A likely paleo-autotetraploidization event shaped the high conservation of Nyssaceae genome

Scientific knowledge about the ancestral genome of core eudicot plant kingdom can potentially have profound impacts on both basic and applied research,including evolution,genetics,genomics,ecology,agriculture,forestry,and global climate.To investigate which plant conserves best the core eudicots common ancestor genome,we compared Arcto-Tertiary relict Nyssaceae and 30 other eudicot plant families.The genomes of Davidia involucrata(a known living fossil),Camptotheca acuminata and Nyssa sinensis,one per existent genus of Nyssaceae,were performed comparative genomic analysis.We found that Nyssaceae originated from a single Nyssaceae common tetraploidization event(NCT)-autotetraploidization 28-31 Mya after the core eudicot common hexaploidization(ECH).We identified Nyssaceae orthologous and paralogous genes,determined its chromosomal evolutionary trajectory,and reconstructed the Nyssaceae most recent ancestor genome.D.involucrata genome contained the entire seven paleochromosomes and 17 ECH-generated eudicot common ancestor chromosomes and was the slowest in mutation among the analyzed 42 species of 31 plant families.Combing both its high retention of paleochromosomes and its low mutation rate,D.involucrata provides the best case in conservation of the core eudicot paleogenome.

Differences in leaf cuticular wax induced by whole-genome duplication in autotetraploid sour jujube

Drought-resistant plants exhibit strong water retention capability.In this regard,the autotetraploid sour jujube leaves exhibit better water retention than diploid leaves.Morphological comparisons and physiological comparisons of diploid and autotetraploid leaves showed that the autotetraploid leaves had thicker leaf cuticles and more leaf wax accumulation than the diploid leaves,which could reduce cuticle permeability and improve the drought tolerance of leaves.In this study,the cuticular wax crystalloids on the adaxial and abaxial sides of young and mature jujube leaves were observed in the two ploidy types,and unique cuticular wax crystalloids covering a large area of the cuticle on autotetraploid sour jujube leaves may provide an advantage in reducing leaf non-stomata transpiration and improving plant drought tolerance.Based on the transcriptome,115 differentially expressed genes between diploids and autotetraploids were further analyzed and found to be involved in the accumulation of cuticular wax components,including terpenoids,fatty acids,and lipids,as well as ABC transporter and wax biosynthetic process.Finally,14 genes differentially expressed between glossy autotetraploid leaves and nonglossy diploid leaves,such as LOC107414787,LOC107411574 and LOC107413721,were screened as candidate genes by qRT-PCR analysis.This findings provided insights into how polyploidization improved drought tolerance.

A likely autotetraploidization event shaped the Chinese mahogany(Toona sinensis)genome

Chinese mahogany(Toona sinensis) is of considerable medical and economic importance, and its genome has been deciphered. However, the process underlying its polyploidy is unclear, and the chromosomal evolutionary trajectory is poorly understood. Here, by reanalysing the T.sinensis genome, we found evidence of a tetraploidization event(T. sinensis special tetraploidization, TST) that occurred approximately 15-17 million years ago(MYA) after the core eudicot-common hexaploidization(ECH or gamma) event. We characterized the synonymous nucleotide substitution rates(Ks values) of collinear genes and found that T. sinensis genes affected by the TST evolve at a slower rate than Acer yangbiense genes. Furthermore, we identified homologous genes related to polyploidization and speciation and constructed multiple alignments with different reference genomes. Notably, the significant balance of gene retention and loss characterized in the two TST-derived subgenomes suggests an autopolyploid nature of the TST. Moreover, we deduced the chromosomal karyotypes of the two subgenomes and identified 7chromosomal fusions that have shaped the T. sinensis genome;more information is available on a newly constructed karyotype platform(http://www.cgrpoee.top/Toona_sinensis/index.html). The T. sinensis genome preserves the ancestral chromosome structure of dicotyledons well and could serve as a good reference for understanding genomic changes in other Meliaceae and Sapindales plants. In addition, we verified that tandem duplication and the ECH have promoted the expansion of terpene synthase(TPS) genes;conversely, the TST seems to have inhibited expansion of these genes. This present effort has clarified the polyploidy events of the T. sinensis genome, filled gaps in the history of karyotype evolution, and laid a solid foundation for further genomic studies in the Meliaceae research community and beyond.

