Ovule Development, Formation of Pollination Drop and Pollination Process in Taxus chinensis (Taxaceae)

Pollen morphology and hydration, ovule development, formation of pollination drop and the pollination process of Taxus chinensis (Pilg.) Rehd were investigated in detail. The results showed that mature pollen was one_celled, irregular in shape, nonsaccate, and the surface was covered with orbicules. When pollen became hydrated, the intine swelled and the exine bursted. The exine usually remained on the surface of water or on the pollination drop all the time, while the rest portion of the pollen sank into the drop of water or the pollination drop. In the late August, the down_curved female buds could be observed in the field trees. The downturning of the female buds was an characteristic feature discriminating from the vegetative buds. At this stage ovular primordium was present in the female bud with a dome_shaped nucellar primordium encircled by a rudimentary integument. The integument grew fast and overgrew the nucellus. In the March of the following year, the ovule formed a micropylar canal about 1?000 μm in length, 200-300 μm in width. Except the micropyle, the other part of the ovule was enclosed by 20-25 scales. During the early stage of the ovule development, the nucellar surface clearly showed cell configuration; but a membrane_like structure covering the nucellar apex appeared in the late stage of the ovule development, and subsequently broke down with nucellar cell disintegration before pollen shedding. The disintegrated cell products might partake in the formation of the pollination drop. Before and during pollination, the nucellar cells contained abundant mitochondria, endospermic reticulum and dictysomes with vesicles; the plasma membrane was undulated and there was no accumulation of electron_dense substances in the pericytoplasm. Pollination drops usually occurred at night, and disappeared by noon in the field tree. 15 days before pollination, the in vitro ovules from the field trees already had the capability of producing pollination drops, which could last for 4-5 days indoors. However, after these drops received pollen, they did not withdraw noticeably compared with the unpollinated ones. The retraction of the pollination drop was presumed not to be a nonmetabolic outcome but rather a result of evaporation.

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinensis

Taxol production of Taxus chinensis (Pilger) Rehd. var. mairei (Lemee et Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f. vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide_treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L _phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.

Chemical Constituents of One Species of Endophytic Fungus in Taxus chinensis

Three compounds were obtained from the mycelia of an endophytic fungus Gliocladium sp. (designated as strain F) of Taxus chinensis (Pilg.) Rehd. growing in Fujian Province, China. Their structures were determined on the basis of spectral analysis. (20S,22S)-4a-homo-22-hydroxy-4-oxaergosta-7,24(28)-dien-3-one was a novel compound. 4,8,12,16-tetramethyl-1,5,9,13-tetraoxacyclohexadecane-2,6,10,14-tetraone was firstly isolated from the genus ofGliocladium and 6,9-epoxyergosta-7,22-dien-3-ol was firstly obtained from the strain F.

Taxol Production in Suspension Cultures of Taxus chinensis as Affected by the Combination of Nutrient Feed with Dissolved Oxygen Control

Influence of the combination of nutrient feed with dissolved oxygen control on taxol production in suspension cultures of Taxus chinensis (Pilg.) Rehd. was investigated in bioreactors. Addition of feeding medium with 20 g/L sucrose on day 16 enhanced both the biomass and taxol production in 5-L bioreactors. Further investigation of the fed-batch cultures in a 20-L bioreactor showed that cultivation under a low dissolved oxygen tension (DOT) of 20% for the entire culture resulted in the highest taxol content of 0.98 mg/g DW, while the taxol production was lower than that with 40% and 60% DOT. Moreover, taxol accumulation was remarkably improved by the cultivation of cells initially under DOT of 60% for 20 d followed by changing the DOT to 20% for another 12 d. By combining the use of these two strategies, a maximum taxol content of 18.7 mg/L was obtained in a 20-L bioreactor.

Optimization of Cell Line Building for Taxus chinensis var. mairei

[Objective] The paper aims to optimize the abduction and domestication conditions of Taxus chinensis var.Mairei callus.[Method] We compared the efficiencies of callus between different explants and investigated the multiplication conditions of callus and suspended cell culture conditions with the buds,young stems and young leaves from T.chinensis var as the explants.[Results] The effect was the best with the bud as the explants; the best way for sterilizing the explants of T.chinensis var mairei was：streptomycin detergent for 2 h ＋ suds for 3 h ＋ 75% alcohol disinfection for 30 s ＋ 10% sodium hypochlorite solution for 25 min ＋ 1‰ mercury chloride for 10 min; the optimum formula of callus subculture was：B5 ＋ 4.0 mg/L NAA ＋ 0.5 mg/L 2,4-D ＋ 0.2 mg/L GA ＋ 0.5 mg/L 6-BA ＋ 2 g/L AC.[Conclusion] This research built the high efficient regeneration system of T.chinensis var.

