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Find video protocols related to scientific articles indexed in Pubmed.
Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis.
Sci Rep
PUBLISHED: 07-14-2014
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Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.
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Identification of differentially expressed genes in Chrysanthemum nankingense (Asteraceae) under heat stress by RNA Seq.
Gene
PUBLISHED: 07-10-2014
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The RNA-Seq platform was used to characterize the high-temperature stress response of Chrysanthemum nankingense. A set of 54,668 differentially expressed unigenes was identified. After a threshold of ratio change ? 2 and a q-value of <0.05 were applied, the number of differentially transcribed genes was reduced to 3955, of which 765 were up-regulated and 3190 were down-regulated in response to heat stress. The differentially transcribed genes were predicted to participate in 26 biological processes, 4 cellular components, and 13 molecular functions. Among the most differentially expressed genes between the two libraries were well-recognized high-temperature responsive protein families, such as heat shock factors and heat shock proteins, various transcription factor families, and a number of RNA metabolism-related genes. Overall, the RNA-Seq analyses revealed a high degree of transcriptional complexity in early heat stress response. Some of these high-temperature responsive C. nankingense genes may prove useful in efforts to improve thermotolerance of commercial chrysanthemum.
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Phylogenetic and transcription analysis of chrysanthemum WRKY transcription factors.
Int J Mol Sci
PUBLISHED: 07-07-2014
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WRKY transcription factors are known to function in a number of plant processes. Here we have characterized 15 WRKY family genes of the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of 15 distinct sequences were isolated; initially internal fragments were amplified based on transcriptomic sequence, and then the full length cDNAs were obtained using RACE (rapid amplification of cDNA ends) PCR. The transcription of these 15 genes in response to a variety of phytohormone treatments and both biotic and abiotic stresses was characterized. Some of the genes behaved as would be predicted based on their homology with Arabidopsis thaliana WRKY genes, but others showed divergent behavior.
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A bHLH transcription factor regulates iron intake under Fe deficiency in chrysanthemum.
Sci Rep
PUBLISHED: 06-26-2014
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Iron (Fe) deficiency can represent a serious constraint on crop growth and productivity. A number of members of the bHLH transcription factor family are known to be involved in the plant Fe deficiency response. Plants have evolved two distinct uptake strategies when challenged by Fe deficiency: dicotyledonous and non-graminaceous species rely mostly on a reduction strategy regulated by bHLH transcription factors, whereas rice relies on a chelation strategy, also regulated by bHLH transcription factors. CmbHLH1, a bHLH transcription factor which is localized within the nucleus, was isolated from chrysanthemum. Its transcription was up-regulated both by Fe deficiency and by the exogenous application of abscisic acid. The roots of transgenic chrysanthemum plants in which CmbHLH1 was up-regulated were better able than those of the wild type chrysanthemum cultivar to acidify their immediate external environment by enhancing the transcription of the H(+)-ATPase encoding gene CmHA. However, there was no effect of the transgene on the efficiency of uptake of either manganese or zinc. Here, Chrysanthemum CmbHLH1 contributed to Fe uptake via H(+)-ATPase mediated acidification of the rhizosphere. ABA may be positively involved in the process.
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A transcriptomic analysis of Chrysanthemum nankingense provides insights into the basis of low temperature tolerance.
BMC Genomics
PUBLISHED: 05-12-2014
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A major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. Yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. Herein, we used RNA-Seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of Chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation.
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A chrysanthemum heat shock protein confers tolerance to abiotic stress.
Int J Mol Sci
PUBLISHED: 03-12-2014
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Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.
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Chrysanthemum CmNAR2 interacts with CmNRT2 in the control of nitrate uptake.
