| Secondary metabolites from medicinal plants are important source for innovative pharmaceuticals.Both the indole alkaloids from Catharanthus roseus and the prenylated products from Morus alba own great bioactivities,while the indole alkaloids and their derivatives have been widely applied to clinical cancer treatment.Nevertheless,the content of these compounds in natural-growing plants is fairly low and their chemical synthesis is tough,which hindered large-scale development and application.Previous research indicated that,as an effective elicitor combing with dark treatment,ultraviolet-B(UVB)radiation induced protein changes in C.roseus and M.alba,which resulted in accumulation of indole alkaloids and Diels-Alder type adducts together with their precursors,respectively.These changes increased the pharmaceutical values of medicinal plants,however,the underlying activation and regulatory mechanisms towards secondary metabolism related to signaling,energy generation,etc.,remain unclear.Based on systems biology,the present thesis integrated data from proteomics,metabolomics,and molecular experiment with the study focus of molecular mechanisms in response to UVB radiation of C.roseus and M.alba,which provided foundation for elucidation of biosynthesis mechanism of active compounds in medicinal plants.The main work and findings are as follows:(1)Study of phosphorylation signaling pathway and primary metabolism alteration in C.roseus under UVB radiationFor investigation of response mechanism in C.roseus under UVB radiation,ATP content measurement,phosphoproteomic analysis,GC-MS-based metabolomic analysis,and Western-Blotting experiment were performed.Under UVB radiation,ATP content significantly increased.Phosphoproteomic analysis identified 242 altered phosphorylated proteins under UVB radiation,which were mainly related to protein synthesis/modification/degradation and signaling;among them,calmodulin,calciumdependent kinase,and heat shock proteins significantly increased.GC-MS-based metabolomic analysis identified 110 altered metabolites under UVB radiation,which were mainly related to carbohydrates,organic acids,amino acids,and alcohols;among them,pentoses,aromatic amino acids,and phenylpropanoids significantly increased.Integrative analysis of omic data and Western-Blotting result indicated that pathways involved in glycolysis and reactive-oxygen species scavenging system were significantly changed.These findings suggested that UVB radiation caused oxidative stress on C.roseus,which activated calcium dependent protein phosphorylation/dephosphorylation.On the one hand,glycolysis altered as precise regulation,tricarboxylic acid cycle up-regulated,which promoted production of ATP,which fueled synthetic steps in secondary metabolism;on the other hand,up-regulated proteins that related to redox reactions took part in secondary metabolism,which promoted the accumulation of secondary metabolites.(2)Study of energy regulation and accumulation of secondary metabolites in C.roseus under UVB radiationFor further investigation of the mitochondrial participation in energy regulation and underlying correlation with secondary metabolism in C.roseus under UVB radiation,enzymatic inhibitory test of mitochondrial complex,subcellular proteomic analysis,LC-MS-based targeted/ untargeted metabolomic analysis,q RT-PCR experiment were performed.Enzymatic inhibitory test of mitochondrial complex indicated that the inhibition of mitochondrial ATP synthase blocked ATP increase under UVB radiation.Subcellular proteomic analysis identified 1051 proteins related to mitochondria;among them,proteins related to mitochondria complex I decreased,while proteins related to mitochondria complex II,complex IV increased;proteins related to methylerythritol phosphate(MEP)pathway increased,geranyl pyrophosphate(GPP)synthase increased,GPP reductase decreased;q RT-PCR analysis confirmed that the gene expression levels of correspondent proteins were consistent with the proteomic alterations.LC-MS-based metabolomics indentified 126 altered metabolites,which were mainly related to alkaloids,organic acids,carbohydrates,phenylpropanoids,fatty acids;among them,8 indole alkaloids significantly increased.Integrative analysis of multi-omic data indicated that several pathways related to amino acid metabolism down-regulated,while biosynthesis of tryptophan up-regulated.These findings suggested that mitochondria paricipated in regulatory mechanisms under UVB radiation.On the one hand,dynamic alterations in mitochondria complexes guaranteed high ATP supply,GPP synthase increased by activation of MEP pathway,which regulated the alteration from GPP to monoterpene backbones and induced accumulation of monoterpene precursors;on the other hand,within mitochondrial nitrogen network,down-regulation in several amino acid metabolism pathways regulated nitrogen flux into biosynthesis of tryptophan,which induced the biosynthesis of another precursor for indole alkaloids.(3)Study of regulatory mechanisms in M.alba under UVB radiation combined with different durations of dark treatmentFor exploration of common features and characteristics of regulatory mechanisms in medicinal plants under UVB radiation,M.alba was taken as an example.Proteomic analysis,metabolite fingerprint chromatogram differential analysis,in vitro antioxidant activity measurement,metabolites' content measurement,and q RT-PCR experiment were performed towards M.alba under UVB radiation combined with different durations of dark treatment and from different tissue part of untreated plants.Before UVB radiation,the content of Diels-Alder type adducts and their precursors in root was higher than other parts;the contents of kuwanone H,morusin,and chalcomoracin in roots were ten fold higher than other parts.During dark treatment period after UVB radiation,the contents of 5 compounds including chalcomoracin which belonged to Diels-Alder type adducts and their precursors significantly increased.Protein changes under 30 h dark treatment were the most significant,from when 253 proteins were altered;proteins related to flavonoid synthesis such as chalcone synthase,dihydroflavonol-4-reductase,naringenin-2-oxoglutarate 3-dioxygenase,and phenylcoumaran benzylic ether reductase increased.Similar to C.roseus,MEP pathway in M.alba was activated under UVB radiation,3 proteins at the pathway that were responsible for the catalyzation of prenyl monomeric endproduct increased,which promoted the accumulation of the prenyl donor,might activate downstream reactions which taking them as substrates and lead to the accumulation of prenylated compounds.However,as the pathway of secondary metabolism in M.alba has not been clarified,leaving speculations that prenyl transfer catalyzed by aromatic prenyltransferase(Aromatic PT)was responsible for the regulation on the biosynthesis of Diels-type adducts and their precursors.(4)Study of functional prenyltransferase in M.alba under UVB radiationFor further investigation of prenyl transfer mechanism in M.alba,novel Aromatic PT in M.alba was studied.After BLAST-based homologous screening,gene cloning,vector construction,heterologous expression in yeast/tabacco,and functional confirmation experiment,one transcript encoding Aromatic PT with novel functions was identified.Its RPKM value consistently increased under UVB radiation followed by dark treatment.The encoding protein,coined as Ma OGT,catalyzed the transfer of GPP to oxyresveratrol.Biochemical analysis and subcellular localization experiment confirmed that Ma OGT shared similar features with other characterizaed Aromatic PTs,which has conserved asparate-rich motifs as functional domains and targeted to plastids.As the first stilbenoid-specific Aromatic PT accepting GPP as the prenyl donor,the discovery of Ma OGT enriched the knowledge about Aromatic PT in moraceae,which benefitted further investigation of unidentified Aromatic PT in moraceae as important reference.Present thesis focused on the common features and characteristics of the regulatory mechanisms in two medicinal plants under UVB radiation,which elucidated their molecular alterations under UVB radiation and indicated the importance of prenyl backbones synthesis from MEP pathway and their transfer towards the activation of down-stream specialized secondary metabolism.This thesis laid solid foundantion for further manipulation on biosynthesis of active compounds derived from MEP pathway. |
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