Effects of Piriformospora indica on the Respiration of Taxus chinensis var.mairei under Water Stress

Seedlings of Taxus chinensis var.mairei were used as experimental materials to study the adaptation of Piriformospora indica to this plant under water stress.The materials were divided into two groups,namely,with or without inoculation with P.indica.Each group was subjected to four different levels of water stress.Vitality and physiological and biochemical indexes of the roots of T.chinensis var.mairei were regularly measured.Under water stress,T.chinensis var.mairei had significantly decreased root vitality;root vitality was higher in inoculated roots than in uninoculated roots.Under intense water stress,the inoculated roots had a higher soluble sugar content than the uninoculated roots.Under water stress,T.chinensis var.mairei experienced decreased activity of aerobic respiratory metabolic enzymes.The activity of anaerobic respiratory metabolic enzymes and alcohol dehydrogenase initially increased and then decreased,whereas that of lactate dehydrogenase increased.The inoculated roots had a higher activity of respiratory metabolic enzymes than the uninoculated roots.As water stress was further intensified,the roots had significantly decreased activity of aerobic respiratory metabolic enzymes and significantly increased activity of anaerobic respiratory metabolic enzymes.The activity of respiratory metabolic enzymes decreased faster in the uninoculated roots than in the inoculated roots.This study demonstrated that Piriformospora indica plays a positive role in enhancing the antihypoxic ability of T.chinensis var.mairei,thereby alleviating plant damage due to water stress.

Improved Production of Paclitaxel from Suspension Culture of Taxus chinensis var. mairei by in situ Extraction with Organic Solvents

The production of paclitaxel from suspension culture of Taxus chinensis var,mairei was improved by in situ extraction with organic solvents to avoid feedback repression and product degradation.Oleic acid and dibutyl phthalate were proved to be suitable solvents .The optimal volumetric percentage of organic solvents in the culture medium was found to be around 8%,and the favorable time for their introduction was at the exponential phase of cell growth,Paclitaxel production with the in situ extraction was ca 3-fold of that without extraction.

Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+ of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

A New Taxoid from Leaves and Branches of Taxus chinensis

A new taxoid, 2-deacetyl-2a, 14b-dihydroxybaccatin IV (1), was isolated from the leaves and branches of Taxus chinensis together with the known compound baccatin IV (2). The structure of the new compound was elucidated by spectroscopic techniques. The detailed 13C NMR assignments of baccatin IV are reported for the first time.

NEW TAXANES FROM TAXUS CHINENSIS.2

Tow new taxane diterpenes were isolated from the leaves and stems of Taxus chinensis.Thcir structures were established as 2-deacctyl-5-decinnamatetaxagifine and taxagifinc Ⅲ on the basis of spectroscopic analysis.

Comparison of Morphological and Anatomical Characteristics of Taxus chinensis var.mairei Seedlings Root under Waterlogging Stress in Different Substrates

Four different ratios of river sand,ceramic pellets,vermiculite and perlite(1:1),and field soil were selected as the substrates in this experiment,and four gradient levels of root waterlogging,half waterlogging,full waterlogging and normal were set to investigate the effects of different gradients of waterlogging stress on the root morphology of Taxus chinensis var.mairei seedlings under different substrates.In this study,the root anatomical structure of Taxus chinensis var.mairei under different waterlogging stress was observed by the paraffin section method.The roots of T.chinensis var.mairei were diarch,with no pith and resin canals.There was a large number of tannins in the pericycle of the aerial adventitious roots of seedlings adapted to waterlogging.Also,the endodermis has obvious casparian strip thickening,and there were 4-5 layers of large parenchymatous cells in the close to the inner side of the pericycle in the vascular cylinder,which could increase the storage capacity,and transport capacity of the root.Under the treatment of root waterlogging stress,the development of plant roots in the mixed substrate of vermiculite and,perlite was the earliest.Under half waterlogging stress,T.chinensis var.mairei seedlings treated with various substrates all could better adapt to the environment of waterlogging stress.Under the stress of fully waterlogging,the roots of seedlings planted in river sand substrate developed secondary growth.

