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.

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.

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.

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.

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.

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.

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.

Technical Regulations for Under-forest Cultivation of Paris polyphylla Smith in Hunan Province

In this paper,the seedling raising,cultivation,field management,pest and disease control,harvesting and processing of Paris polyphylla Smith under the forest in Hunan Province were scientifically standardized,to provide technical support for standardized cultivation and sustainable development of Paris polyphylla Smith industry in Hunan Province.

Unraveling the serial glycosylation in the biosynthesis of steroidal saponins in the medicinal plant Paris polyphylla and their antifungal action

Sugar-sugar glycosyltransferases play important roles in constructing complex and bioactive saponins.Here,we characterized a series of UDP-glycosyltransferases responsible for biosynthesizing the branched sugar chain of bioactive steroidal saponins from a widely known medicinal plant Paris polyphylla var.yunnanensis.Among them,a 2'-O-rhamnosyltransferase and three 6'-O-glucosyltrasferases catalyzed a cascade of glycosylation to produce steroidal diglycosides and triglycosides,respectively.These UDP-glycosyltransferases showed astonishing substrate promiscuity,resulting in the generation of a panel of 24 terpenoid glycosides including 15 previously undescribed compounds.A mutant library containing 44 variants was constructed based on the identification of critical residues by molecular docking simulations and protein model alignments,and a mutant UGT91AH1^(Y187A)with increased catalytic efficiency was obtained.The steroidal saponins exhibited remarkable antifungal activity against four widespread strains of human pathogenic fungi attributed to ergosterol-dependent damage of fungal cell membranes,and 2'-O-rhamnosylation appeared to correlate with strong antifungal effects.The findings elucidated the biosynthetic machinery for their production of steroidal saponins and revealed their potential as new antifungal agents.

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.

四川地区华重楼(Paris polyphylla var.chinensis)主要病害调查及病原菌鉴定

华重楼是重要的中药材,近年来,华重楼病害已成为制约四川华重楼产量和品质提高的重要因素之一。本研究对四川种植区华重楼病害进行了调查,共调查到主要危害华重楼病害有4种。通过组织分离法对相应的病原菌进行分离,并对病原菌进行致病性测定、形态学观察和分子生物学鉴定。试验结果显示,结合病原菌形态特征及r DNA-ITS、HSP60、G3PDH、RPB2、TEF、ACT、LSU、Alt琢1、TUB基因序列分析,并构建多基因分子系统发育树,鉴定出引起华重楼4种真菌病害病原菌,分别为华重楼叶枯病(Epicoccum sorghinu)、褐斑病(Alternaria tenuissima)、灰霉病(Botrytis cinerea)和黑斑病(Alternaria alternata)。目前,危害华重楼的最为严重的病害是灰霉病、褐斑病和叶枯病。华重楼主要病害的调查及病原菌种类的鉴定为华重楼病害的防治提供理论依据。

Extract of Paris polyphylla Simth Protects Cardiomyocytes from Anoxia-Reoxia Injury through Inhibition of Calcium Overload

Objective：To assess any direct effect of extract of Paris polyphylla Simth（EPPS）,a Chinese plant,on a cardiomyocyte subject to ischemia-reperfusion injury and to further elucidate its protective effect against myocardium ischemia on the cellular level.Methods：Neonatal rat cardiomyocytes were isolated and subjected to an anoxia-reoxia injury simulating the ischemia-reperfusion injury in vivo in the presence or absence of EPPS or diltizem,a positive control.The lactate dehydrogenase（LDH） activities in culture supematants and cell viabilities were analyzed using the enzymatic reaction kinetics monitoring-method and MTT method, respectively.Free intracellular calcium concentrations and activities of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase in cells were also measured with laser confocal microscopy and the inorganic phosphorus-transformation method,respectively.Results：In cardiomyocytes subject to anoxia-reoxia injury,EPPS at 50-400 mg/L showed a concentration-dependent inhibition on LDH leakage and maintenance of cell viability,and the effect was significant at 275 and 400 mg/L（both P0.01）.In addition,EPPS at 275 and 400 mg/L significantly inhibited the increase in intracellular free calcium（both P0.01） as well as decreased the activities of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase（P0.01,P0.05）.Conclusions：EPPS prevents anoxia-reoxia injury in neonatal rat cardiomyocytes in vitro by preservation of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase activities and inhibition of calcium overload.The direct protective effect on cardiomyocytes may be one of the key mechanisms that underlie the potential therapeutic benefit of EPPS against myocardium ischemia.