Sci Rep
PUBLISHED: 03-06-2014
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Nitrate transporters are an important component of plant growth and development. Chrysanthemum morifolium is an important ornamental species, for which a sufficient supply of nitrogenous fertilizer is required to maintain economic yields. In this study, the full-length cDNA of the nitrate transporter genes CmNRT2 and CmNAR2 were isolated. CmNRT2 transcript accumulation was inducible by both nitrate and ammonium, but the latter ion down-regulated the transcript accumulation of CmNAR2. CmNRT2 might be a plasma membrane localized protein, while CmNAR2 was distributed throughout the cell. CmNAR2 was shown to interact with CmNRT2 by in vitro and in vivo assays. Arabidopsis thaliana plants heterologously expressing CmNRT2 showed an increased rate of nitrate influx, while this trait was unaltered in plants expressing CmNAR2. Double transformants (CmNRT2 plus CmNAR2) exhibited an enhanced rate of nitrate influx into the root. Our data indicated that the interaction of CmNAR2 with CmNRT2 contributed to the uptake of nitrate.
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Microsatellite polymorphism among Chrysanthemum sp. polyploids: the influence of whole genome duplication.
Sci Rep
PUBLISHED: 02-24-2014
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Polyploidy is common among flowering plants, including the Asteraceae, a relatively recent angiosperm group. EST-SSRs were used to characterize polymorphism among 29 Chrysanthemum and Ajania spp. accessions of various ploidy levels. Most EST-SSR loci were readily transferable between the species, 29 accessions were separated into three groups in terms of the number of fragments. It inferred that the formation from tetraploid to hexaploid and from octoploid to decaploid may be a recent event, while from the diploid to the tetraploid may be an ancient one in the Chrysanthemum lineage. EST-SSR polymorphism was found and some transcripts containing an SSR were transcribed differently in the de novo autotetraploid C. nankingense and C. lavandulifolium than in their progenitor diploid. EST-SSR could provide a potential molecular basis of adaptation during evolution, while whole genome duplication has a major effect on the mutational dynamics of EST-SSR loci, which could also affect gene regulation.
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The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum.
Mol. Biol. Rep.
PUBLISHED: 02-13-2014
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Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na(+)/H(+) antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum 'Jinba'. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na(+) into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K(+)/Na(+) ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na(+) accumulation. This study is the first to report the transgenic expression of a Na(+) efflux carrier in chrysanthemum.
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The constitutive expression of a two transgene construct enhances the abiotic stress tolerance of chrysanthemum.
Plant Physiol. Biochem.
PUBLISHED: 02-02-2014
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Various abiotic stresses downgrade the quality and productivity of chrysanthemum. A construct carrying both CcSOS1 (from Chrysanthemum crassum) and CdICE1 (from Chrysanthemum dichrum) was constitutively expressed in the chrysanthemum variety 'Jinba'. The transgenic plants were superior to the wild type (WT) ones with respect to their sensitivity to low temperature, drought and salinity, as measured by visible damage and plant survival. Salinity stressed transgenic plants accumulated more proline, and their level of superoxide dismutase and peroxidase activity was higher than in WT plants. At the physiological level, they suffered less loss of viable leaf area, maintained a lower leaf electrolyte conductivity and retained more chlorophyll (a+b). The ratio between the K(+) and Na(+) content was higher in the root, stem and median leaves of salinity stressed transgenic plants than in those of WT plants.
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Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species.
Mol. Biol. Rep.
PUBLISHED: 01-12-2014
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Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT50) decreased from -7.3 to -23.5 °C in Chrysanthemum dichrum and -2.1 to -7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.
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Rapid genetic and epigenetic alterations under intergeneric genomic shock in newly synthesized Chrysanthemum morifolium x Leucanthemum paludosum hybrids (Asteraceae).
Genome Biol Evol
PUBLISHED: 01-11-2014
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The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (?45%) than in the parental lines (51.5-50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.
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A putative high affinity phosphate transporter, CmPT1, enhances tolerance to Pi deficiency of chrysanthemum.
BMC Plant Biol.
