Effects of Arbuscular Mycorrhizal Fungi on the Physiology and Saponin Synthesis of Paris polyphylla var. yunnanensis at Different Nitrogen Levels

Arbuscular mycorrhizal fungi(AMF)are important members of the plant microbiome and affect the uptake and transfer of mineral elements by forming a symbiotic relationship with plant roots.Nitrogen(N),as an important min­eral element,can directly affect plant growth and development at different N levels.It has been confirmed that inoc­ulation with AMF can improve the efficiency of N utilization by plants.However,there are still fewer reports on the dynamic relationship between arbuscular mycorrhizal and plant secondary metabolites at different nitrogen levels.In this experiment,the physiological indexes and genes related to saponin synthesis were determined by applying differ­ent concentration gradients of nitrogen to the medicinal plant P.polyphylla var.yunnanensis infested by AMF as the test material.It was found that nitrogen addition increased the biomass,chlorophyll content,and nutrient content of above-and below-ground plant parts and increased the content of saponin content of P.polyphylla var.yunnanensis to some extent,but AMF inoculation increased the saponin content of P.polyphylla var.yunnanensis more significantly.AMF inoculation also promoted the expression of genes related to the saponin synthesis pathway,including 3-hy­droxy-3-methylglutaryl coenzyme A synthase(HMGS),squalene epoxidase 1(SE1),and cycloartenol synthase(CAS),which is in according with the accumulation of saponin in plants.It also may increase the saponin content of AMF plants by altering the expression of P450s and UGTs related to saponin synthesis.

Isolation of the Endophytic Fungi of Paris polyphylla var. yunnanensis and Their Effects on the Embryo Development of P. polyphylla var. yunnanensis Seeds

[Objective] This study aimed to isolate the endophytic fungi of Paris polyphylla var. yunnanensis and investigate their effects on the embryo development of P. polyphylla var. yunnanensis seeds. [Method] The endophytic fungi of P. polyphylla were isolated and identified morphologically, and their effects on the embryo development of P. polyphylla var. yunnanensis seeds were studied by using paraffin sectioning and microphotography. [Result] Nine endophytic fungi, i.e. P. polyphylla var. yunnanensis endophytic fungi PPYEF-1, PPYEF-2, PPYEF-3, PPYEF-4, PPYEF-5, PPYEF-6, PPYEF-7, PPYEF-8 and PPYEF-9 belonging to seven genera in five families, three orders were isolated from the rhizomes. Except PPYEF-4 (Cladosporium sp.), other fungi could promote the embryo development of the P. polyphylla var. yunnanensis seeds, mostly reaching the extremely significant or significant level. PPYEF-9 (Trichoderma sp.) resulted in the highest embryo length and embryo-emerging ratio. [Conclusion] This paper could provide a reference for the application of the endophytic fungi of P. polyphylla var. yunnanensis in the dormancy-breaking of P. polyphylla var. yunnanensis seeds.

A novel phenylpropanoid glycosides and a new derivation of phenolic glycoside from Paris Polyphylla var. yunnanensis

A novel phenylpropanoid glycosides 1, named parispolyside E and a novel derivation of phenolic glycoside 2, named parispolyside G, as well as two known flavonoid glycosides were isolated from the rhizome of Paris polyphylla var. yunnanensis. Their structures were elucidaed by spectroscopic methods.

滇重楼(Paris polyphylla var. yunnanensis)中产皂苷内生真菌的分离及筛选

药用植物内生真菌在植物生长发育、抵御不良环境甚至繁育生殖过程中都扮演着重要的角色,尤其在宿主的次生代谢产生中具有十分重要的作用。内生真菌的代谢产物具有多种生理活性,部分内生真菌发酵液还可产生与宿主植物中相同的活性成分。鉴于此,本研究以传统药用植物滇重楼(P.polyphylla var.yunnanensis)为实验材料,通过组织分离法及分子测序鉴定法,共鉴定出18种内生真菌,并对这些真菌进行了物理发泡及薄层层析色谱法(TLC)实验,目的是筛选出能够产重楼皂苷的内生真菌。通过对菌丝菌液皂苷的提取,最终获得产皂苷类物质的菌株数共有3株,且皂苷类物质主要存在于内生真菌的发酵液中。

Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var.yunnanensis as plants age

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and-independent(metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes(four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments(90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum(46.55% frequency of occurrence) was the predominant endophyte,followed by Leptodontidium sp.(8.66%) and Trichoderma viride(6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes(33.33%) when compared with four-year-old(21.21%) and six-year-old(15.15%) rhizomes. Certain fungal species were present only at particular ages. For example,Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of(metagenomics) community DNA extracted from different-aged samples revealed that, at the class level,the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.

Mycorrhizas Affect Polyphyllin Accumulation of Paris polyphylla var.yunnanensis through Promoting PpSE Expression

Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.

RNA-seq analysis of Paris polyphylla var.yunnanensis roots identified candidate genes for saponin synthesis

Paris polyphylla Smith var.yunnanensis(Franch.) Hand.-Mazz.is a rhizomatous,herbaceous,perennial plant that has been used for more than a thousand years in traditional Chinese medicine.It is facing extinction due to overharvesting.Steroids are the major therapeutic components in Paris roots,the commercial value of which increases with age.To date,no genomic data on the species have been available.In this study,transcriptome analysis of an 8-year-old root and a 4-year-old root provided insight into the metabolic pathways that generate the steroids.Using Illumina sequencing technology,we generated a high-quality sequence and demonstrated de novo assembly and annotation of genes in the absence of prior genome information.Approximately 87,577 unique sequences,with an average length of 614 bases,were obtained from the root cells.Using bioinformatics methods,we annotated approximately 65.51% of the unique sequences by conducting a similarity search with known genes in the National Center for Biotechnology Information's non-redundant database.The unique transcripts were functionally classified using the Gene Ontology hierarchy and the Kyoto Encyclopedia of Genes and Genomes database.Of 3082 genes that were identified as significantly differentially expressed between roots of different ages,1518(49.25%) were upregulated and 1564(50.75%) were downregulated in the older root.Metabolic pathway analysis predicted that 25 unigenes were responsible for the biosynthesis of the saponins steroids.These data represent a valuable resource for future genomic studies on this endangered species and will be valuable for efforts to genetically engineer P.polyphylla and facilitate saponin-rich plant development.

Mycorrhizal Fungal Effects on Growth,Antioxidant Capacity,and Medicine Quality of Paris polyphylla var.yunnanensis

A field experiment was conducted to determine the effects of two commercial strains composed of mulple arbuscular mycorrhizal fungi(AMF)species on plant growth,antioxidant capacity,and medicine quality of Paris polyphylla var.yunnanensis in three subtropical soils from Wanzhou,Anshun and Baoshan in fields.The results showed that AMF inoculation enhanced the fungal colonization rate and activities of both succinate dehydrogenase and alkaline phosphatase,thereby,enhancing the mycorrhizal viability of P.polyphylla var.yunnanensis.The concentrations of photosynthetic pigments(chlorophyll a,b,a+b and carotenoids),soluble sugar,soluble protein and photosynthetic capacity were higher in AMF-inoculated plants than in non-AMF-treated plants in field.AMFtreated plants recorded higher activities of catalase,peroxidase and superoxide dismutase,and caused the reduction in malondialdehyde content,indicating lower oxidative damage,compared with non-AMF plants.Polyphyllin I,Polyphyllin II,Polyphyllin III,Polyphyllin IV and total polyphyllin contents were increased by AMF treatment.In conclusion,AMF improved the plant growth,antioxidant capacity and medicinal quality of P.polyphylla var.yunnanensis seedlings.Hereinto,AMF effects on the soil from Wanzhou was relatively greater than on other soils.

