Study On The Effects Of AMF Colonization On The Accumulation Of Polygonatum Kingianum Functional Components Under Different Phosphate Levels And Its Regulation Mechanism

ObjectivePolygonatum kingianum Coll.et Hemsl is a perennial herbaceous plant belonging to the genus Polygonatum the Liliaceae family.It is one of the bulk medicinal materials in Yunnan Province that government focuses on.Rhizomes of P.kingianum is commonly used both food process and drug therapy from ancient times to now.Arbuscular mycorrhizal fungi（AMF）is a kind of mutually beneficial symbiotic endophytic fungi that extensively colonize at plant roots.AMF play important roles in promoting the absorption of mineral elements,increasing biomass,accumulating metabolites,and improving stress resistance of medicinal plants.Phosphorus is a major element in the growth and development of plants,which effective concentration in the soil directly affects the accumulation of biomass and functional components of medicinal plants to a large extent.In this paper,AMF was used as a treatment strategy to investigate the effects of AMF on the growth and accumulation of main functional components of P.kingianum and its regulation mechanism under different effective phosphorus levels,in which different AMF agents would be inoculated at the roots of P.kingianum seedlings.This study has an important theoretical and practical significance for clarifying the mechanism of AMF,implementing ecological planting of P.kingianum,and improving quality and yield.Methods1.Test design and determination of physical and chemical indicators:factors of low phosphateate（LP,10 mg/kg）,normal phosphateate（NP,50 mg/kg）and AMF spores（AMF1,Glomus intraradices,AMF2,Glomus.etunicatum,AMF3,Glomus.mosseae,AMFs,G.intraradices+G.etunicatum+G.mosseae）were chosen as variates.As a result,a total of8 treatment groups were set,inclduing LP＿AMF1,LP＿AMF2,LP＿AMF3,LP＿AMFs,NP＿AMF1,NP＿AMF2,NP＿AMF3,NP＿AMFs and LP＿Non-AMF（negative control group）and NP＿Non-AMF（non-inoculated blank group,CK）.The effect of the interaction of phosphorus and AMF on the growth and accumulation of functional components of P.kingianum would be clarified by determining biomass and the contents of polysaccharides,monosaccharide components,steroidal saponins and other functional components in P.kingianum.The regulatory mechanism of AMF are going to be revealed via determination of phosphate absorption efficiency and transfer factor,and the expression level changes of key enzyme genes involved in polysaccharides or steroidal saponins biosynthesis.Biomass indicators were mainly about total fresh weight,tuber fresh weight,root weight,root length and plant height.The content of total polysaccharides and small molecule total sugars were determined with the method of sulfuric acid phenol,while reducing sugars were measured by DNS method.the content of monosaccharide composition in rhizome would be clear by liquid chromatography mass spectrometry（LC-MS）.The phosphorus content in the cultivation medium was determined by Na HCO₃extraction-molybdenum antimony colorimetric,and available phosphate concentration in P.kingianum was determined by molybdenum blue colorimetric method.2.Transcriptome sequencing analysis:Fresh fibrous materials were used for transcriptome sequencing analysis with the help of biological company,in which 4 groups in total,including NP＿AMF1,LP＿AMF1,LP＿Non-AMF and NP＿Non-AMF.Then,the heat map of the differential expression of key enzyme genes involved in steroidal saponins biosynthesis based on a better transcriptome quality.3.Screening of target genes involved in steroidal saponins synthesis,and their bioinformatics,tissue expression characteristics and heterologous expression vector construction:According to the transcriptome results,the key steroidal saponins synthesis genes with significantly different expressions were screened as target genes for further study.Bioinformatics analysis would be carried out via various online software for predicting isoelectric points,molecular weights,affinity,etc.Amino acid sequence alignment and phylogenetic tree were constructed with software of DNAMAN and MEG.6.0,meanwhile the heat map of differential expression genes involved in steroidal saponins biosynthesis was drew using Hem I 1.0.3.7.The analysis of tissue expression characteristics adopted real-time fluorescence quantitative 2^-△△Ctmethod.Besides,a prokaryotic expression vector with target gene was constructed,and translated into E.coli BL 21,that target protein could be used to study the protein action mechanism.Results1.Under normal phosphorus or low phosphorus levels,the AMF colonization rates in the roots of P.kingianum seedlings reached to 60%after 6 months of cultivation and treatment,of which the range were from 60.05%to 75.06%among different treatment groups.AMF colonization can increase the total fresh weight,tuber fresh weight,fibrous root fresh weight,plant height and root length.Under normal phosphate and low phosphate levels,G.intraradices（AMF1）has the most significant increase in total fresh weight with growth-promoted rates of 104.46%（NP）and 127.07%（LP）,respectively,followed by G.etunicatum（AMF2）with parameters of 23.51%and 65.67%.Besides,the colonization of AMF can promote the increase of tuber fresh weight,fibrous root fresh weight,plant height and root length to varying degrees.whatever the treatment is either low phosphate or normal phosphate,the colonization of AMF significantly improved the phosphorus absorption efficiency of P.kingianum.2.The colonization of AMF increased of the total polysaccharide content of rhizome,and the promotion effect is more significant under the stress of low phosphorus.Under normal phosphorus level,G.intraradices+G.etunicatum+G.mosseae（AMFs）have the most significant effect on the increase of total polysaccharide content with a