xylem.It is speculated that flavonoids mainly exist in parenchyma cells.Through the study of different tissues of “withered skin” and “hollow heart”,the results showed that the distribution of saponins increased,while the flavones in dead cells did not show significant decrease.25 compounds were identified in “withered skin”,including 6 flavonoids and 15 saponins.28 compounds were detected in the “hollow heart” structure,including 10 flavonoids and 15 saponins.The distribution of saponins in the newly periderm or interxylary cork increased significantly,13 and 15,respectively,which were more abundant than other tissues.[Conclusion](1)In the root of A.membranaceus,“withered skin” is the structure of rhytidome.The “hollow heart” in the roots of A.membranaceus is due to the formation of the abnormal structure of the interxylary cork.The occurrence and development of “withered skin” and “hollow heart” are all PCD events,which are typical PCD characteristics such as nuclear deformity,nucleolus disappearance,degradation of various organelles in cells,DNA fragmentation,etc.(2)The formation of “withered skin” and “hollow heart” in the root resulted in the increase of saponins and flavonoids.That is to say,the appearance of “withered skin” and “hollow heart” affected the ratio of saponins and flavonoids in the root of A.membranaceus.