Metabolomics-based Germplasm Evaluation And High-temperature Stress Studies Of Polygonatum Cyrtonema Hua In Wuyi Mountains

Polygonatum cyrtonema Hua(P.cyrtonema)is a perennial herb of the genus Polygonatum in the family Liliaceae,often found in the understory of forests,thickets,or shady places on mountain slopes,with important ornamental and medicinal value.Polygonatum species germplasm resources,including Polygonatum cyrtonema Hua(P.cyrtonema),Polygonatum kingianum Coll.et Hemsl,and Polygonatum sibiricum Red et al.,are abundant in China,and Polygonatum species are naturally distributed in Fujian,Anhui,Zhejiang,and Jiangxi Provinces.Among these species,P.cyrtonema,including pointed-leaved green stem,pointed-leaved purple stem,broad-leaved green stem,and broad-leaved purple-green germplasm,is a key constituent of herbal medicines in Fujian Province and is mainly distributed near the Wuyi Mountains.P.cyrtonema is rich in various active components,and the traditional methods for the analysis of some medicinal components,such as total polysaccharides,total saponins,and total flavonoids,do not allow a detailed evaluation of P.cyrtonema germplasms and age sections.Widely targeted metabolomics analysis is a technology that combines the advantages of untargeted and targeted metabolite assays,which allows comparison of the metabolite species and abundance between samples,the screening for biologically significant metabolites between groups,and has been applied in a variety of species(turmeric,sesame,and radish,etc.).However,a widely targeted metabolomics analysis of P.cyrtonema has not been reported in the literature.Extreme heat problems caused by global warming have posed a great threat to P.cyrtonema cultivation,and it has become increasingly important to breed resistant germplasm.Ethyl methyl sulfonate(EMS),a commonly used chemical mutagen,can quickly obtain mutants.TAG plays an important role in alleviating stress,and its content can be used as an important indicator for screening resistant germplasm.At present,the P.cyrtonema industry is in the development stage,and there is a lack of comprehensive and objective evaluation and research on the breeding of good germplasm and high temperature resistance.In this study,different metabolites from these three P.cyrtonema germplasms(pointed-leaved green stem(JL),pointed-leaved purple stem(JZ),and broad-leaved green stem(GK))and three age sections(one year(AT),two years(BT),and three years(CT))of the GK germplasm were analyzed by widely targeted metabolomics.This study identified lipids as a key differential metabolic component.As triacylglycerol(TAG)is an important component of lipids,the identification of three key genes for TAG synthesis(glycerol-3-phosphate acyltransferase(GPAT),lysophosphatidic acid acyltransferase(LPAT),and diacylglycerol acyltransferase(DGAT))was subsequently carried out.The relationship between the expression of Pc GPAT,Pc DGAT,and Pc LPAT in the three germplasms and the three age sections was analyzed.The effect of high temperature stress on P.cyrtonema was analyzed and the trend of TAG content under high temperature stress was also explored.The effect of heat stress mitigation on P.cyrtonema was explored using exogenous melatonin(MT)and diphenyl methylphosphate(DMP)treatments.In addition,an ethyl methanesulfonate(EMS)mutagenesis system was established for the rhizomes of P.cyrtonema tissue culture seedlings,and mutant germplasm with high TAG content was screened,so as to obtain high quality resistant germplasm and lay a foundation for the cultivation of resistant germplasm.The main results are as follows:1.Metabolic characterization and evaluation of different germplasms and age sections of P.cyrtonema by widely targeted metabolomicsIn this study,three major cultivated P.cyrtonema germplasms(JL,JZ,and GK)from the Wuyi Mountains were used as test material.Different metabolites from these three P.cyrtonema germplasms(JL,JZ,and GK)and three age sections(AT,BT,and CT)of the GK germplasm were analyzed by widely targeted metabolomics.The results showed that a total of 685 metabolites in 13 classes and 291 differential metabolites were identified in the three P.cyrtonema germplasms.By comparing differential metabolites,it was found that the GK germplasm is rich in polysaccharides,alkaloids,and lipids;the JL germplasm is rich in flavonoids,steroids,and amino acids,while the JZ germplasm contains more phenolic acids.Additionally,this study identified a total of 41 key differential metabolites,which can be used as markers to distinguish the three P.cyrtonema germplasms.To further understand the metabolic differences between the different age sections of P.cyrtonema,the three age sections of GK germplasms were analysed by widely targeted metabolomics analysis,which resulted in the identification of 977 metabolites in 13 classes and 331 differential metabolites.It was found that the AT age section is rich in polysaccharides,steroids,organic acids,and lipids;the CT age section contains more flavonoids,alkaloids,and amino acid metabolites.Differences in the pentose phosphate pathway(ko00030)and metabolic pathways(ko01100)may be the main causes of the metabolite differences between the age sections.In summary,the different germplasms and age sections of P.cyrtonema were rich in different nutritional and medicinal components.Furthermore,TAG,which is associated with resistance,was found at the highest level in the GK germplasm and AT age section in this study.2.Identification and expression analysis of families of genes related to P.cyrtonema triacylglycerol synthesisBased on the above findings,TAG content was found to differ significantly among the three germplasms and age sections,and was highest in the GK germplasm and AT age section.Therefore,to investigate the expression patterns of TAG synthesis-related genes in P.cyrtonema,the families of TAG synthesis-related genes(GPAT,DGAT,and LPAT)were identified,and their expression patterns in different germplasms and age