promotion rate of90.47%,followed by G.etunicatum（AMF2）with a value of 59.76%.Under low phosphorus level,the total content of polysaccharide3.Compared with CK group,both G.intraradices（AMF1）and AMFs presented significantly difference on the increase of total polysaccharide with the promotion rates of176.32%and 108.97%.Analysis of the carbohydrate composition of polysaccharides found that the contents of polysaccharides included sucrose,fructose,glucose and inositol.Under normal phosphorus level,AMF agents significantly promoted the accumulation of sucrose and fructose,particular in AMF2 with promoting rates of 143.36%（sucrose）,61.39%（fructose）and 79.21%（inositol）,respectively.Under low phosphorus level,the colonization of AMF2 had a significant effect on the accumulation of fructose and glucose.with a fructose-promoted rate of 180.06%,glucose-promoted rate of 79.10%.Besides,either normal or low phosphate treatment,AMF1 and AMF2 could also significantly promote the accumulation of diosgenin,and the promotion rates of 29.25%,146.74%（NP）,58.28%and 140.57%（LP）.4.The transcriptome sequencing results of treatment groups（NP＿AMF1,LP＿AMF1）,negative control group（LP＿Non-AMF）and blank group（NP＿Non-AMF）showed that the quality was good and reliable based on the Q20 and Q30 value ranges from 97.70 to98.12%and 93.62 to 94.37%,respectively.Under the same phosphate level,the colonization of AMF induced more genes up-regulated expression.According to GO,KOG,and KEGG analysis,it’s found that differential genes were mainly concentrated in cells and cell parts in terms of cell composition,molecular function and biological processes.The analysis of KEGG metabolic pathways showed that differential genes were mainly enriched in ribosomes,secondary metabolite biosynthesis,metabolism and other pathways under the interaction of phosphorus levels and AMF.The heat map of the differential key-enzyme genes involved in steroidal saponin biosynthesis showed that the DXR,DXS,FPS,CAS,SS and SE family genes were up-regulated in response to AMF induction,especially for low phosphate stress.5.Further research results of key-enzyme genes involved in steroidal saponin biosynthesis revealed that five target genes of Pk FPS1,Pk CAS1,Pk SE5,Pk SS1,Pk SS2 belonged to the4 families of CAS,FPS,SS,and SE.The length of ORFs were 1,050 bp,2,286 bp,1,077bp,1,230 bp and 1,233 bp,which encoded 349 aa,761 aa,358 aa,409 aa and 410 aa.Correspondingly,the isoelectric points and protein molecular weights were 6.27/110.02k Da,5.59/64.06 k Da,7.97/62.41 k Da,6.83/70.22 k Da and 6.48/70.30 k Da,respectively.The q PCR results showed that the 5 target genes had different responses to AMF and low-phosphorus induction at different tissues of rhizome,roots and leaves.Under normal phosphate level treatment,the expression levels of Pk FPS1,Pk CAS1,Pk SS1 and Pk SS2 in rhizome were significantly up-regulated under the induction of G.mosseae（AMF3）,and their expression levels were 1.83,3.38,2.39,and 1.99 folds,respectively.Under low phosphate treatment,the expression levels of Pk FPS1,Pk CAS1,Pk SS1 and Pk SS2 in roots were significantly up-regulated under the induction of G.intraradices（AMF1）with folds of 2.51,1.43,1.84,and 1.62.The expression of Pk FPS1,Pk CAS1,Pk SS1 and Pk SS2 in rhizome were significantly up-regulated under the induction of G.intraradices+G.etunicatum+G.mosseae（AMFs）with expression levels of 1.46,1.51,4.17,and 5.86 times.Under normal or low phosphate level treatment,the expression of the whole 5 genes in leaves were significantly up-regulated under the induction of AMF1,and the expression levels of the normal phosphate treatment group were higher than the low phosphate treatment group.It should be pointed that the expression of Pk CAS1,Pk SS1 and Pk SS2 in rhizome were significantly up-regulated under the induction of AMF3 and AMFs,and the expression levels of Pk SS1 and Pk SS2 in the low phosphate treatment group are higher than the normal phosphate group.The expression of Pk FPS1,Pk CAS1,Pk SS1 and Pk SS2in roots were up-regulated under the induction of AMF1,and the folds were higher under the low phosphate.By the comparison of different tissue parts,under the same treatment conditions,the expression of the same gene in the leaves is the highest,followed by rhizome.6.Through prokaryotic heterologous expression assay of Pk FPS1,recombinant fusion protein was obtained with a molecular weight size of 64 k Da,which was consistent with the predicted.During the induction process,as the induction time increasing,the amount of protein expression also increased.The target protein only appeared in the precipitate,indicating that the protein is an inclusion body protein.ConclusionAs above described,AMF inoculants could promote the increase of the biomass of P.kingianum under normal or low phosphorus treatment conditions,particular in G.intraradices（AMF1）and G.etunicatum（AMF2）.In addition,the contents of total polysaccharides,steroidal saponins,sucrose,fructose,and glucose were also significantly improved by inoculating AMF agents,and phosphate deficiency strengthened the effect.Generally,G.etunicatum（AMF2）had the most significant promotion effect on the accumulation of the functional components of P.kingianum by horizontal comparison.For the analysis of mechanism,we found that the improvement of effective phosphorus absorption efficiency is a directly main reason for promoting the growth and biomass-improved of P.kingianum.The differential expression of key enzyme genes in the biosynthetic pathway of functional components is an important link for AMF to regulate the accumulation of polysaccharides and steroidal saponins.This study lays the foundation for further in-depth research on the response mechanism of the accumulation of polysaccharides,steroidal saponins and other functional components of P.kingianum to AMF induction.