PUBLISHED: 01-06-2014
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Inorganic phosphate (Pi) is essential for plant growth, and phosphorus deficiency is a main limiting factor in plant development. Its acquisition is largely mediated by Pht1 transporters, a family of plasma membrane-located proteins. Chrysanthemum is one of the most important ornamental plants, its productivity is usually compromised when grown in phosphate deficient soils, but the study of phosphate transporters in chrysanthemum is limited.
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RNA-Seq derived identification of differential transcription in the chrysanthemum leaf following inoculation with Alternaria tenuissima.
BMC Genomics
PUBLISHED: 01-04-2014
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A major production constraint on the important ornamental species chrysanthemum is black spot which is caused by the necrotrophic fungus Alternaria tenuissima. The molecular basis of host resistance to A. tenuissima has not been studied as yet in any detail. Here, high throughput sequencing was taken to characterize the transcriptomic response of the chrysanthemum leaf to A. tenuissima inoculation.
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Ambient temperature enhanced freezing tolerance of Chrysanthemum dichrum CdICE1 Arabidopsis via miR398.
BMC Biol.
PUBLISHED: 11-02-2013
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ICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance.
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Anatomical and physiological differences and differentially expressed genes between the green and yellow leaf tissue in a variegated chrysanthemum variety.
Mol. Biotechnol.
PUBLISHED: 06-26-2013
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The leaves of the chrysanthemum variety NAU04-1-31-1 are variegated with distinct green and yellow sectors. The chlorophyll content of the yellow leaf tissue is less than that in the green one. The chloroplasts in the yellow leaf tissue were vacuolated, lacked thylakoid membrane structure and contained clusters of plastoglobuli with few or no starch grains. The yellow leaf tissue was more sensitive to photo-inhibition than the green leaf tissue. Suppression subtractive hybridization (SSHs) libraries were constructed to identify genes which were differentially transcribed in the two tissue types. The sequencing of 339 SSH clones identified 150 unigenes (93 singletons and 57 contigs), of which 85 were differentially transcribed in the green leaf tissue and 65 in the yellow leaf tissue. Unigenes associated with photosynthesis were particularly frequent, and many of these genes were up-regulated in the yellow leaf tissue. Both CmChlH which encodes the large subunit of Mg-chelatase and CmFtsH (ATP-dependent metalloprotease) were up-regulated in the yellow leaf tissue, and their transcription was regulated by light.
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Rapid genomic and transcriptomic alterations induced by wide hybridization: Chrysanthemum nankingense?×?Tanacetum vulgare and C. crassum?×?Crossostephium chinense (Asteraceae).
BMC Genomics
PUBLISHED: 06-14-2013
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Hybridization is a major driver of evolution in plants. In a number of plant species, the process of hybridization has been revealed to be accompanied by wide-ranging genetic and epigenetic alterations, some of which have consequences on gene transcripts. The Asteraceae family includes a number of polyploid species, and wide crossing is seen as a viable means of genetically improving ornamental species such as Chrysanthemum spp. However, the consequences of hybridization in this taxon have yet to be characterized.
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Ethylene promotes induction of aerenchyma formation and ethanolic fermentation in waterlogged roots of Dendranthema spp.
Mol. Biol. Rep.