Production of autotetraploid plants by in vitro chromosome engineering in Allium hirtifolium

Persian shallot has been propagated vegetatively through daughter bulbs and has a narrow genetic base.In the present study,to create genetic diversity in terms of ploidy level,the effects of in vitro polyploidization were investigated on morphological,physiological and phytochemical traits.Different colchicine treatments(0,0.3%,0.5% and 0.7% concentrations)for 24,36 and 48 h and oryzalin treatments(0,0.001%,0.002% and 0.004% concentrations)for 4,6 and 8 h were used.A sterile basal plate of bulbs was used for the induction of polyploidy in liquid and solid media.After obtaining plantlets,root tip chromosomes were counted for the determination of ploidy levels.Flow cytometry was performed to confirm the chromosome counting results.The regenerated diploid and tetraploid plantlets were transferred to ex vitro conditions.Colchicine and oryzalin were both successful in inducing polyploids and the polyploids induced were tetraploids and mixoploids.The highest induction of polyploidy was obtained in solid media using 0.5%(w/v)colchicine for 36 h(35.0% polyploidy induction)and in 0.001%(w/v)Oryzalin for 8 h(45.5% polyploidy induction).Differences in plantlet height and weight,bulblet weight,density,stomatal width and length,and chlorophyll content were observed between tetraploid and diploid plants.Chromosome duplication,as a result of tetraploid induction,significantly influenced the antioxidant content and enzyme activities.The results showed that total phenolic content,allicin,and antioxidant capacity were significantly higher in the tetraploid plantlets than that in the diploid plantlets.SRAP loci polymorphisms indicated that the 12 autotetraploid plants(with high allicin content)had different genotypes from the parental diploid plant.Besides,in vitro polyploidy induction not only duplicated chromosomes but also altered the DNA sequence in Allium hirtifolium.In vitro induction of tetraploids in A.hirtifolium can be a reliable way to obtain suitable plant material for breeding programs to generate new genotypic variations.

Evaluation of the effect of shodhana(detoxification)process using chromatographic profiling(HPTLC,HPLC,LC-MS,and GC-MS)and estimation of the content of toxic colchicine in Gloriosa superba Linn.tubers

Shodhana(purification/detoxification)is an important aspect of Ayurveda.It has been described in several important pieces of literature.In this study,we performed shodhana process on Langali(Gloriosa superba Linn.)tubers of family Liliaceae,a semi-woody herbaceous climber with significant therapeutic properties.We compared the unprocessed(before purification)and processed(after purification)samples based on the phytochemical,physicochemical,and chromatographic study,including HPTLC,HPLC,GC-MS and LC-MS.Significant changes were observed in profiling after the shodhana process.This plant contains colchicine,known for its toxic effects,particularly cardiotoxicity.Therefore,we quantified colchicine in the unprocessed and processed samples of Gloriosa superba Linn.tubers using HPLC method.Results showed that the percentage of colchicine was reduced by 13.67%in chloroform extract and 20.97%in ethanol extracts after the shodhana process.

Colchicine-induced Melilotus Research

[Objective] The aim was to identify the ploidy of sweet clover induced by colchicine. [Method] The sweet clover germinating seeds and colchicine solution were used for materials to research colchicine-induced Melilotus. [Result] The results showed that the concentration of 0.2% colchicine solution could induced double sweet clover which showed corresponding features of polyploidy plants as a whole showed great features. [Conclusion] Sweet clover induced by colchicine was polyploidy.

Autotetraploidy induced in Nianmaohuangqin (Radix Scutellariae viscidulae) with colchicine in vitro