Determination of 10 Kinds of Inorganic Elements in Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(and Polygerm Varieties)

[Objectives] To determine the content of 10 kinds of inorganic elements(Pb,Cu,As,Cd,Hg,Zn,Fe,Mn,Sr and Cr) in samples of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( and polygerm varieties) produced in different areas. [Methods] The wet digestion and technique of inductively coupled plasma optical emission spectrometry( ICP-OES) were used. [Results] Under the experimental conditions,elements were not related to each other,and several kinds of elements could be measured at the same time. The content of Pb exceeding the standard only in sample S4 of P. polyphylla Smith var. yunnanensis( and polygerm varieties),Hg was not detected in all samples,and the rest heavy metals did not exceed the limit. [Conclusions] This study was expected to provide a certain reference for the development and utilization of P. polyphylla Smith var. yunnanensis( and polygerm varieties).

Investigation on the Resources of Paris Polyphylla var.yunnanensis

In this study,the resource distribution,morphological characteristics,artificial cultivation and market circulation of Paris Polyphylla var.yunnanensis and its polygerm varieties in Yunnan were investigated by referring to the research literature,field investigation and our planting experience.It is found that P.polyphylla var.yunnanensis is widely distributed in Yunnan Province,realizing large-scale artificial cultivation,while the wild resources of polygerm varieties are almost on the verge of extinction and are in urgent need of protection.

Identification of Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(Polygerm Varieties) Produced in Gaoligong Mountain Areas

[Objectives] To make original plant and microscopic identification and study the traits of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( polygerm varieties) produced in Gaoligong Mountain areas,and provide experimental data for their identification and application. [Methods] The original plant powder was used,and paraffin sections and physicochemical properties were identified. [Results]P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas showed obvious microscopic features and physicochemical properties. [Conclusions] This study was expected to provide certain reference for identification of original plant and medicine and formulation of quality standard for P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas,and provided basis for their further research and development.

Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in <i>Paris polyphylla</i>var. Yunnanensis

Paris polyphylla var. yunnanensis (Franch.), one of the best-known medicinal plants in China, has a dehiscent anther which physiologically work in pollination, however, the dehiscent anther always closes in response to darkness every day, and watering or raining every time. To explore this frequently closing and its unkown physiology, next-generation sequencing was performed, and the transcriptome was de novo assembled. RNA-sequencing was carried out in 15 samples including seven openning samples, four closed samples owing to darkness or watering, and tissue samples (leaf, petal, calyx, and stigma) were used for control. We obtained 72.75 GB data, assembled into 79,815 unigenes. Differentially expressed unigenes (DEGs) between opened and closed anther samples were 6231 and the DEGs between anther and control samples were 2831. Comparation between the two DEGs by KEGG enrichment showed that “plant hormone signal transduction” pathway is the most significant pathway for DEGs from closing anther vs. opening anther, and expression model of DEGs in the pathway might elicit change in germination and seed dormancy. Further examination of the action of the signal pathway on physiology showed “chromatin binding” function was prominent in “DNA binding” function of annotated DEGs between opened and closed anthers, of the 215 “chromatin binding” unigenes, 120 were involved in epigenetic silencing, and 50 of the epigenetic unigenes were directly related to germination or seed dormancy, strongly correlating anther closing to epigenetic modification and seed dormancy. These results were verified that at least three auxins involved in seed dormancy showed same expression patterns occurred in abnormal closing anther and seed embryo in Paris polyphylla var. yunnanensis. In conclusion, the information from transcriptome point out that frequent abnormal closing of dehiscent anthers possibly transfer the impact on seed dormancy, and epigenetic modification happened in closing may be the cause.