sections were analysed using bioinformatics analysis and quantitative real-time PCR(q RT-PCR),respectively.19,14,and 13 members of the glycerol-3-phosphate acyltransferase(GPAT,LPAT,and DGAT)gene families,respectively,involved in triacylglycerol synthesis were also identified.Transcriptome data from different tissue sites revealed that DGAT1,DGAT2.4,GPAT9.1,GPAT9.3,LPAT1.1,LPAT2.9,and LPAT4.3 genes were specifically highly expressed in rhizomes.The q RT-PCR results further suggested that the differentially expressed Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3 genes may play important roles in TAG synthesis in P.cyrtonema.The q RT-PCR results showed that the gene expression of Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3 genes in GK germplasm and AT age section followed the same trend as the TAG content,suggesting that these genes may be key genes for TAG synthesis in P.cyrtonema.3.The effect of high temperature stress on P.cyrtonema tissue culture seedlings and rhizomes triacylglycerolP.cyrtonema prefers a cool and humid environment.The extreme high temperatures caused by global warming have posed a great threat to the P.cyrtonema planting industry.TAG is closely related to plant resistance and can be used as an important indicator for germplasm resistance evaluation.Therefore,the effect of high temperature at 40 ℃and 45 ℃ on the TAG content of P.cyrtonema seedlings and rhizomes was investigated using histoculture seedlings of broad-leaved green stalk(GK)germplasm screened in part 1.The results showed that high temperature stress inhibited the accumulation of chlorophyll content and increased the relative conductivity of P.cyrtonema leaves.It also promoted the accumulation of TAG content and the increase of SOD and POD activities in rhizomes.The above indicates that P.cyrtonema actively resists heat stress on its own.The q RT-PCR revealed that the expression of TAG synthesis-related genes(Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3)increased significantly with increasing stress time under high temperature stress at 45 ℃.It is speculated that the above genes may further resist high temperature stress by promoting TAG synthesis.4.Relieving effect of exogenous melatonin and diphenyl methylphosphate on high temperature stress of P.cyrtonemaPre-laboratory studies have found that both MT and DMP can promote TAG accumulation.The accumulation of TAG can promote the formation of lipid droplets and plastids,playing an important role in the tolerance of plants to environmental stress.It is speculated that MT and DMP can alleviate the high temperature stress of P.cyrtonema.To investigate the alleviating effect of MT and DMP on P.cyrtonema under high temperature stress,different concentrations of MT and DMP were applied externally to the tissue culture seedlings of broad-leaved green stalk(GK)germplasm,and the tissue culture seedlings treated with the best MT and DMP concentrations were subjected to high temperature treatment.It was found that both 15 μM MT and 35 μM DMP significantly promoted rhizome proliferation and TAG synthesis under different concentrations of MT and DMP treatment in P.cyrtonema cultivated seedlings,and the best effect was observed.The seedlings were then subjected to high temperature stress at 40 ℃ and 45 ℃ using 15 μM MT and 35 μM DMP treatments,respectively.Both MT and DMP promoted the accumulation of chlorophyll content and reduced the relative electrical conductivity of leaves compared to the control.It also promoted the accumulation of TAG content and increased SOD and POD activities in rhizomes.The above results indicate that MT and DMP alleviated P.cyrtonema heat stress.Furthermore,under high temperature stress at 45 ℃,the Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3 genes were significantly up-regulated in both the MT and DMP treated groups compared to the control group.Therefore,it was hypothesized that MT and DMP could respond to high temperature stress by inducing the Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3 genes.5.Establishment of EMS mutagenesis system in rhizomes and selection of resistant P.cyrtonema germplasmsIn this study,rhizomes from tissue culture seedlings were treated with different concentrations of EMS for different times to explore the optimal EMS mutagenesis system,on the basis of which the TAG content was used for preliminary screening and evaluation of the mutagenic germplasm.The results showed that EMS inhibited the growth and reduced the survival rate of P.cyrtonema.In addition,it was found that the mutagenic concentration and mutagenic time of EMS were proportional to the lethality of P.cyrtonema.The best mutagenic conditions for P.cyrtonema rhizomes were found to be 0.6% EMS treatment for 3 h,with a lethality rate of 53.33%,using half lethality as a screening criterion.A total of 150 EMS mutagenic germplasms were obtained based on optimal mutagenic conditions.In addition,34 EMS mutagenic germplasms were screened for TAG content,and six germplasms with high TAG content(> 1000μg/g)were obtained,with a view to laying the foundation for the screening of resistant germplasm.In conclusion,based on widely targeted metabolomics,this study found that three germplasms and age sections were rich in different metabolic components,and found that TAG content was the highest in the broad-leaved green stalk germplasm and one year section,suggesting that they might have higher stress resistance.It was found that Pc DGAT1,Pc DGAT2.4,Pc GPAT9.1,Pc LPAT2.9,and Pc LPAT4.3 genes may be key genes for TAG synthesis in P.cyrtonema.It was also found that MT and DMP may promote TAG synthesis by upregulating the expression of these genes,there by alleviating high temperature stress.Finally,the optimal EMS mutation system for the rhizome of P.cyrtonema was discovered,and six samples with high TAG content(> 1000 μg/g)were preliminarily screened out resistant germplasms.The results of this study will provide a basis for breeding quality resistant germplasms.