Effects of drought on non-structural carbohydrates and C,N,and P stoichiometric characteristics of Pinus yunnanensis seedlings

To study non-structural carbohydrate character-istics and nutrient utilization strategies of Pinus yunnanen-sis under continuous drought conditions,2-year-old seed-lings were planted in pots with appropriate water,light and moderate and severe drought treatments[(80±5),(65±5),(50±5),and(35±5)%of field water-holding capacity].Non-structural carbohydrates,carbon(C),nitrogen(N),and phosphorus(P)concentrations were measured in each plant component.The results show that:(1)With increasing drought,non-structural carbohydrates gradually increased in leaves,stems,and coarse roots,while gradually decreased in fine roots;(2)C concentrations of all were relatively stable under different stress levels.Phosphorous utilization of each component increased under light and moderate drought conditions,while N and P utilization efficiency of each plant component decreased under severe drought.Growth was mainly restricted by N,first decreasing and then increasing with increased drought;(3)There was a correlation between the levels of non-structural carbohydrates and C,N,and P in each component.Changes in N concentration affected the interconversion between soluble sugar and starch,which play a regulatory role in the fluctuation of the concentration of non-structural carbohydrates;and,(4)Plasticity analysis showed that P.yunnanensis seedlings responded to drought mainly by altering starch concentration,the ratio of soluble sugar to starch in leaves and stems,and further by alter-ing N and P utilization efficiencies.Overall,these results suggest that the physiological activities of all organs of P.yunnanensis seedlings are restricted under drought and that trade-offs exist between different physiological indicators and organs.Our findings are helpful in understanding non-structural carbohydrate and nutrient adaptation mechanisms under drought in P.yunnanensis seedlings.

The Identification of Phenylalanine Ammonia-Lyase(PAL)Genes from Pinus yunnanensis and an Analysis of Enzyme Activity in vitro

Phenylalanine ammonia lyase(PAL)is the rate-limiting and pivotal enzyme of the general phenylpropanoid path-way,but few reports have been found on PAL genes in Pinus yunnanensis.In the present study,three PAL genes were cloned and identified from P.yunnanensis seedlings for thefirst time,namely,PyPAL-1,PyPAL-2,and PyPAL-3.Our results indicated that the open-reading frames of PyPAL genes were 2184,2157,and 2385 bp.Phylogenetic tree analysis revealed that PyPALs have high homology with other known PAL genes in other plants.In vitro enzymatic analysis showed that all three PyPAL recombinant proteins could catalyze the deamination of L-phenylalanine to form trans-cinnamic acid,but only PAL1 and PAL2 can catalyze the conversion of L-tyrosine toρ-coumaric acid.Three PyPAL genes were expressed in different tissues in 1-year-old P.yunnanensis,and such genes had different expression patterns.This study lays a foundation for further understanding of the biosynthesis of secondary metabolites in P.yunnanensis.

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.

On the Identity and Taxonomic Status of Coluber nuthalli Theobald, 1868, with Redescription of the Type Specimens of Coluber nuthalli and Elaphis yunnanensis Anderson, 1879(Reptilia, Squamata, Colubridae)

The taxaColuber nuthalli Theobald, 1868 andElaphis yunnanensis Anderson, 1879 are compared and re-described, based on the examination of their type material. The morphological similarities of these two taxa revealed them as conspeciifc.Elaphis yunnanensis Anderson is thus declared as a protected name （nomen conservandum） with priority overC. nuthalli Theobald （nomen oblitum）. A lectotype forElaphis yunnanensis is designated and described. The validity ofyunnanensis as subspecies name in combination withOrthriophis taeniurus （Cope） will be discussed. A key to the subspecies ofO. taeniurus is provided.

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.

New Macrocyclic Diamide from Rauvolfia Yunnanensis Tsiang

A new macrocyclic diamide, 22-membered macrocyclic diamide, named cyclodicaprylamide（2）, and five known compounds, bis（2-ethylhexyl） phthalate（1）, ethyl 3,4,5-trimethoxybenzoate（3）, ethyl 3,4,5-trimethoxycinnamate （4）, （＋）-syringaresinol（5）, loliolide（6）, were isolated from the roots of Rauvolfia yunnanensis Tsiang. Their structures were elucidated based on NMR, 2D NMR, and MS spectrum, respectively. They were obtained from it for the first time.

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.