PUBLISHED: 04-29-2013
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The role of ethylene in induction of aerenchyma formation and ethanolic fermentation in waterlogged roots of Dendranthema zawadskii and D. nankingense, two species that differ with respect to waterlogging tolerance, was examined. In the more tolerant D. zawadskii, but not in D. nankingense, ethylene accelerated programmed cell death and promoted formation of lysigenous aerenchyma, both of which were inhibited by treatment with the ethylene inhibitor 1-methylcyclopropene. Waterlogged D. zawadskii roots generated a higher quantity of endogenous ethylene than did those of D. nankingense. In waterlogged D. zawadskii roots, transcription of the genes encoding alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) increased rapidly but transiently, whereas expression of these genes in D. nankingense increased gradually and over a longer period. In D. nankingense, waterlogging elevated both alcohol dehydrogenase and pyruvate decarboxylase activity, and the production of ethanol and acetaldehyde was increased in the presence of exogenous ethylene and inhibited by 1-methylcyclopropene. In D. zawadskii, in contrast, after a prolonged episode of waterlogging stress, exogenous supply of ethylene suppressed the production of ethanol and acetaldehyde, whereas exogenous 1-methylcyclopropene enhanced their production. In the more tolerant Dendranthema species, ethylene appeared to signal an acceleration of both waterlogging-induced programmed cell death and aerenchyma formation and to alleviate ethanolic fermentation, whereas in the more sensitive species ethylene activated fermentation and increased the release of ethanol and acetaldehyde, which are by-products probably responsible for the collapse of the waterlogging-damaged root.
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Proteomic changes in the base of chrysanthemum cuttings during adventitious root formation.
BMC Genomics
PUBLISHED: 03-30-2013
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A lack of competence to form adventitious roots by cuttings of Chrysanthemum (Chrysanthemum morifolium) is an obstacle for the rapid fixation of elite genotypes. We performed a proteomic analysis of cutting bases of chrysanthemum cultivar Jinba during adventitious root formation (ARF) in order to identify rooting ability associated protein and/or to get further insight into the molecular mechanisms controlling adventitious rooting.
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An isoform of eukaryotic initiation factor 4E from Chrysanthemum morifolium interacts with Chrysanthemum virus B coat protein.
PLoS ONE
PUBLISHED: 01-18-2013
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Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in plant virus infection as well as the regulation of gene translation.
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Genetic Mapping of Quantitative Trait Loci Underlying Flowering Time in Chrysanthemum (Chrysanthemum morifolium).
PLoS ONE
PUBLISHED: 01-01-2013
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Flowering time is an important trait in chrysanthemum, but its genetic basis remains poorly understood. An intra-specific mapping population bred from the cross between the autumn-flowering cultivar Yuhualuoying and the summer-flowering Aoyunhanxiao was used to determine the number and relative effect of QTL segregating for five measures of flowering time. From flowering time data recorded over two consecutive seasons, 35 additive QTL were detected, each explaining between 5.8% and 22.7% of the overall phenotypic variance. Of these, 13 were detected in both years. Nine genomic regions harboring QTL for at least two of the five traits were identified. Ten pairs of loci epistatically determined the flowering time, but their contribution to the overall phenotypic variance was less than for the additive QTL. The results suggest that flowering time in chrysanthemum is principally governed by main effect QTL but that epistasis also contributes to the genetic architecture of the trait, and the major QTL identified herein are useful in our ongoing efforts to streamline the improvement of chrysanthemum via the use of molecular methodology.
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The abundance and diversity of soil fungi in continuously monocropped chrysanthemum.
ScientificWorldJournal
PUBLISHED: 01-01-2013
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Chrysanthemum is an important ornamental plant which is increasingly being monocropped. Monocropping is known to affect both fungal abundance and species diversity. Here, quantitative PCR allied with DGGE analysis was used to show that fungi were more abundant in the rhizosphere than in the bulk soil and that the fungal populations changed during the growth cycle of the chrysanthemum. The majority of amplified fragments appeared to derive from Fusarium species, and F. oxysporum and F. solani proved to be the major pathogenic species which are built up by monocropping.
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Chrysanthemum cutting productivity and rooting ability are improved by grafting.