华重楼(Paris polyphylla Smith var. chinensis)无性繁殖试验研究

华重楼为我国一味珍贵的民族药,本文以华重楼根茎为材料开展其无性繁殖试验,研究植物生长调节剂6-BA不同浓度对其潜伏芽萌发、生长的影响。试验结果显示5mg/L的6-BA可促进华重楼切段的繁殖系数,提高其双芽率、多芽率及有根率。

Temporal transcriptomics reveal the molecular mechanism of dormancy and germination regulated by temperature in Paris polyphylla seed

The mature seed of Paris polyphylla var.chinensis(PPC)is morphophysiologically dormant and develops differently under warm and cold temperatures.To elucidate the molecular mechanisms underlying temperature-dependent regulation of PPC seed dormancy and germination,we investigated the dynamic changes in PPC seed transcript levels under warm and cold temperature stratifications(WS and CS,respectively)by time-resolved RNA sequencing,focusing on genes related to hormone metabolism and signaling and cell wall remodeling(CWRM)and encoding transcription factors/regulators(TFs/TRs).A total of 48765 and 47836 differentially expressed genes(DEGs)were associated with WS and CS,respectively.Of these,17581 and 16652 DEGs,respectively,unique to WS and CS,and 5386 were common to both temperature stratifications across nine analyzed growth stages.The expression of hormone metabolism and signaling,TF/TR,and CWRM genes were closely associated with temperature.More genes related to gibberellin(GA),cytokinin,auxin,and brassinosteroid biosynthetic were upregulated in WS as compared to CS seeds,while genes related to dormancy release and germination were downregulated in WS seeds.However,only GA and abscisic acid levels were altered in PPC seeds breaking morphophysiological dormancy(MPD).Overall,37 TF and five TR families were upregulated whereas 24 TF and 16 TR families were downregulated in WS as compared to CS seeds.Most CWRM families were highly expressed under WS as compared to CS,suggesting that they promote endosperm weakening and embryo growth of WS seeds and facilitate MPD release and germination.A coexpression analysis revealed positive correlations between TFs/TRs and DEGs involved in hormone metabolism and signaling and CWRM.These results provided a basis for investigating the interaction between the endosperm and underdeveloped embryo in the regulation of PPC seed germination and seedling emergence.

Two New Highly Oxygenated Spirostanol Saponins from Paris polyphylla var.stenophylla

Phytochemical investigation of the rhizomes of Paris polyphylla var.stenophylla led to the isolation of two new highly oxygenated spirostanol saponins,named paristenosides A(1)and B(2),together with seven known compounds.Their structures were established mainly on the base of NMR spectroscopic techniques and mass spectrometry,as well as chemical methods.In addition,the cytotoxicity of the two new saponins was tested.Graphical Abstract Two new highly oxygenated spirostanol saponins,paristenosides A(1)and B(2),were isolated from the rhizomes of Paris polyphylla var.stenophylla.Their structures were established mainly based on NMR spectroscopic techniques and mass spectrometry,as well as chemical methods.

Comprehensive Evaluation on Impacts of Diseases on Quality of Paris polyphylla var. chinensis Based on Ultraviolet and Infrared Spectra

[Objectives] The research aimed to evaluate impacts of diseases on quality of Paris polyphylla var. chinensis. [Methods]Ultraviolet spectrophotometry was used to determine and contrast content of total saponins in root,stem and leaf from healthy and diseased plants. Infrared characteristic absorption peaks of healthy and diseased plants were found by infrared fingerprint method for comparative analysis.[Results]Total saponins content in root sample J4 of healthy plant reached 4. 89% and was the highest,while it was 0. 13% in stem sample J11 of healthy plant and was the lowest. Total saponins content in root sample B1 of diseased plant was 1. 68% and was the highest,while it was 0. 1% in stem sample B7 of diseased plant and was the lowest. In healthy and diseased plants,total saponins content in root was significantly higher than that in stem and leaf. Content of total saponins in root from healthy plant was significantly higher than that from diseased plant,and there was little difference in stem and leaf of healthy and diseased plants. Root sample Jg of healthy plant had obvious C-O characteristic vibration of polysaccharides and glycosides and absorption peak of O-H bending vibration in steroidal saponin near 1 160. 02 cm-1,while diseased plant had no obvious absorption peak. Near 861. 97 and 768. 07 cm-1,root sample Jg of healthy plant had significant C-C stretching vibration in sugar ring and characteristic absorption peak of characteristic vibration of steroidal saponin,while root sample Bg of diseased plant had no obvious characteristic peak. [Conclusions] Diseases significantly affected composition and content of total saponins and steroidal saponins from P. polyphylla var. chinensis,further affecting quality of P. polyphylla var. chinensis.

