Inducement Of Polyphenols Synthesis In Suspension Culture Of Cyclocarya Paliurus Cells And Its Mechanism

Polyphenols are a large variety of secondary metabolites in plants.Studies showed that polyphenols have many beneficial bioactivities such as antioxidant,antibiosis,anti-inflammatory,anti-tumor,anti-diabetes and prevention of cardiovascular and cerebrovascular diseases,and therefore they have been widely used in medicines,cosmetics and foods.Polyphenols are an important class of bio-active components in Cyclocarya paliurus（C.paliurus）,however these compounds haven’t yet attracted the interest of researchers,and there are few researches and applications about these substance at present.In our laboratory,a variety of calluses of C.paliurus with different phenotypes have been successfully screened and cultivated,and a stable cell suspension culture technology has been developed using superior calluses with high total polyphenols content.In order to further increase the accumulation of polyphenols in the cultured cells,five elicitors were screened in the present paper.An induction technology of 5-ALA combined with SA was established to promote the biosynthesis of polyphenols in C.paliurus cells.Furthermore,integrated decection and analysis of transcriptome and metabolome were used to investigate the induction mechanism of 5-ALA and SA on the polyphenols biosynthesis in the cultured C.paliurus cells.The main results and conclusions were as follows:（1）Elicitors were screened to promote the biosynthesis of polyphenols in the cultured suspension C.paliurus cells.Five elicitors,including 5-ALA,SA,MeJA,SNP and ROE,were used to increase the content and yield of polyphenols in the cultured cells of C.paliurus.Result indicated that all the five elicitators promoted the biosynthesis of polyphenols in cultured cells,among which SA showed the best promotion,followed by5-ALA,ROE,MeJA and SNP.Compared with the untreated cells,the polyphenol content and yield of the cells treated by SA increased by 0.78 and 0.9 times,and the content and yield in the 5-ALA treated cells also enhanced 72%and 63%respectively.Both of SA and5-ALA showed good application prospects.（2）The induction technology of 5-ALA combined with SA to stimulate the biosynthesis of polyphenols in C.paliurus cells was established.A comprehensive experimental design was used to optimize the concentration of 5-ALA and SA in the combined induction.The results showed that the optimized conditions were as following:50μM 5-ALA was added on the 0^th day and 50μM SA was added on the 4^th day,and the cells were harvested on the 6^th day after inoculation.Under the optimized parameters,the content and yield of total polyphenols reached 8.0 mg/g and 147.12 mg/L,which were 3.4and 3.58 times that of the control,respectively.（3）Transcriptome detection and analysis of the cultured C.paliurus cells treated by5-ALA and SA were performed.RNA-seq of the 5-ALA and SA treated cells was carried out with Illumina Hi Seq sequencing platform,using the untreated cells as control.A total of 72795281 high quality unigenes with average length of 1023.25 bp and N50 of 1595 bp were obtained,which were further searched against six public databases and 32465,15349,11379,15187,31397 and 21412 unigenes were annotated in NR,GO,KEGG,Pfam,egg NOG and Swissprot databases,respectively.By comparison analysis differentially expressed genes（DEGs）between the treated and the untreated cells,a total of 15 DEGs related to SA biosynthesis and signal transduction pathway were screened out,among which 13 genes up-expressed and 2 genes were down-expressed,respectively.Eleven DEGs in polyphenols biosynthesis pathway were identified,among the identified 11 DEGs,9 genes（such as pal,chs,fls）were up-regulated,and 2 genes（such as f3＇5＇h and anr）were down-regulated.In addition,a total of 8 regulatory genes involved in the regulation of SA signal transduction were identified,among which TGA1,TGA6 and WRKY28 were up-regulated and 6 regulatory genes（such as TGA2,TGA4,WRKY46）were down-regulated.A total of 6 regulatory genes associated with phenylpropane biosynthesis pathway were identified,and 3 regulatory genes（ERF105,MYB12）were up-regulated,while MYB44 and MYC2 expression was down-regulated.（4）Metabolome detection and analysis of the cultured C.paliurus cells treated by5-ALA and SA were performed using LC-MS/MS.A total of 970 and 958 substances were identified from the C.paliurus cell extracts in positive and negative ion mode,respectively.Under the positive mode,the content of 138 substances was up-regulated and 39substances were down-regulated.Under the negative mode,the content of 110 substances was up-regulated and 34 substances were down-regulated.PCA（Principal Component Analysis）analysis results showed that the two groups of treated cells and untreated cells in positive and negative ion mode have obvious separation trend on PC1 and PC2two-dimensional diagram,which indicated that the induction effect is obvious.Moreover,the results of OPLS-DA（Orthogonal Partial Least Square-Discriminate Analysis）showed that the established model has high reliability and can better distinguish treated cells and untreated cells.In addition,all the substances identified in positive ion mode were analyzed by significant clustering hierarchy analysis,which showed that samples in the same group can appear in the same cluster by clustering,and the substances clustered in the same cluster have similar expression patterns.The results indicated that 5-ALA combined with SA elicitation has a significant influence on the metabolism of C.paliurus cells.Through KEGG enrichment analysis,a total of 256 different metabolites were enriched in 70 metabolic pathways.The pathways related to polyphenols metabolism including flavonoid biosynthesis pathway,phenylalanine biosynthesis pathway,phenylpropanol biosynthesis pathway,flavonoid and flavonol metabolism pathway and plant hormone signal transduction pathway.In addition,a total of 13 polyphenolic differential metabolites were screened out,such as Dihydrokaempferol,quercetin and naringin,among which the content of 11 metabolites were up-regulated and 2 metabolites were down-regulated.（5）Integrated detection and analysis of transcriptome and metabolome were conducted to interpret the induction mechanism of 5-ALA and SA on the polyphenols biosynthesis in C.paliurus cells.Through the analysis of KEGG database,a total of 13differential metabolites and 42 related transcripts were obtained,among which 5metabolites related to the polyphenol biosynthesis pathway including naringin,phenylalanine and queretin and there are 26 transcripts associated with them.Further significant difference analysis showed that the contents of salicylic acid,phenylalanine,quercetin and caffeic acid and the expression of 10 transcripts related to them changed significantly after induction.Through the correlation analysis of transcripts and metabolites,we speculated that the polyphenol biosynthesis pathway in suspension cultured C.paliurus cells treated by 5-ALA and SA was mainly through dihydrokaempferol branched pathway and dihydroquercetin branched pathway.In these two branched pathways,the up-regulation of key enzyme genes in polyphenol biosynthesis pathway,such as pal,f3’h,chs,fls,lar,ans,which leads to the content of metabolites in its downstream increasing,and finally promotes the accumulation of polyphenols in suspension cultured C.paliurus cells.In conclusion,5-ALA and SA were synergistically used as combined elicitors to improve the polyphenols yield of the cultured suspension C.paliurus cells,which increased the production by 3.58 times.Moreover,integrated detection and analysis of transcriptome and metabolome were conducted to interpret the induction mechanism of5-ALA and SA on the polyphenols biosynthesis in the cultured C.paliurus cells.This study provides a basis for the regulation of polyphenols production by suspension culture of C.paliurus cells.