Effect Of Bacillus Megaterium On Physiological Characteristics Of Glycyrrhiza Uralensis Under Phosphorus Deficiency Stress And Transcriptome Analysis

Licorice（Glycyrrhiza uralensis Fisch.）is a perennial herb belonging to the legume family,and its roots and rhizomes are widely used for clinical treatment in traditional Chinese medicine.Phosphorus availability can affect the nitrogen fixation ability of leguminous plants,thereby affecting their growth and development,while large areas of arable land in China are in a phosphorus deficit state.Therefore,the growth of licorice is often limited by the effectiveness of soil phosphorus.In this study,licorice seedlings was taken as the research object,using 0.2%calcium phosphate[Ca₃（PO4）₂]as insoluble phosphorus source and Bacillus megaterium as as a phosphate solubilizer.The water culture method was used,with control（CK,Hoagland’s nutrient solution）,phosphorus deficient（-P,Hoagland nutrient solution without phosphorus）,CK+B(addition of 2.5×10⁵cfu·m L^-1B.megaterium to Hoagland’s nutrient solution),and-P+B(addition of 2.5×10⁵cfu·m L^-1B.megaterium to Hoagland’s nutrient solution without phosphorus).The purpose of this study is to investigate the effects of B.megaterium on the physiological characteristics,secondary metabolites accumulation,and molecular mechanisms of licorice seedlings under phosphorus deficiency stress.The research results are as follows:1.Phosphorus deficiency inhibited the growth of licorice seedlings,decreased the total biomass and the content of hormones auxin（IAA）,gibberellin（GA）in different tissues,significantly increased the content of abscisic acid（ABA）,proline（Pro）,total protein（TP）,glutathione（GSH）and inorganic phosphorus levels in different tissues,increased the activity of antioxidant enzymes（SOD,POD,CAT）in roots and leaves,and the activity of acid phosphatase（ACP）in roots,as well as root length.Under phosphorus deficiency,B.megaterium decreased the activities of SOD,POD,CAT and ACP in the roots of licorice seedlings,and increased the contents of Pro,TP,GSH,and inorganic phosphorus in different tissues.It also increased the contents of hormones（IAA,GA）in different tissues,and increased the total biomass of licorice.2.Phosphorus deficiency significantly reduced leaf photosynthetic indicators（P_n,G_s,C_i,T_r）,chlorophyll fluorescence parameters（F_v/F_m,F_v/F₀）and photosynthetic pigment contents in leaves,and significantly increased chlorophyll fluorescence parameters（NPQ,DI₀/RC,TR₀/RC,ABS/RC）,starch and soluble sugar concentrations.Under phosphorus deficient conditions,B.megaterium significantly increased photosynthetic indexes（P_n,G_s,C_i,T_r）,photosynthetic pigment and chlorophyll parameters（ET₀/RC）,while reducing chlorophyll fluorescence parameters（NPQ,DI₀/RC,DI₀/RC,ABS/RC）,starch content,and soluble sugar content.This indicates that B.megaterium can prevent oxidative damage to the photosynthetic system caused by phosphorus deficiency by changing photosynthetic pigments,maintain high photosynthetic capacity,and prevent oxidative damage to the photosynthetic system caused by phosphorus deficiency.3.Under phosphorus deficient conditions,the addition of B.megaterium significantly increased the content of glycyrrhizic acid,liquiritigenin and liquiritin in roots,while the content of flavonoids in the roots and leaves significantly decreased.In addition,phosphorus deficiency significantly induced the expression of LUS and CHS genes,and the addition of B.megaterium significantly induced the HMGR andβ-AS gene expression.It indicates that phosphorus deficiency can induce the expression of the key enzyme LUS gene,and the B.megaterium can induce the expression of HMGR andβ-AS,thereby increasing the content of the active components of licorice（glycyrrhizic acid,liquiritigenin and liquiritin）.4.Transcriptome analysis by high-throughput sequencing showed that CK+B vs CK had 1093 genes up-regulated expression and 1187 genes down-regulated expression.-P vs CK had 1109 genes up-regulated expression and 1275 genes down-regulated expression.-P+B vs-P group had 572 genes up-regulated expression and 205 genes down-regulated expression.GO functional enrichment analysis identified 8075,7413 and 2568 DGEs annotated in the GO database under-P vs CK,CK+B vs CK and-P+B vs-P comparisons,respectively.KEGG enrichment indicated that-P vs CK,CK+B vs CK and-P+B vs-P phosphorus deficiency response stress was mainly manifested in the metabolic pathways of biosynthesis and secondary metabolites.5.In this study,phosphorus deficiency stress induced the expression of most Pht1gene members in licorice roots and promoted the upregulation of ABA-related genes as well as upregulation of transcription factors such as b HLH and WRKY.Under phosphorus deficiency,the addition of B.megaterium down-regulated the expression of Pht1,while the expression of genes related to water transport was up-regulated,suggesting that B.megaterium could improve the low phosphorus tolerance of plants by helping licorice absorb phosphorus nutrients and regulating the expression of water channel proteins.In summary,under phosphorus deficiency stress,B.megaterium could reduce antioxidant enzyme activity,alter osmoregulatory substances and hormone content,increase photosynthetic pigment and photosynthetic parameters,change chlorophyll fluorescence parameters,and protect the stability of cell membrane structure to alleviate oxidative damage to seedlings under phosphorus deficiency stress and repair damage to the PSII reaction center of leaves.In addition,B.megaterium could also increase the content of liquiritin,glycyrrhizic acid and liquiritigenin under phosphorus deficiency and regulate the expression of key enzyme genes for secondary metabolism synthesis and Pht1 gene,thereby improving its phosphorus deficiency tolerance,and promoting the growth of licorice seedlings.