Comparison on Morphology and Micromorphology of Paris polyphylla Sm. var. polyphylla with Different Year Numbers

[Objectives] The research aimed to identify Paris polyphylla with different growth year numbers and evaluate its resource quality.[Methods]Comparative research on P. polyphylla with different growth year numbers was conducted by using morphological identification and microscopic identification method. [Results] By contrasting original plant morphology,transverse sections of rhizome and fibrous root,powder,it displayed that microstructure of P. polyphylla had regularity difference in cell shape and size,duct quantity,trait and size,size and distribution of calcium oxalate cluster crystal. [Conclusions] The microstructure change of P. polyphylla was related to the number of its growth year,which could provide the reference for identifying the growth year number of P. polyphylla and evaluating its resource quality.

Parisaponin Ⅰ和Parisvietnaside A的NMR数据分析

从滇重楼中分离得到2个甾体皂苷,利用1H NMR、13C NMR、1H-1H COSY、HSQC、HMBC、1D和2D TOCSY等多种核磁共振方法鉴定其结构分别为(25R)-26-O-β-D-吡喃葡萄糖基-3β,22α,26-三羟基-呋甾-5-烯-3-O-α-L-吡喃鼠李糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖苷(1,Parisaponin I)和(25R)-3β,5α,6β-三羟基-△7-螺甾烯-3-O-β-D-吡喃葡萄糖基-(1→3)-[α-L-吡喃鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷(2,paris-vietnaside A).对化合物1和2的1H NMR和13C NMR信号分别进行了归属和详细分析,并纠正了文献中的核磁数据归属错误.化合物2为首次从滇重楼中分离得到.

Steroid and triterpenoid saponins from the rhizomes of Paris polyphylla var.stenophylla

Five new saponins,including three steroid saponins,paristenoids A-C(1-3),and two triterpenoid saponins,paristenoids D-E(4-5),along with four known ones(6-9)were isolated from the rhizomes of Paris polyphylla var.stenophylla.The structures of the isolated compounds were identified nainly by detailed spectroscopic analysis,including extensive 1D and 2D NMR,MS,as well as chemical methods.Compound 3 is a new cyclocholestanol-type steroidal saponin with a rare 6/6/6/5/5 fused-rings cholestanol skeleton,and this skeleton has been first found from the genus Paris.The cytotoxicities of the isolated compounds against three human three glioma cell lines(U87MG,U251MG and SHG44)were evaluated,and compound 7 displayed certain inhibitory effect with ICso values of 15.22±1.73,18.87±1.81 and 17.64±1.69μmol^(-1),respectively.

滇重楼(Paris polyphylla var.yunnanensis)的内生细菌的分离与鉴定

滇重楼(P. polyphylla var. yunnanensis)是中国传统药用植物,多年生草本植物。很多研究已经证明,植物内生菌在植物次生代谢过程中起着十分关键的作用。鉴于此,本研究利用栽培于云南大理的滇重楼为实验材料,对其全株组织(叶,茎,根状茎)内生细菌进行了分离、纯化,并通过测序方法对其种类进行鉴定。结果显示滇重楼叶中内生细菌含有8属11种,茎中含有2属5种,根茎中含有9属11种。滇重楼不同组织中的内生细菌种属分布差异大,叶与根状茎中的内生细菌种类只共有鞘氨醇杆菌属,其余都不共有,而茎与叶、根状茎中的内生细菌都不相同,表明滇重楼内生细菌在不同组织中所体现的多样性及不同内生菌在分布上的差异性。本研究结果为后续滇重楼内生菌的多样性开发利用提供了科学依据,为未来滇重楼内生细菌深度挖掘与利用提供研究基础。