The Redox Mechanism Of Glutathione Regulating The Proliferation Of Korean Pine Embryogenic Callus

Korean pine(Pinus koraiensis Sieb.et Zucc.)somatic embryogenesis technology system was used as the carrier to optimize the key technical links of somatic embryogenesis.induce cell lines with different proliferation potential,and cryopreservation them.And the two cell lines with different proliferation capabilities[001#-001(F)and 001#-010(S)cell line]were used as the test material,with the help of transcriptomics and metabolomics techniques.to reveal the metabolites and genes that regulated the embryonic callus(EC)proliferation difference,then to understand the molecular regulation mechanism that led to the difference in EC proliferation potential,and to find the key marker substances that caused the difference in EC proliferation potential of Korean pine.By adding key substances glutathione(GSH)and Lbuthionine-sulfoximine(BSO)to the EC proliferation medium,the cell morphology,glutathione and ascorbic acid synthesis and relative oxygen species(ROS)metabolic response of exogenous GSH and BSO affecting the proliferation of Korean pine EC were explored.The main results are as follows:(1)The method of complete sterilization of cones is beneficial to the EC induction of Korean pine and taking zygotic embryo(ZE)as the explant is better than megagametophyte(MG).Low temperature storage at 4? before inoculation of explants can significantly increase the EC induction percentage,and the EC induction percentage of 5#family increased from 10.00%to 62.77%.There were significant differences in proliferation potential among different cell lines.And 11 cell lines with different proliferation potential were cryopreservation,including high proliferation potential cell line F and low proliferation potential cell line S.(2)Transcriptome sequencing analysis between F and S cell lines revealed a total of 1883 differentially expressed genes,870 genes showed up-regulated expression,and 1013 genes showed down-regulated expression.A total of 17 differential genes related to glutathione have changed significantly between F and S cell lines.Among them,there were 8 genes of Glutathione S-transferase(GST)family,3 genes of Gulonolactone oxidase(GULO)family,1 gene of Leucyl aminopeptidase(CARP),Ornithine decarboxylase(ODC1)and Dehydroascorbate reductase(DHAR)family,2 genes of Ascorbate oxidase(AAO)family and 1 gene of 6-Phosphogluconate dehydrogenase(PGD)family.A total of 14 genes were downregulated and 3 genes were up-regulated.A systematic analysis of these differential genes showed that the intracellular GSH content in the F cell line may be higher than S.It is speculated that the intracellular GSH content plays an important role in regulating the difference of proliferation capacity between F and S cell lines.Moreover,12 differentially expressed genes of Korean pine were selected for qRT-PCR verification,and the results showed that the differential genes obtained by sequencing in this study were credible.(3)A total of 893 metabolites were obtained between F and S cell lines,including 412 differentially expressed metabolites,the relative content of 183 metabolites increased,and the relative content of 229 metabolites decreased.The differential metabolites were classified into 27 categories.There were 9 different metabolites related to glutathione between F and S cell lines,all showed that the relative content of F cell line was greater than S.Metabolomics further confirmed the transcriptomic findings,that intracellular GSH content might be the reason for the difference of proliferation ability between F and S cell lines.(4)Exogenous GSH promoted the proliferation of F and S cell lines,while exogenous BSO inhibited the proliferation of F and S cell lines.From a cytological point of view,exogenous GSH promotes cell division,and exogenous BSO inhibits cell division.On the 35 days of F-CK proliferation and culture,the EC proliferation efficiency was 827.85%,F-GSH was 1160.65%,and the EC proliferation efficiency increased by 40.20%.This chapter further verified the results speculated in Chapter 3-4,that,the content of GSH in cells is the reason for the difference of EC proliferation potential.(5)Exogenous GSH promoted the synthesis of intracellular glutathione and ascorbic acid in F and S cell lines,while exogenous BSO inhibited the synthesis of intracellular glutathione and ascorbic acid in F and S cell lines.GSH content in the F-CK was higher than that of the S-CK(0.161 ?mol/gFW and 0.089 ?mol/gFW,respectively).Exogenous GSH increased the content of total glutathione(T-GSH),GSH,glutathione oxidized(GSSG),Total ascorbic acid(T-ASA),ASA and the ratio of GSH:T-GSH and ASA:T-ASA in F and S cell lines,exogenous BSO further confirmed this conclusion;Exogenous GSH could increase the activity of glutathione reductase(GR)and dehydroascorbate reductase(DHAR)of two cell lines and reduced the activity of ascorbate peroxidase(APX),exogenous BSO further confirmed this conclusion;Exogenous GSH promoted the contents of intracellular GSH and ASA and the ratio of GSH:T-GSH and ASA:T-ASA,so that the culture environment was in a reduced state,which was beneficial to the proliferation of Korean pine EC.(6)Exogenous GSH reduced the level of intracellular ROS in F and S cell lines,while exogenous BSO increased the level of intracellular ROS in F and S cell lines.Exogenous GSH increased the activity of intracellular superoxide dismutase(SOD),reduced the amount of cell death,and decreased intracellular hydrogen peroxide(H2O2),malonaldehyde(MDA),nitric oxide(NO)level,exogenous BSO further confirmed this conclusion.Exogenous GSH can promote ROS metabolism in Korean Pine EC cells and reduce the intracellular ROS level.In this study,Found the key substance(GSH)causing the difference of embryonic cell proliferation potential from the perspective of molecular biology.It not only promotes EC proliferation,but also reveals the redox regulation mechanism of exogenous GSH and BSO on EC proliferation.It lays the foundation for improving the proliferation efficiency of Korean pine embryonic cells and the large-scale breeding of excellent planting resources,and provides a reference for the optimization of other coniferous species' somatic embryogenesis technology system.