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.

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.

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.

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.

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).

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.

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.

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.

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。

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。

云南重楼种子育苗技术研究

目的：缩短云南重楼种子萌发和出苗所需时间，提高其出苗率；提出在生产中切实可行的云南重楼种子有性繁殖育苗方法。方法：考察不同温度、不同播种方式（播种时间、覆土厚度、地膜覆盖）等因素对云南重楼种子萌发和出苗的影响。结果：云南重楼种子胚后熟和萌发的最适温度在18～20℃，在此温度下处理3～4个月以后，转至0～10℃低温处理2～4个月，可在翌年4月出苗；适宜的播种时间是4月份以前，育苗土覆盖厚度为1cm左右，播种后用黑色地膜进行覆盖，可提高出苗率。结论：上述试验结果可为云南重楼种子育苗提供操作参考。

超声波对滇重楼种子萌发能力的影响

研究超声波处理时间、温度、功率对滇重楼(Paris polyphylla var.yunnanensis)种子萌发的影响,寻找其萌发的最佳条件。通过室内常规发芽试验,统计滇重楼种子在不同超声波处理时间、温度、功率条件下的发芽率和发芽指数。结果表明,滇重楼种子经适当的超声波处理,缩短了萌发时间,提高了发芽率和发芽指数。40 min、50℃、200 W的超声波处理条件最适宜于滇重楼种子萌发。

滇重楼的化学成分研究

目的研究滇重楼Paris polyphylla var.yunnanensis根茎中的化学成分。方法利用正相硅胶柱色谱、葡聚糖凝胶Sephadex LH-20、反相制备色谱等手段进行分离纯化,并通过1H-NMR、13C-NMR、ESI-MS等波谱技术进行结构鉴定。结果从醋酸乙酯层中分离得到了7个化合物,分别鉴定为豆甾醇-3-O-β-D-葡萄糖苷(Ⅰ)、1-O-(β-D-葡萄糖基)-(2S,3S,4E,8E)-2-[(2′R)-2′-羟基十六酰氨基]-4(E),8(E)-十八二烯-1,3-二醇(Ⅱ)、β-蜕皮激素(Ⅲ)、薯蓣皂苷元-3-O-α-L-呋喃阿拉伯糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅳ)、薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅴ)、薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅵ)和偏诺皂苷元-3-O-α-L-呋喃阿拉伯糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅶ)。结论化合物Ⅱ为首次从该属植物中分离得到。

滇重楼的psbA-trnH条形码分子鉴定研究

为探讨滇重楼药材鉴定新方法,本研究通过对重楼样品提取基因组DNA,PCR扩增叶绿体psb A-trn H序列并进行双向测序,所得序列经Codon Code Aligner拼接后,采用MEGA5.0软件进行序列比对,计算种内和种间遗传距离(K2P),构建邻接数(neighbor-joining tree)进行结果分析。研究表明,叶绿体psb A-trn H片段在滇重楼种内变异较小,平均K2P遗传距离为0.007,而滇重楼与其它重楼种间平均K2P遗传距离为0.025,滇重楼种间最大K2P遗传距离明显小于滇重楼与重楼属其它种内的最小K2P遗传距离。中药材DNA条形码鉴定系统比对和NJ树鉴定结果均表明psb A-trn H序列可区分滇重楼及其同属物种,且具有较好的稳定性和准确性,为保障临床安全用药提供了新的技术手段。

氮、磷、钾对滇重楼产量及皂苷含量的影响

滇重楼是名贵道地中药材,过度采挖已使其野生资源濒临灭绝,人工栽培是解决滇重楼资源危机的必然途径。合理施用氮、磷、钾肥可提高其药用部位产量和品质。本文通过"3414"田间小区肥料效应试验,设N:23 kg/667 m^2、P2O5:12 kg/667 m^2、K2O:30 kg/667 m^2为常规施肥水平,研究不同水平氮、磷、钾对滇重楼产量及皂苷含量的影响。结果表明:土壤氮、磷、钾的贡献率分别为77.97%,64.93%和136.52%,土壤供钾量丰富,供氮量中等,供磷量不足;增施氮肥和磷肥,不施钾肥可提高滇重楼的产量。由滇重楼根茎增产率的单因素、双因素和三因素肥料效应函数方程可知:单施氮、磷、钾肥对滇重楼根茎增产率影响较小,氮、磷、钾配施对滇重楼根茎增产率影响最大;三因素肥料效应方程推荐的最佳施肥量(N:34.5 kg/667 m^2,P2O5:18 kg/667 m^2,K2O:0 kg/667 m^2)所得滇重楼根茎增长率最高,为87%;适量的氮肥和磷肥可提高滇重楼新、老根茎的皂苷含量,增施钾肥会使滇重楼新根茎皂苷含量降低。当氮肥和磷肥施用量分别为23 kg/667 m^2和18 kg/667 m^2时,滇重楼新、老根茎总皂苷含量最高;钾肥施用量为0 kg/667 m^2时,滇重楼新根茎总皂苷含量最高。

滇重楼寄生菌的研究

从滇重楼（Ｐａｒｉｓｐｏｌｙｐｈｙｌａｖａｒ．ｙｕｎｎａｎｅｎｓｉｓ）地下茎中分离和鉴定出两种细菌———蜡状芽孢杆菌（Ｂａｃｉｌｕｓｃｅｒｅｕｓ）和产碱假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓａｌｃａｌｉｇｅｎｅｓ），以及三种真菌———黑团孢霉（Ｐｅｒｉｃｏｎｉａｓｐ．）、白色厚顶孢霉（Ｐａｃｈｎｏｃｙｂｅａｌｂｉｄａ）和重楼索霉（Ｈｏｒｍｏｍｙｃｅｓｐａｒｉｄｉｐｈｉｌｕｓ）。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量，结果表明重楼索霉可分泌大量胞外多糖，这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。

云南重楼种子萌发过程内源激素含量及酶活性变化研究

为探讨云南重楼种子萌发不同阶段的酶和内源激素的变化,本研究采用生理生化方法测定云南重楼种子萌发不同阶段超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、苹果酸脱氧酶(MDH)和6-磷酸葡萄糖脱氢酶(G-6-PDH)活性,以及吲哚乙酸(IAA)、赤霉素(GA_3)、脱落酸(ABA)、水杨酸(SA)、反玉米素(TZ)、茉莉酸(JA)6种内源激素的含量,并分析比较其差异。结果表明,云南重楼种子在即将出苗阶段的SOD、POD活性迅速升高,CAT、MDH和G-6-PDH活性降低;IAA、GA_3含量均在即将出苗阶段显著高于其他阶段,种子休眠解除后ABA含量迅速下降。胚根刚突破种皮阶段,其SA、TZ、JA含量均最高,SOD、POD、IAA、GA_3间呈极显著正相关(P<0.01),MDH与G-6-PDH呈极显著正相关(P<0.01),ABA与GA_3呈显著负相关(P<0.05),SA、TZ、JA间呈极显著正相关(P<0.01),IAA/ABA、GA_3/ABA、SA/ABA在种子休眠解除和出苗阶段均呈上升趋势。综上,SOD、POD、GA_3是种子出苗阶段的关键物质,TZ、SA和JA对种子休眠解除具有重要的影响,IAA/ABA、GA_3/ABA及SA/ABA增大对种子休眠解除和出苗均有促进作用。本研究结果为云南重楼种子繁殖提供了理论依据和实践指导。