OBJECTIVE： To establish and optimize the propaga- tion of Nianmaohuangqin （Radix Scutellariae Viscid- ulae） and induce and characterize polyploidy of Nianmaohuangqin （Radix Scutellariae Viscidulae）. METHODS： Buds from germinating seed-derived explants were induced by tissue culture. With an or- thogonal test, different concentrations of 6-benzyl- aminopurine （BAP）, indole-3-acetic acid （IAA） and kinetin （KT） were used to determine the optimal concentrations for the propagation of Nianmaohuangqin （Radix Scutellariae Viscidulae）. The differ- ent concentrations of IAA and rooting powder （ABT） were used to induce rooting. A 0.3% w/v col- chicine solution was used to induce polyploidy and the induced buds was identified by root-tip chromosome determination and stomatal apparatus ob- servation. RESULTS： A large number of buds could be in- duced directly from epicotyl and hypocotyl ex- plants on Murashige and Skoog （MS） （Murashige and Skoog 1962） medium supplemented with 1.1-1.3 mg/L BAP and 0.2 mg/L IAA. Root induction and development could be observed within 20 days of inoculation on 1/2 MS medium supplemented with 0.2 mg/L IAA and 0.1 mg/L ABTo Furthermore, 27 lines of autotetraploid individuals were ob- tained with a plantlet chromosome number of 2n= 4x=36. CONCLUSION： Autotetraploid lines could be ob- tained through induction with colchicine in vitro, proving that this method might be used for plant selection and breeding.

Colchicine reduces procollagen Ⅲ and increases pseudocholinesterase in chronic liver disease

AIM:To test whether colchicine would be an effective antif ibrotic agent for treatment of chronic liver diseases in patients who could not be treated with α-interferon.METHODS:Seventy-four patients(46 males,28 females) aged 40-66 years(mean 53±13 years) participated in the study.The patients were affected by chronic liver diseases with cirrhosis which was proven histologically(n=58);by chronic active hepatitis C(n=4),chronic active hepatitis B(n=2),and chronic persistent hepatitis C(n=6).In the four patients lacking histology,cirrhosis was diagnosed from anamnesis,serum laboratory tests,esophageal varices and ascites.Patients were assigned to colchicine(1 mg/d) or standard treatment as control in a randomized,double-blind trial,and followed for 4.4 years with clinical and laboratory evaluation.RESULTS:Survival at the end of the study was 94.6% in the colchicine group and 78.4% in the control group(P=0.001).Serum N-terminal peptide of type Ⅲ procollagen levels fell from 34.0 to 18.3 ng/mL(P=0.0001),and pseudocholinesterase levels rose from 4.900 to 5.610 mU/mL(P=0.0001) in the colchicine group,while no signif icant change was seen in controls.Best results were obtained in patients with chronic hepatitis C and in alcoholic cirrhotics.CONCLUSION:Colchicine is an effective and safe antifibrotic drug for long-term treatment of chronic liver disease in which fi brosis progresses towards cirrhosis.

苯并螺环β-溴代酮Wagner-Meerwein重排反应研究及其在(±)-Colchicine形式合成中的应用

报道了在AgBF4促进下苯并螺环β-溴代酮的Wagner-Meerwein重排反应,从而发展了一条简捷新颖的构筑天然产物中广泛存在的n,7,6-三环骨架的方法.作为该反应的应用,合成了(±)-colchicine的7,7,6-三环母核,完成了(±)-colchicine的形式合成.

Tolerance of nestin^+ cholinergic neurons in the basal forebrain against colchicine-induced cytotoxicity

In the present study we injected colchicine into the lateral ventricle of Sprague-Dawley rats to investigate the effects of colchicine on the number of different-type neurons in the basal forebrain and to search for neurons resistant to injury. After colchicine injection, the number of nestin^＋ cholinergic neurons was decreased at 1 day, but increased at 3 days and peaked at 14-28 days. The quantity of nestincholinergic neurons, parvalbumin-positive neurons and choline acetyl transferase-positive neurons decreased gradually. Our results indicate that nestin^＋ cholinergic neurons possess better tolerance to colchicine-induced neurotoxicity.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Acute and recurring pericarditis: More colchicine,less corticosteroids

Acute and recurring pericarditis are frequently encountered clinical entities.Given that severe complications such as tamponade and constrictive pericarditis occur rarely,the majority of patients suffering from acute pericarditis will have a benign clinical course.However,pericarditis recurrence,with its painful symptoms,is frequent.In effect,recent studies have demonstrated a beneficial role of colchicine in preventing recurrence,while also suggesting an increase in recurrences with the use of corticosteroids,the traditional first-line agent.