ScientificWorldJournal
PUBLISHED: 01-01-2013
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Chrysanthemum has been commercially propagated by rooting of cuttings, whereas the quality will decline over multiple collections from a single plant. Therefore, we compared the vigour, rooting ability, and some physiological parameters between cuttings harvested from nongrafted "Jinba" (non-grafted cuttings) with those collected from grafted "Jinba" plants onto Artemisia scoparia as a rootstock (grafted cuttings). The yield, length, node number, stem diameter, fresh weight, and dry weight of the grafted cuttings were superior to the non-grafted cuttings. Also grafted cuttings "Jinba" rooted 1 day earlier, but showing enhanced rooting quality including number, length, diameter, and dry weight of roots, where compared to the non-grafted. The physiological parameters that indicated contents of soluble protein, peroxidase activity, soluble sugar, and starch, ratios of soluble sugar/nitrogen ratio, and carbohydrate/nitrogen (C/N), as well as contents of indole-3-acetic acid (IAA) and abscisic acid (ABA), and IAA/ABA ratio were significantly increased in the grafted cuttings. This suggested their important parts in mediating rooting ability. Results from this study showed that grafting improved productivity and rooting ability related to an altered physiology, which provide a means to meet the increasing demand.
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The Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB1 Alters Lignin Composition and Represses Flavonoid Synthesis in Arabidopsis thaliana.
PLoS ONE
PUBLISHED: 01-01-2013
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Plant R2R3-MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of many secondary metabolites at the transcriptional level. In this study, a chrysanthemum subgroup 4 R2R3-MYB transcription factor gene, designated CmMYB1, was isolated through screening chrysanthemum EST (expressed sequence tag) libraries and using rapid application of cDNA ends (RACE) methods and functionally characterized. CmMYB1 is expressed in the root, stem, leaf and flowers, but most strongly in the stem and most weakly in the root. Its heterologous expression in Arabidopsis thaliana reduced the lignin content and altered the lignin composition. The heterologous expression also repressed the flavonoids content in A. thaliana. Together, these results suggested that CmMYB1 is a negative regulator of genes involved in the lignin pathway and flavonoid pathway, it may be a promising gene for controlling lignin and flavonoids profiles in plants.
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Next-generation sequencing of the Chrysanthemum nankingense (Asteraceae) transcriptome permits large-scale unigene assembly and SSR marker discovery.
PLoS ONE
PUBLISHED: 01-01-2013
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Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low.
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Heterologous expression of a Nelumbo nucifera phytochelatin synthase gene enhances cadmium tolerance in Arabidopsis thaliana.
Appl. Biochem. Biotechnol.
PUBLISHED: 07-26-2011
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Phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins, which are thought to play important roles in heavy metal detoxification. The sacred lotus (Nelumbo nucifera), one of the most popular ornamental species, has been shown to be a potential phytoremediator of heavy metal polluted water. However, the phytochelatin synthase gene in N. nucifera has not been identified yet. Here, we report the isolation and function characterization of a N. nucifera homologue of phytochelatin synthase. The sequence obtained shares a high degree of similarity with PCSs from other plant species and was named as Nelumbo nucifera phytochelatin synthase1 (NnPCS1). By using quantitative RT-PCR, we found that the expression of NnPCS1 in leaves of N. nucifera was dramatically increased in response to Cadmium (Cd) treatment. We further showed that, when exposed to Cd stress, Arabidopsis transgenic plants heterologous expressing NnPCS1 accumulated more Cd when compared with wild type. These results suggest that NnPCS1 involved in the response of N. nucifera to Cd stress and may represent a useful target gene for the phytoremediation of Cd-polluted water.
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The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance.
