Ymechanism Of Endophytic Bacteria Bacillus Cereus G2 Regulated Synthesis And Accumulation Of Active Components In Glycyrrhiza Uralensis Fisch. By Carbon Metabolism Under Salt Stress

ObjectiveIn the present study,Glycyrrhiza uralensis Fisch.was used as research material,and by studying the growth,active components,carbon metabolism and flavonoid biosynthesis of G.uralensis seedlings,the regulatory effects of Bacillus cereus G2 on the carbon metabolism and flavonoid biosynthesis in G.uralensis seedlings under salt stress were clarified at the physiological and molecular levels,and the mechanisms of G2 regulation of carbon metabolism and flavonoid accumulation of G.uralensis seedlings under salt stress were revealed.On this basis,we used correlation,PCA,path and stepwise regression analysis to reveal the mechanism of G2 regulating the synthesis and accumulation of active ingredients through regulating carbon metabolism in G.uralensis under salt stress.The main findings and conclusions are as follows.MethodsG.uralensis were seedlings were cultured by indoor sand-bearing pot experiments,collected and measured growth parameters,and determined the activities of key enzymes and contents of substances in carbon metabolism and flavonoid synthesis and the contents of active ingredients by HPLC,ultraviolet spectrophotometry and ELISA;Transcriptomics and PCR techniques were used to study the expression of genes related to carbon metabolism and flavonoid synthesis in G.uralensis seedlings.Results1.Salt stress inhibited growth,decreased the contents of total saponins,total polysaccharides and isoliquiritigenin,but increased liquiritin content,which might be a protective mechanism of G.uralensis in response to salt stress.However,G2 not only promoted growth and increased biomass,but also increased glycyrrhizic acid and other active components in G.uralensis seedlings under salt stress.Specifically,G2 significantly increased stem diameter,plant height,fresh weight,dry weight and the the contents of total saponins,total polysaccharides,isoliquiritigenin and glycyrrhizic acid,but decreased liquiritin content in G.uralensis seedlings,indicating that G2 could alleviate the inhibitory effect of salt stress on growth and active ingredient accumulation of G.uralensis seedlings.2.Salt stress reduced carbohydrates contents in G.uralensis seedlings,while G2 accumulated more carbohydrates by balancing carbon metabolism in G.uralensis under salt stress.Specifically,(1)In photosynthesis,chlorophyll accumulation was increased and correspondingly to the leaves was greener in salt-treated G.uralensis seedlings,and then,maybe,leading to highly efficient photosynthesis in terms of light reaction;G2 further increased chlorophyll a and carotenoid content in G.uralensis exposed to salinity environments,which possibly further increase the photosynthetic rate of G.uralensis seedlings.(2)In terms of carbohydrate transformation,salt stress reduced the contents of TSS,starch,sucrose and fructose in G.uralensis seedlings,while G2 increased the contents of TSS,starch,sucrose and fructose in G.uralensis seedlings exposed to salinity environments.The reasons for this are as follows: G2 promoted the elongation of a-1,4-glycosidic bonds on amylopectin molecules in G.uralensis seedlings exposed to salinity environments by increasing SSS activity,which in turn promoted starch synthesis and consequently activated AM and BM activity,which accelerated the hydrolysis of starch to a mixture of smaller oligosaccharides,thus providing sufficient substrate for subsequent carbon metabolism.G2 increased the SS activity of G.uralensis seedlings exposed to salinity environments by up-regulating the gene Glyur001957s00039090 encoding SS,which promoted sucrose synthesis;meanwhile,G2 promoted sucrose decomposition in G.uralensis seedlings exposed to salinity environments by increasing NI and AI activity and up-regulating the gene Glyur000005s00001105 encoding α-glucosidase,which led to fructose accumulation,which in turn provided sufficient substrate for the subsequent EMP-TCA cycle.(3)During EMP pathway,salt stress inhibited HK activity and increased PFK and PK activity in G.uralensis seedlings,while G2 reversed this phenomenon.Specifically,G2 promoted the phosphorylation of hexose and induced sugar signal transduction in G.uralensis seedlings exposed to salinity environments by increasing HK activity;while G2 reduced PFK and PK activity and inhibited the conversion of fructose-6-phosphate to pyruvate in G.uralensis seedlings exposed to salinity environments.This phenomenon indicated that G2 provided sufficient energy and carbon source for G.uralensis seedlings exposed to salinity environments by increasing carbohydrate content and promoting the first step of EMP process.(4)In the TCA cycle,salt stress decreased PDH and SDH activities and CA content,but increased MDH activity in G.uralensis seedlings.Interestingly,G2 increased PDH activity and thus increased CA content in G.uralensis seedlings exposed to salinity environments,which subsequently activated ICDH activity to promote CA conversion,thereby accelerating the TCA cycle.3.Salt stress inhibited the biosynthesis and accumulation of flavonoids,while G2 reversed this phenomenon to some extent.Specifically,salt stress inhibited the synthesis of p-coumaroyl Co A and cinnamoyl-Co A by reducing PAL and 4CL activities,which in turn inhibited phenylpropanoid and flavonoid biosynthesis at the substrate level.Further,in phenylpropanoid and flavonoid biosynthesis pathways,salt stress inhibited the synthesis of phenylpropanoids,flavonoids,isoflavonoids,flavones and flavonols in G.uralensis seedlings by decreasing 4CL,CHS,and CHI activities and down-regulating the genes encoding CSE,COMT,CYP75 A,and CYP81E1.Interestingly,in phenylpropanoid biosynthesis pathway,G2 significantly increased PAL activity,decreased C4 H and HCT activities,up-regulated the gene Glyur000217s00014848 encoding COMT,and had complicated regulation of the gene encoding POD,suggesting that G2 possibly inhibited phenylpropanoids biosynthesis in G.uralensis seedlings exposed to salinity environments,thus reduced lignin content.In flavonoid biosynthesis pathway,G2 promoted flavonoid synthesis and inhibited isoflavonoid synthesis by increasing CHS and CHI activities and down-regulating the gene Glyur000721s00027409 encoding CYP93C2 in G.uralensis seedlings exposed to salinity environments,thus increased isoliquiritigenin content.In brief,G2 could decrease metabolic flux of phenylpropanoid in the lignin synthesis pathway in G.uralensis seedlings exposed to salinity environments by reducing the activities of C4 H and HCT,and increasing the activities of PAL,CHS and CHI,thereby promoting the flow of more phenylpropanoid metabolic flux towards flavonoids,ultimately led to the decrease of lignin content and the increase of isoliquiritigenin content in G.uralensis seedlings exposed to salinity environments.ConclusionG2 provided carbon and energy for subsequent life activities in G.uralensis seedlings exposed to salinity environments by increasing photosynthetic pigment content,promoting carbohydrate accumulation,and regulating the EMP-TCA cycle;G2 regulated the conversion of phenylpropanoid between lignin synthesis pathway and flavonoid synthesis pathway by affecting key enzymes and related genes in phenylpropanoid and flavonoid biosynthesis,thereby decreasing lignin accumulation and increasing isoliquiritigenin accumulation.