不同处理方法对重楼种子萌发的Meta分析

应用Meta分析方法,通过中英文数据库筛选了1990—2019年关于重楼(Paris polyphylla)种子萌发的文献24篇,得到78个独立试验组,分析温度、机械、生长素、复合处理对重楼种子萌发的影响,并探讨较高海拔下重楼种子繁育的可能性。结果表明,恒温处理促进重楼种子萌发是一种简单可靠的方法,恒温、变温、沙藏方式的交互处理能够有效提高重楼种子发芽率(相对发芽率为0.61)。生长素使用不当会抑制重楼种子萌发。在相同处理条件下,滇重楼[Paris polyphylla Sm.var.yunnanensis(Franch.)Hand.-Mazz]比华重楼[Paris polyphylla Sm.var.chinese(Franch.)H.Hara]具有更好的发芽效率和稳定性。针对适宜重楼生长的较高海拔生境下种子发芽相关研究缺乏,需要针对较高海拔重楼繁育技术进行研究。

滇重楼辣椒轻斑驳病毒分离物的鉴定及全基因组序列分析

滇重楼(Paris polyphylla var.yunnanensis)是多年生草本植物,其药用部位为地下根茎,是一种重要的中药材。为明确侵染滇重楼的病毒病原,对采自云南省曲靖市的滇重楼进行透射电镜观察、DASELISA和RT-PCR技术检测。在透射电镜下观察到杆状病毒粒子,大小为(200~300)nm×(20~36)nm。对采集到的7份病样进行DAS-ELISA检测,结果表明,其中5份病样呈阳性。通过RT-PCR技术扩增、克隆获得1个全长为6357 bp的曲靖分离物PMMoV-QJ的全基因组序列。将PMMoV-QJ的全基因组序列与国内外已经报道的10个PMMoV分离物进行同源性分析比对,其同源性为99.42%~99.81%。基于全基因序列的系统发育分析表明,PMMoV-QJ与韩国分离物亲缘关系密切。本研究明确了该分离物的亲缘关系,从分子水平鉴定该分离物,为后续滇重楼病毒病的预防提供理论依据。

Untiring Researches for Alternative Resources of Rhizoma Paridis

Rhizoma Paridis(RP,M®),a traditional Chinese medicine,is the rhizoma of Paris polyphylla var.yunnanensis(PPY)or P.polyphylla var.chinensis which are widely used as important raw materials for several Chinese patent drugs.However,the wild resources of these herbs have become less and less due to their slow-growing characteristics and previously excessive excavation.This review covers untiring investigations on alternative resources of RP by our research group over the past decades,including non-medicinal parts of PPY as well as other plants of Liliaceae and Liliflorae families.The arial parts of PPY and the whole plants of Trillium kamtschaticum might be alternative resources for RP based on the fact that they shared the same or similar saponins and bioactivities.

滇重楼新病毒ParV1的分子变异分析及致病性测定

为明确云南省引起滇重楼(Paris polyphylla var.yunnanensis)病毒病的病原,以昆明市发现的花叶、斑驳症状的滇重楼叶片为样品,进行透射电镜观察、高通量测序和反转录(RT-PCR)检测。结果表明,观察到大小约(700~1000)nm×13 nm的线状病毒粒子;基于高通量测序结果设计特异性引物进行RTPCR扩增并克隆,得到1条全长为10066个核苷酸(nt)的病毒序列,将该病毒暂命名为Paris virus 1(ParV1);BLAST比对分析表明,ParV1与马铃薯Y病毒属(Potyvirus)中的浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)具有最高为65.1%、68.9%的核苷酸及氨基酸(aa)序列同源性;多序列比对分析显示,ParV1多聚蛋白与potyviruses具有49.6%~65.1%的nt序列同源性及47.1%~68.9%的aa序列同源性;基于多聚蛋白氨基酸序列构建的系统发育树表明,该病毒与TFMV亲缘关系较近且聚为一个单独的分支。综上所述,确定ParV1属于Potyvirus的一种新病毒。将含ParV1的病毒汁液以机械摩擦的方法接种到心叶烟、本氏烟上对ParV1进行致病性初步测定。接种后的心叶烟植株叶片表现出明显病毒病症状,经RT-PCR及分子克隆在接种的心叶烟中检测到ParV1的存在,表明心叶烟植株受到ParV1的侵染,而接种的本氏烟上暂未检测到该病毒的存在。

Changes in Active Components before and after Microorganism Fermentation of Rhizoma Paridis(Chonglou)

[Objectives] To study the changes in active components before and after microorganism fermentation of Rhizoma Paridis( Chonglou). [Methods]Analytical methods using total saponins and total flavonoids as indicators were established. UV spectrophotometry was used to determine the changes in active components before and after fermentation of Rhizoma Paridis. [Results] The content of total saponins decreased after fermentation,while the content of total flavonoids increased. [Conclusions]The content of total saponins decreased after fermentation and the content of total flavonoids increased. The fermentation process of Monascus purpureus Went. needs further optimization.