Ypsiyunnosides A-E,Five New Cholestanol Glycosides from Ypsilandra yunnanensis

Phytochemicalinvestigation onthe whole plants of Ypsilandra yunnanensiswas carried out forthe firsttime andledtothe isolation of five new cholestanol glycosides,ypsiyunnosides A–E(1–5),and one known analogue.Their structures were determined mainly by detailed spectroscopic analysis,including extensive 1D and 2D NMR,MS and UV,as well as chemical methods.Among them,compound 1 possessed a rare 6/6/6/5/5 fused-rings cholestanol sketelon,which was identified as(23R,25R)-3β,16α,26-triol-16,23-cyclocholest-5,17(20)-dien-22-one.Their induced platelet aggregation activities and cytotoxicities were evaluated.Graphical Abstract Five new cholestanol glycosides,ypsiyunnosides A–E(1–5),were isolated from the whole plants of Ypsilandra yunnanensis.Compound 1 possessed a rare 6/6/6/5/5 fused-rings cholestanol sketelon.Their structures were elucidated by a combination of 1D and 2D NMR,MS and chemical analysis.

High regional genetic differentiation of an endangered relict plant Craigia yunnanensis and implications for its conservation

Of the genus Craigia,widespread in the Tertiary,only two relict species survived to modern times.One species is now possibly extinct and the other one,Craigia yunnanensis,is severely endangered.Extensive surveys have located six C.yunnanensis populations in Yunnan province,southwest China.Using fluorescent amplified fragment length polymorphism(AFLP),the genetic diversity and population structure of these populations were examined.It was found that genetic diversity of C.yunnanensis was moderate at the species level,but low at regional and population levels.Analysis of population structure showed significant genetic differentiation between Wenshan and Dehong regions,apparently representing two geographically isolated for long time refuges.There are also clear indications of isolation between populations,which,together with anthropogenically caused decline of population size,will lead to general loss of the species genetic variation with subsequent loss of adaptive potential.To conserve the genetic integrity of C.yunnanensis,we recommend that ex-situ conservation should include representative samples from every population of the two differentiated regions(e.g.Wenshan and Dehong).The crosses between individuals originated from different regions should be avoided because of a high risk of outbreeding depression.As all the extant populations of C.yunnanensis are in unprotected areas with strong anthropogenic impact,there is no alternative to reintroduction of C.yunnanensis into suitable protected locations.

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.

Novel indole and quinoline alkaloids from Melodinus yunnanensis

6/7-Seco rearranged spiro-indolone alkaloids,meloyunines A(1)and B(2)and a monoterpenoid quinoline alkaloid meloyunine C(3)together with its possible intermediate 14,15-dehydromelohenine B(4),and their precursorΔ14-vincamenine(5)were isolated from Melodinus yunnanensis.All structures were elucidated based on NMR,FTIR,UV,and MS spectroscopic data.The isolation of monoterpenoid indole,quinoline,and its immediate from the same plant chemically supported the biosynthesis of quinoline from indole.Compound 2 was cytotoxic against several human cancer cell lines.

Variation in seed and seedling traits and their relations to geo-climatic factors among populations in Yunnan Pine(Pinus yunnanensis)

Differences in seed germination and seedling growth among populations of Pinus yunnanensis Franch. were quantified to provide information for use in the regeneration, reforestation, and improvement of this species. Seed germination and seedling growth traits at the nursery stage were investigated at the population level. We evaluated relationships between pairs of measured traits as well as the association between measured traits and geo- climatic variables including longitude, latitude, elevation, temperature and precipitation. Seedling growth showed significant differences although a low magnitude of variation was found in the seed germination traits among the populations. Some populations with strong and stable growth （e.g., the Baoshan population） may be selected as superior seed sources for regeneration and reforestation. Germination percentage was negatively correlated with elevation, and positively with precipitation. In addition, germination index, vigor index and seedling height were positively correlated with precipitation. Seed germination index, cotyledon number and length, and seedling diameter were significantly and positively correlated with seed weight. Strong correlations between pairs of traits will be helpful in early evaluations for the selection of certain prominent traits.

Selective logging enhances ecosystem multifunctionality via increase of functional diversity in a Pinus yunnanensis forest in Southwest China

Background:The impacts of selective logging on ecosystem multifunctionality(EMF)remain largely unexplored.In this study,we analyzed the response of nine variables related to four ecosystem functions(i.e.nutrient cycling,soil carbon stocks,decomposition,and wood production)to five selective logging intensities in a Pinus yunnanensisdominated forest.We included a control group with no harvest to evaluate the potential shifts in EMF of the P.yunnanensis forests.We also assessed the relationship between above-and belowground biodiversity and EMF under these different selective logging intensities.Additionally,we evaluated the effects of biotic and abiotic factors on EMF using a structural equation modeling(SEM)approach.Results:Individual ecosystem functions(EFs)all had a significant positive correlation with selective logging intensity.Different EFs showed different patterns with the increase of selective logging intensity.We found that EMF tended to increase with logging intensity,and that EMF significantly improved when the stand was harvested at least twice.Both functional diversity and soil moisture had a significant positive correlation with EMF,but soil fungal operational taxonomic units(OTUs)had a significant negative correlation with EMF.Based on SEM,we found that selective logging improved EMF mainly by increasing functional diversity.Conclusion:Our study demonstrates that selective logging is a good management technique from an EMF perspective,and thus provide us with potential guidelines to improve forest management in P.yunnanensis forests in this region.The functional diversity is maximized through reasonable selective logging measures,so as to enhance EMF.