Taxus chinensis ameliorates diabetic nephropathy through down-regulating TGF-β1/Smad pathway

Diabetic nephropathy(DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin(30 mg·kg^(-1) body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L^(-1) were selected for experiments. The DN rats were treated with Taxus chinensis orally(0.32, 0.64, and 1.28 g·kg^(-1)) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-β1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-β1/Smad signaling pathway.

Microsatellite loci from Taxus chinensis var. mairei (Taxaceae), an endangered and economically important tree species in China

Taxus chinensis var.mairei is an endangered and economically important tree species in China.Four polymorphic microsatellite markers were developed and characterized using the fast isolation by amplified fragment length polymorphism(AFLP)of sequences containing repeats(FIASCO)protocol.Polymorphism of each locus was quantified for five natural populations from Hubei Province,China.The number of alleles per locus varied from 6 to 24.The ranges of observed and expected heterozygosity were 0.493–1.000 and 0.440–0.845,respectively.These are the first microsatellites reported for the family of Taxaceae and will be helpful for the ongoing population and conservation genetics research of Taxus chinensis var.mairei.

Neural networks modeling signal responses and taxol production of cultured Taxus chinensis cells induced by bio-elicitor

Quantitatively describing the signal transduction process is important for understanding the mechanism of signal regulation in cells,and thus,poses both a challenge and an opportunity for chemical and biochemical engineers.An artificial neural network(ANN),in which we took the signal molecules as neural nodes,was constructed to simulate the generation of active oxygen species(AOS)in Taxus chinensis cells induced by a bio-elicitor.The relative contents of AOS in cells predicted by the ANN model agreed well with the experimental data and three notable stages of AOS increase were observed from the 3D figure of AOS generation.The robustness of AOS trajectories indicated that signal regula-tion in vivo was an integral feedback control model that ensured the adaptation of Taxus chinensis to environmental stress.The artificial neural network was able to predict taxol production as well as determine the optimal concentration of oligosaccharides needed for it.

Bulbuls and crows provide complementary seed dispersal for China's endangered trees

Background: Different functional frugivores generally exhibit unequal contributions,both in terms of quantity(seed removal) and quality(seedling recruitment),to effective seed dispersal of plant species.However,variations in this dispersal pattern generated by frugivores across different regions are still unknown.Methods: In our study,we evaluated the contributions of two functional frugivore bird groups,the bulbuls(Pycnon?otidae) and crows(Corvidae),in both the seed removal and seedling recruitment of the endangered Chinese yew tree(Taxus chinensis) across three different geographical regions in eastern China.Results: The dominant crow species,Urocissa erythrorhyncha,was the most common disperser crow species at all sites,while the dominant bulbul species varied across the three sites.Furthermore,the two functional groups of dispersers diverged in the aspects of seed removal(quantity) and seedling recruitment pattern(quality).While bulbuls outperformed crows in seed removal(quantity),crows took more seeds to a safer site for seedlings(quality).Conclusions: Our results highlight the importance of variations in the effective seed dispersal patterns of different functional disperser groups across different regions in the conservation and management of endangered tree species.

Importance of microhabitat selection by birds for the early recruitment of endangered trees in a fragmented forest

Background:Remnant microhabitats are important for bird habitat selection and plant regeneration in the fragmented habitat.However,empirical information on the consequences of how microhabitat use by birds affects the early recruitment of plants is lacking.Methods:In this study,we evaluated whether microhabitat selection by the Black Bulbul(Hypsipetes leucocephalus)(J.F.Gmelin,1789)impacts the early recruitment of the endangered tree species,the Chinese Yew(Taxus chinensis(Pilger)Rehd),in a fragmented forest over a 4-year period(2011–2012,2018–2019).Results:Our results showed the main factors affecting H.leucocephalus microhabitat selection were distance to the nearest T.chinensis mature tree,herb cover and density,leaf litter cover,and vegetation type.Moreover,the results of logistic regression also highlighted the importance of elevation,distance to light gap and roads,tree cover in bird microhabitat selection.Furthermore,the seed emergence rate in microhabitats used by birds did not differ from the natural forest,which was related to five factors of bird microhabitat.The Random Forest model showed that seedling emergence rate was increased with leaf litter cover and distance to fallen dead trees,but decreased in relation to herb cover,slope,and elevation.Conclusion:Our results highlight the importance of remnant microhabitats in fragmented forests for sustaining forest ecology and optimal management.The contribution of microhabitats used by birds to plant recruitment provides insights into how frugivore species contribute to plant regeneration,which should be incorporated in future conservation and management practices of fragmented forests.