Colchicine Inhibited the Expression of Tissue Inhibitor of Metalloprotenase-1 and Interleukin-6 in Cultured Activated Hepatic Stellate Cells

Cultured HSCs were treated colchicine with different concentrations for 12 h, respectively. The effects of eolehicine on HSCs growth were measured by MTT assay. Effects of eolchicine on gene expression of HSCs were analysed by using a self-made oligonucleotide mieroarray. Colehieine inhibited HSCs growth in a dose-dependent manner. After 12 h of treatment with 6.25 mg/L of colehicine, the expression of tissue inhibitor of metalloprotenase-1 （TIMP-1） and the expression of interleukin-6 （IL-6） in HSCs were downregulated by 2.3 folds and 2.1 folds, respectively. These results suggest that colchieine＇s beneficial effects may, at least in part, owe to the inhibitory to the proliferation of HSCs and down regulation of the expression of both TIMP1 and IL-6 in HSCs.

Extraction and Anti-inflammatory Effects of Colchicine from Sagittaria sagittifolia

[Objective] This study was to study the optimal extraction technology and anti-inflammatory effects of colchicine from Sagittaria sagittifolia. [Method] The ef- fects of ethanol concentration, extraction time, extraction temperature and solid-liquid ratio on the extraction rate of colchicine from S. sagittifolia were investigated. On the basis of single-factor experiments, an L9 （34） orthogonal test was carried out to optimize the extraction process. According to the optimal extraction process, the content of colchicine in S. sagittifolia was determined by high performance liquid chromatography. The anti-inflammatory ability of colchicine was evaluated through an anti-inflammatory test in vitro. [Result] The optimal extraction process of colchicine from S. sagittifolia was as follows： ethanol concentration of 60%, extraction temper- ature of 50℃, extraction time of 30 min, and solid-liquid ratio of 1：25 （g/ml）. The content of colchicine in S. sagittifolia was determined as 40.58 μg/100 mg. Com- pared with the control, the colchicine from S. sagittifolia （9.0 and 4.5 g/kg） inhibited the increase in PGE2, TNF-α and IL-1β contents in pleural fluid （P〈0.05）. High-dose colchicine inhibited the increase in TNF-α, IL-1β and MDA contents in lung tissue （P〈0.01）, and middle-dose colchicine inhibited the increase in IL-1β content in lung tissue （P〈0.01）. [Conclusion] The colchicine in S. sagittifolia has a good anti-inflam- matory effect, which may be achieved through hindering the production of inflam- matory mediators and antioxidation.

Colchicine sensitizes human hepatocellular carcinoma cells to damages caused by radiation

AIM： We studied the effect of colchicine combined with radiation on the survival of human hepatocellular carcinoma （HCC） HA22T/VGH cells.METHODS： Twenty-four hours after treatment with 0-8 ng/mL colchicine, HA22T/VGH cells were irradiated at various doses （0, 1, 2, 4, and 8 Gy）. Colony assay was performed to assess the surviving cell fraction. Survival curves were fitted by using a linear-quadratic model to estimate the sensitizer enhancement ratio （SER）. Flow cytometry was used for cell cycle analysis.RESULTS： Colchicine at lower concentrations （1 and 2 ng/mL） had obvious synergy with radiation to inhibit HCC cell growth, whereas higher concentrations （4 and 8 ng/mL） had only additive effect to radiation. Pretreatment with 1 and 2 ng/mL colchicine for 24-h enhanced cell killing by radiation with SERs of 1.21 and 1.53, respectively.G2/N arrest was only observed with higher colchicine doses （8 and 16 ng/mL） after 24-h treatment, this effect was neither seen with lower doses （1, 2, and 4 ng/mL）nor with any dose after only 1 h of treatment.CONCLUSION： Our results suggest that colchicine has potential as an adjunct to radiotherapy for HCC treatment.Lower doses of colchicine possess radiosensitizing effects via some mechanism other than G2/M arrest. Further study is necessary to elucidate the mechanism.