Effects of flavonoids derived from Taxus yunnanensis on p-glycoprotein and cytochrome P450 3A4

The intestinal uptake of paclitaxel is hampered by trans-membrane efflux transporters such as P-glycoprotein(P-gp),and paclitaxel is mainly metabolized by cytochrome P4503A4(CYP3A4)presented in the liver.Our previous results demonstrated that flavonoids extracted from Taxus yunnanensis could improve the oral absorption of paclitaxel.The current study was purposed to investigate the effects of the flavonoid extracts on P-gp and CYP3A4 in vitro.The expression and activity of P-gp were detected by western blotting and intracellular rhodamine 123 accumulation assay in Caco-2 cells treated with the flavonoids extract.The expression of CYP3A4 was investigated by western blotting in mouse primary hepatocytes and the activity of CYP3A4 was detected by LC-MS/MS method using rat liver microsomes.Our results showed that the flavonoid extracts from T.yunnanensis could inhibit P-gp activity and concurrently decrease the expression and activity of CYP3A4.In conclusion,activity of P-gp and CYP3A4 could be inhibited by flavonoids extracted from T.yunnanensis which might be potential candidates for development of oral formulation of paclitaxel.

A New Iridoid Glucoside from Lagotis yunnanensis

Phytochemical investigation of Lagotis yunnanensis led to the isolation and identification of a new iridoid glucoside 1, named as 10-O-(3,4-dimethoxy-(E)-cinnamoyl)aucubin. Its structure was elucidated by spectroscopie methods.

TWO NEW OLEANOLIC ACID SAPONINS FROM ASTER YUNNANENSIS

Two new oleanolic acid saponins named asteryunnanoside F and G were isolated from the roots. of Aster yunnenensis. The chemical structures were determined as oleanolic acid-28-O-βD-glucopyranosyl-(1→6)-β-D-glucO and 3-O-β-Dglucopyranosyl-oleanolic acid-28-O-βD-glucopyranosyl-(1→6)[-rhamn by speCtral data,especially 2D NMR anajysis (COSY, HETCOR, HOHAHA and ROESY), and Chemical transformations.

A NEW TRITERPENOID SAPONIN, ASTERYUNNANOSIDE H, FROM ASTER YUNNANENSIS

A new echinocystic acid saponin, asteryunnanoside H (1), has been isolated from the roots of Astsr yunnanensis (Compositae). The structure and the unambiguous assignments of the 1H- and 13C NMR data of 1 were obtained based on extensive use of 1D and 2D NMR techniques (COSY. TOCSY, ROESY, HMQC and HMBC), and chemical transformations.

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.

Alkaloids from Stems of Ervatamia yunnanensis （Ⅱ）

Six indole alkaloids were isolated from the stems of Ervatamia yunnanensis. Among them, 10-hydroxy-19, 20-dihydroisositsirikine （I） is new. The other five are known alkaloids, namely： matrine （Ⅱ）, 19, 20-dihydroisositsirikine （Ⅲ）, 19-s-voacangarine （Ⅳ）, 1 l＇-methoxyl-19s- heyneanine （Ⅴ）, conodurine （Ⅵ）. The structural elucidation of the alkaloids was based on spectral means.

ASTERYUNNANOSIDE A AND B，TWO NEW TRITERPENOID SAPONINS FROM ASTER YUNNANENSIS FRANCH

Two new oleanane-type triterpenoid saponins.sateryunn3noside A and B,were isolated from the roots of Aster yunnanensis Franch,and their Structures were elucidated based on spectral evidence and chemical hydrolysis. They have the same aglycone,Which has been assigned as 2a,3 .23-trihydroxyolean-12-en-28-cio acid,and the deference consisted only in 28-O-linked sugar moieties. Asteryunnanoside A is a 28-O-a-L-rhamnopyranosyl-(1-2)- -D-glucopyranoside and B is 28-O- -D-glucopyranosyl-(1 -2),-D-glucopyranoside of the corresponding aglycone.