Effect of frugivorous birds on the establishment of a naturally regenerating population of Chinese yew in ex situ conservation

The Chinese yew Taxus chinensis is catalogued as an endangered species in China because of the small size andsenescent status of most populations. Its lack of natural regeneration is the most important reason for its endangeredstatus. We investigated the regeneration of an ex-situ conservation population, which was introduced intothe Nanjing Botanical Garden Memorial Sun Yat-Sen in the 1950s, and evaluated the role of frugivorous birds on theestablishment of this regenerating population. Two hundred and thirteen individual yew seedlings and saplingswere found on the hillside in 2006, and the nearest seedling was found more than 10 m away from mother trees. Thespatial pattern of all seedlings and saplings occurred as a clumped distribution, which is typical for vertebratedispersedplants. Six bird species were seen ingesting whole “fruits” at yew trees in the present study and 745 visitsby avian frugivores were recorded. Red-billed blue magpie (Urocissa erythrorhyncha), Chinese bulbul (Pycnonotussinensis) and azure-winged magpie (Cyanopica cyana) were the most frequent visitors over the two years of thestudy. Comparing the flights of departure and perching habitats of the three main bird species, we inferred that U.erythrorhyncha would be the most important disperser. This regeneration population has had good developmentover the past 20 or more years;frugivorous birds have removed seeds to the hillside every year until now, and seedgermination and seedling growth continue to develop well under natural conditions. We suggest that the conservationsystem of the Chinese yew should be composed of yews, avian dispersers and habitats for seed germinationand seedling growth.

Seed handling by primary frugivores differentially influence post-dispersal seed removal of Chinese yew by ground-dwelling animals

Seed handling by primary frugivores can influence secondary dispersal and/or predation of post-dispersal seeds by attracting different guilds of ground-dwelling animals.Many studies have focused on seeds embedded in fe­ces of mammals or birds;however,less is known about how ground-dwelling animals treat seeds regurgitated by birds(without pulp and not embedded in feces).To compare the effect of differential seed handling by pri­mary dispersers on secondary seed removal of Chinese yew(Taxus chinensis var.mairei),we conducted a series of exclosure experiments to determine the relative impact of animals on the removal of defecated seeds(handled by masked palm civet),regurgitated seeds(handled by birds)and intact fruits.All types of yew seeds were con­sistently removed at a higher rate by rodents than by ants.Regurgitated seeds had the highest removal percent­age and were only removed by rodents.These seeds were probably eaten in situ without being secondarily dis­persed.Defecated seeds were removed by both rodents and ants;only ants might act as secondary dispersers of defecated seeds,whereas rodents ate most of them.We inferred that seeds regurgitated by birds were subject­ed to the highest rates of predation,whereas those dispersed in the feces of masked palm civets probably had a higher likelihood of secondary dispersal.Seeds from feces attracted ants,which were likely to transport seeds and potentially provided a means by which the seeds could escape predation by rodents.Our study highlighted that primary dispersal by birds might not always facilitate secondary dispersal and establishment of plant popu­lations.

不同湿度条件下红豆杉种子生理生化特性变化规律研究

以红豆杉(Taxus wallichiana var.chinensis)种子为试材,研究不同湿度条件下低温层积处理过程中红豆杉种子生理生化特性的变化规律。结果表明:低温变湿层积处理能有效打破红豆杉种子休眠。不同湿度层积处理的红豆杉种子可溶性糖含量变化呈现先降低后升高的趋势,粗脂肪含量呈现逐渐下降的趋势;脱氢酶和葡萄糖六磷酸脱氢酶含量都呈先下降后升高的趋势。在低温层积处理过程中第1阶段高湿表现出较好的促进作用,在第2、3阶段低湿表现出较好的促进作用。