Mechanism Studies Of Chitosan In Regulating G Triterpenoid Saponin Biosynthesis In Psammosilene Tunicoides Hairy Roots

Psammosilene tunicoides is an endangered medicinal plant,belonging to the Caryophyllaceae family,Psammosilene tunicoides.It is mainly distributed in southwestern China.Its main active ingredient is the triterpenoid saponin,which has the wide market demand as a medicinal ingredient.At present,the wild resources of Psammosilene tunicoides are scarce and cannot meet the market demand.In this study,the hairy roots of Psammosilene tunicoides（PTHRs）were used as materials to screen the optimal conditions for chitosan to elicit the accumulation of triterpene saponins.And the experiment determined the change of reactive oxygen species（ROS）,antioxidant enzymes and nitric oxide（NO）elicited by chitosan,combined with the transcriptome sequencing technology to analyze the regulatory mechanism of chitosan-elicited triterpene saponins synthesis.The results are as below:（1）The treatment of 200 mg/L chitosan is the most conducive to the biosynthesis of triterpene saponins in PTHRs.And compared with the control group,Chitosan at 200 mg/L for 9 days improved both the total saponin production in the hairy root tissue（up to 4.55-fold）and the secretion of these compounds to the liquid medium（up to 1.38-fold）in PTHRs.Simultaneously,chitosan increased the production of quillaic acid（QA）and gypsogenin-3-O-β-D-glucuronopyranoside（G3P）in PTHRs,which showed the largest enhancements by 1.76-fold and 6.18-fold compared to the control,respectively.（2）Application of exogenous chitosan dramatically triggered the ROS burst,enhanced antioxidant enzyme activities,and increased the NO content in hairy roots.The H₂O₂ contents increased under all concentrations of chitosan at one and five days.The level of H₂O₂ then decreased in both control and chitosan-treated groups,while no significant difference was observed at nine days.Superoxide dismutase（SOD）,peroxidase（POD）and glutathione reductase（GR）exhibited the highest antioxidant enzyme activity after nine days of chitosan elicitation.And under the treatment of 200 mg/L chitosan,the activities of SOD and POD reached the largest enhancements by 2.27-fold and 5.57-fold compared to the control,respectively.But there was a negative correlation between GR activities and chitosan concentrations.The GR activities in hairy roots were induced by 50 mg/L chitosan treatment,and reached the maximum value of 20.88 nmol/min/mg protein at nine days.Compared to the control group,the NO content in hairy roots dramatically enhanced after chitosan treatments.（3）Pac Bio third-generation sequencing produced a total of 40.53 Gb original data,and finally obtained 16,375 high-quality non-redundant gene sequences with an average length of2776 bp.Among them,51.06%of genes were annotated in 7 databases（NR,NT,Pfam,KOG/COG,Swiss Prot,KEGG and GO databases）.Based on the analysis of transcriptome sequencing data,7 antioxidant enzyme genes（SOD,PODs,and GR）were significantly up-regulated after 1 d of 50 mg/L chitosan treatments（CS1）,and 5 nitrate reductase genes（NRs）were significantly up-regulated after 9 d of 200 mg/L chitosan treatment（CS9）.55transcription factor families were obtained in RNA-seq of PTHRs.Among these families,the NAC,WRKY and AP2/ERF families showed the three largest numbers of significant up-regulated genes.And the NACs exhibited the most enormous numbers of up-regulated genes in CS1 and CS9.Based on the pathway enrichment analysis,71 differentially expressed genes（DEGs）encoding terpenoid biosynthetic enzymes（1-deoxy-D-xylulose-5-phosphate synthase gene（DXS）,geranyl diphosphate synthase gene（GPPS）,squalene epoxidase gene（SE）,etc.）were also screened.And SE（25976/f2p0/1891）showed a extremely correlation with NAC072 and WRKY7.Therefore,we speculated that a regulatory chain of‘NAC/WRKY-SE-saponin biosynthesis’might exist in PTHRs in response to the chitosan elicitation.In summary,this work demonstrated that chitosan could increase the yields of total saponins and representative saponin derivatives in PTHRs.Application of exogenous chitosan dramatically triggered the ROS burst,enhanced antioxidant enzyme activities,and increased the NO content in hairy roots.Comparative transcriptome analysis revealed DEGs encoding antioxidant enzymes（SOD,POD and GR）,NO synthetic enzymes（NR）,transcription factors（WRKY,NAC and AP2/ERF）and terpenoid biosynthetic enzymes（DXS,GPPS and SE）might be the critical regulator in chitosan-elicited saponin biosynthesis.Overall,the physiological and transcriptional roles of chitosan in the elicitation of saponin accumulation were preliminarily elucidated,which lays a foundation for effectively improving the saponin production in Psammosilene tunicoides.