| As a tonic traditional Chinese medicine,Epimedium has become one of the most commonly used traditional Chinese medicine in China.It is widely used in clinic to effectively treat osteoporosis,chronic nephritis and immunoregulation.The clinical efficacy of medicinal plants depends on the unique active ingredients in the medicinal materials,and the levels of the active ingredients are affected by both genetic factor of the plant and environmental factors.Epimedium pubescens Maxim and E.brevicomu Maxim are two of five recorded epimedium species in the Pharmacopoeia of the People’s Republic of China(version 2015).The physiological traits,dynamic changes of metabolites and regulatory mechanism of gene expression in these two species were investigated during the growth and development and under UV-B stress,which will be of guiding significance to improve their quality control system and cultivation conditions in the future.We also studied the absorption characteristics of nitrogen,phosphorus and potassium in E.pubescens plant and the effects of nitrogen,phosphorus,and potassium fertilizer levels on the yield and quality of epimedium folium,which will provide the theoretical basis for designing fertilization scheme during epimedium artificial cultivation in the future.The main results are shown below:1.It was observed that in Beijing,E.pubescens and E.brevicomu reached their maximum dry leaf biomass in late June and the middle of June,respectively.Icariin content was highly accumulated in leaves of both epimedium species at the full flowering stage,however,icariin content in E.brevicomu is much higher than that of E.pubescens.Photosynthetic investigation showed that E.pubescens and E.brevicomu leaves both displayed the M-shaped diurnal variation of net photosynthetic rate and had the obvious photosynthetic noonbreak.Meantime,E.pubescens had higher water use efficiency but weaker photosynthetic capacity than E.brevicomu.Based on dynamic changes of leaf biomass and accumulation of active components during the growth period,the best harvesting time for both epimedium species in Beijing was proposed in the middle and late June.2.In this study,403 and 396 metabolites were identified via a UPLC-ESI-Q TRAP-MS/MS-based widely targeted metabolome analysis separately from the leaves of E.pubescens and E.brevicomu,among which 389 metabolites were shared by two species,indicating that they had the similiar metabolite composition.Pairwise stage comparisons revealed 302/268 differential metabolites in E.pubescen/E.brevicomu leaves through the growth period.Among them,most differential flavonoids and phenolic acids in both species were increased with their growth and development while the differential lipids,nucleotides and amino acids decreased.E.pubescens and E.brevicomu had a quite different metabolic profiling at the same development stage.A total of 295 metabolites were differentialy expressed between two species,of which,20 flavonoid metabolites were lower at all developmental stages in E.pubescens than E.brevicomu,while 20 metabolites mainly belonging to phenolic acid,were higher at all developmental stages in E.pubescens than E.brevicomu.3.196487/134747 Uni genes were obtained in E.pubescens/E.brevicomu leaves via RNA-sequencing.Of them,19542/13639 differential Unigenes were detected during the growth period.Both species possessed the quite different transcript profiling at the full flowering stage than at later three stages.K-means clustering showed that 10658/6469 differential genes in E.pubescens/E.brevicomu were up-regulated after the fully flowering stage.The study revealed that 537 Unigenes in E.pubescens and 449 in E.brevicomu were putatively involved in flavonoid biosynthesis pathways,of which,164 and 154 genes were differentially expressed during the growth period.Most of these flavonoid biosynthetic DEGs in E.pubescens leaves were increased significantly after flowering and reached the highest levels at the harvest stage,while in E.brevicomu,most DEGs reached the highest levels at the reproductive stage and then decreased at the harvest stage.4.The integration analysis of transcriptome and metabolome of E.pubescens/E.brevicomu revealed that almost all the differential metabolites were significantly correlated to the expression of differential of genes.The differential accumulation of a metabolite between two developmental stages was coregulated positively and negatively by multiple genes.25 differential flavonoids in E.pubescens,including catechin and chlorogenic acid showed correlation to the expression of 29 differential genes from 11 flavonoid biosynthetic families such as F3’5’H and CHS,20 differential flavonoids in E.brevicomu,including catechin and kaempferol showed correlation to the expression of 26 differential genes from 16 flavonoid biosynthetic families such as F3’5’H and CHS.The integrated KEGG pathways of differential genes and differential metabolites in E.pubescens/E.brevicomu revealed that the enriched KEGG patwhays were mainly composed of metabolic pathways,biosynthesis of secondary metabolites,phenylpropanoid biosynthetic process,starch and sucrose metabolism,interconversion of pentose and glucuronic acid,flavonoid biosynthesis,amino acid biosynthesis,ABC transporter,carbon metabolism,isoflavone biosynthesis,phosphoinositide metabolism,etc.5.UV-B irradiation can significantly reduce photosynthetic pigments and increase contents of MDA,H2O2 and activities of antioxidant enzymes in both species.However,E.brevicomu were more UV-B tolerant and had better defense ability than E.pubescens under the same days of irriadation.With irradiation going on,the contents of some main icariin-type components including epimedin C and sagittatoside A first increased in E.pubescens and then decreased while they remained a significantly increasing treand in E.brevicomu.Our metabolomic data further revealed that most flavonoid and phenolic acid metabolites were down-regulated by UV-B irradiation in E.pubes cens over radiation time and up-regulated in E.brevicomu,which indicated that E.pubescens was more sensitive to UV-B stress than E.brevicomu.6.In UV-B irradiation experiment,Go enrichment analysis showed that differential genes in E.pubescens were mainly enriched in photosynthetic system including photosystem,photosystem II,photosystem I,and chlorophyll binding,while in E.brevicomu were mainly enriched in cell respiration,flavonoids biosynthesis,flavonoids metabolism,respiratory chain and protein metabolism.KEGG enrichment analysis showed that UV-B stress had significant effects on amino acid metabolism,fatty acid metabolism,flavone and flavonol biosynthesis and other pathways in E.pubescens,while in E.brevicomu mainly on phosphatidylinositol signaling system,arachidonic acid metabolism,glutathione metabolism,ABC transporter,phenylpropanoid biosynthesis,MAPK signaling pathway-plant,oxidative phosphorylation,carbon metabolism and other pathways.12 differential flavonoids including catechin and naringin in UV-B irradiated E.pubescens were in correlation to the expression of four differential genes in FG,CYP98A and DFR families.In E.brevicomu under UV-B irradiation,five differential flavonoids including vitexin,kaempferol,apigenin,acacia and sinapine,were correlated to the expression of five DEGs from CHS,CYP98A,DFR and anthocyanin 3-o-6"-o-coumaroylglucoside:glucosyltransferase families.8.E.pub escens most required fertilizer from seedling emergence to the end of June.The order of accumulation amount of nitrogen,phosphorus,and potassium per plant leaf was N>K>P.All three fertilizers significantly increased chlorophyll content of E.pubescens leaves and the effect rank was N>K>P.The single fertilizer effect analysis showed that E.pubescens leaf yield was gradually increased with the amount of P fertilizer applied,however,increased first and then decreased with the increase of N and K fertilizer.The fertilizer effect on E.pubescens leaf yield was N>K>P.The recommended amounts of nitrogen and potassium fertilizers from the established fertilizer effect models were 185.3 kg·hm-2 and 160.7 kg·hm-2,respectively.Assay of E.pubescens foliar active ingredients showed that K fertilizer inhibited the accumulation of epimedins A,B,C and icariin,while high P level promoted their accumulation,and the medium level of N fertilizer increased the accumulation of epimedin C and icariin.Sagittatosides A and B were significantly increased by all three fertilizers. |
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