Development of a novel rat model of heterogeneous hepatic injury by injection with colchicine via the splenic vein

AIM To develop a novel rat model of heterogeneous hepatic injury.METHODS Seventy male Sprague-Dawley rats were randomly divided into a control group(n = 10) and a colchicine group(n =60). A 0.25% colchicine solution(0.4 mL/kg) was injected via the splenic vein in the colchicine group to develop a rat model of heterogeneous hepatic injury. An equal volume of normal saline was injected via the splenic vein in the control group. At days 3, 7, and 14 and weeks 4, 8, and 12 after the operation, at least seven rats of the colchicine group were selected randomly for magnetic resonance imaging(MRI) examinations, and then they were euthanized. Ten rats of the control group underwent MRI examinations at the same time points, and then were euthanized at week 12. T2-weighted images(T2 WI) and diffusion weighted imaging(DWI) were used to evaluate the heterogeneous hepatic injury. The heterogeneous injury between the left and right hepatic lobes was assessed on liver sections according to the histological scoring criteria, and correlated with the results of MRI study. RESULTS Obvious pathological changes occurred in the hepatic parenchyma in the colchicine group. Hepatic injury scores were significantly different between the left and right lobes at each time point(P < 0.05). There was a significant difference in apparent diffusion coefficient(ADC) of DWI and liver-to-muscle ratio(LMR) of T2 WI between the left and right lobes of rats in the colchicine group(P < 0.05) at each time point, and similar results were observed between the colchicine and control groups. Besides, there was a significant correlation between hepatic injury scores and ADC values or LMR(r =-0.682, P = 0.000; r =-0.245, P = 0.018).CONCLUSION Injection with colchicine via the splenic vein can be used to successfully develop a rat model of heterogeneous hepatic injury. DWI and T2 WI may help evaluate the heterogeneous injury among liver lobes.

Genome-wide analysis of microRNAs induced and regulated by colchicine in mulberry

A variety of plants were colchicine treated to double their chromosome number.Chromosomes are genetic carriers that determine the individual traits of organisms.The doubling of chromosomes will lead to modifications in plant morphology,physiology and genetics.To determine the response of mulberry trees induced by colchicine,using mulberry variety Yu-711 leaves as research materials,two small RNA libraries(control and experimental groups)were constructed.It was found that 45 known miRNA genes and 78 predicted novel miRNA genes in the sequence results.A comparison of data between the control group and the experimental group revealed 37 differentially expressed miRNA genes,19 genes of which were up-regulated and 18 genes of which were down-regulated.Eight miRNAs were selected from 37 differentially expressed miRNA genes.These miRNAs were verified by quantitative real-time PCR,and the expression levels of these miRNAs were found to be consistent with those obtained by sequencing,which proved the accuracy of sequencing results.The PsRNATarget software was used to predict the target genes of 8 miRNAs.The Gene Ontology and Kyoto Encycopedia of Genes and Genomes analysis were used to collect the functions of target genes and confirm that target genes are mainly involved in biological processes,cell components and molecular functions.novel-77 miRNA was selected from 37 differentially expressed miRNAs.What will serve as a fundamental data in understanding mulberry miRNAs function in molecular biology research and further provides the molecular mechanism of mulberry gene regulation as induced by colchicine is that the prediction of novel_77 precursor sequence and the target gene(XM_010104258.2),as well as the secondary structure analysis.

Observation on Double Fertilization and Early Embryonic Development in Autotetraploid Polyembryonic Rice

The process of double fertilization and the characters of embryo and endosperm development in an autotetraploid polyembryonic mutant rice IR36-Shuang were studied with a laser scanning confocal microscopy. Some abnormalities including degenerated ovary, abortive embryo sac, single fertilization, double-ovule and double-embryo and so on. were found dudng double fertilization and embryo development in IR36-Shuang. The rate of the abnormalities was 46.67% in IR36-Shuang, significantly higher than that in the control, an autotetraploid rice line IR36-4X （33.00%）. Cytological and embryonic evidences were provided for seed setting decline and the initiation of additional embryo in IR36-Shuang.

A Novel Approach for Production of Colchicine as a Plant Secondary Metabolite by in Vitro Plant Cell and Tissue Cultures

The secondary metabolites synthesized by plants are economically important chemical compounds in the agricultural and industrial areas such as food, perfumery and pharmaceutical sectors. In recent years, attempts for their production by in vitro plant cell and tissue cultures have been accelerated considerably. Colchicine, the principle secondary metabolite of Colchicum autumnale L. and Gloriosa superba L., is an important alkaloid that has poison effect used for treatment of various diseases and plant breeding studies. Presently, colchicine has been produced by using the seeds of C. autumnale L. and the tubers of G. superba L. through different chemical extraction methods. Applying in vitro plant cell and tissue cultures together with metabolic and genetic engineering techniques, large-scale production of colchicine can be achieved from the above two plant species.