Planta
PUBLISHED: 07-22-2011
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Zinc finger proteins (ZFPs) play an important role in plant growth and development. Here, we describe the isolation of CgZFP1, a Cys2/His2 (C(2)H(2)) ZFP gene, using RACE PCR from chrysanthemum, and the investigation of its function with ectopic expression in Arabidopsis. CgZFP1 contains two conserved C(2)H(2) regions, a nuclear localization domain (B box), a Leu-rich domain (L box) and a conserved DLN sequence (DLN box) close to its C-terminus. Its expression in the chrysanthemum leaf was strongly induced by salinity or drought, but not by ABA. Subcellular localization assay indicated that CgZFP1 protein is localized in nucleus in vivo. Yeast-one hybrid assay showed that CgZFP1 possesses transcriptional activation ability, heterologous expression of CgZFP1 conferred tolerance of transgenic Arabidopsis plants to both salinity and drought stresses. Under salinity stress, genes involved in osmotic adjustment, ROS scavenging, and ion homeostasis: Atlea3, AtP5CS2, AtProT1, and AtMnSOD, AtPOD, AtAPX1, and AtSOS1, AtSOS2, AtSOS3, AtNHX1 were enhanced in CgZFP1 transgenic Arabidopsis plants. Moreover, genes involved in the osmotic adjustment and oxidative stress responses: Atlea3, AtP5CS2, AtProT1, the aquaporin AtPIP2A, and AtMnSOD, AtPOD, AtAPX1 were induced in CgZFP1 transgenic Arabidopsis under drought stress. These results indicate CgZFP1 is an important regulator involved in the salt and drought stress response in plants.
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Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress.
Mol. Biotechnol.
PUBLISHED: 03-19-2011
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Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1 ? (elongation factor 1 ?), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1 ? performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.
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The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance.
Plant Cell Rep.
PUBLISHED: 03-15-2011
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Five intergeneric hybrids between the chrysanthemum cultivar Zhongshanjingui (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While Zhongshanjingui bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from Zhongshanjingui and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.
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The expression of floral organ identity genes in contrasting water lily cultivars.
Plant Cell Rep.
PUBLISHED: 03-01-2011
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The floral organs of typical eudicots such as Arabidopsis thaliana are arranged in four characteristic whorls, namely the sepal, petal, stamen and carpel, and the "ABC" floral organ identity model has been based on this arrangement. However, the floral organs in most basal angiosperms are spirally arranged with a gradual transition from the inside to outside, and an alternative model referred to as "fading borders" was developed to take account of this. The flower morphology of the water lily was tested against the "fading borders" model by determining the expression profile of the six primary floral organ identity genes AP2, AGL6, AP3, PI, AG and SEP1 in two cultivars showing contrasting floral morphology. In addition, to get accurate floatation of the genes expression level from outer to inner, we divided the floral organs into eight whorls according to morphological features. All these genes were expressed throughout all whorls of the flower, but their expression level changed gradually from the outside of the flower to its inside. This pattern was consistent with the "fading borders" model.
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Candidate reference genes for gene expression studies in water lily.
Anal. Biochem.
PUBLISHED: 03-04-2010
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The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1alpha) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.
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Chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation.
J. Plant Physiol.
PUBLISHED: 02-04-2010
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Artificial aphid infestation experiments on the three chrysanthemum cultivars Keiun, Han6 and Jinba showed that the three cultivars vary markedly in their resistance. Of the three cultivars, the most resistant (Keiun) produced the longest, highest and densest trichomes, the largest and fullest gland cells, and the most wax on the lower leaf epidermis. Superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), polyphenol oxidase activity (EC 1.14.18.1) and phenylalanine ammonia lyase (EC 4.3.1.5) were enhanced by aphid herbivory. In the two more resistant cultivars (Keiun and Han6), the activity of superoxide dismutase and ascorbate peroxidase enzymes rapidly increased following infestation, and their levels remained high from seventy-two to one hundred and sixty-eight hours after inoculation. We suggest that these two antioxidant enzymes contribute to aphid resistance of these chrysanthemum cultivars. All three cultivars showed quick responses to aphid infestation by increasing polyphenol oxidase and phenylalanine ammonia lyase activities during the early period after inoculation. Activities of these two defense enzymes were higher in the two resistant cultivars after 72h after inoculation, suggesting involvement of these two enzymes in aphid resistance.
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Production and characterisation of the intergeneric hybrids between Dendranthema morifolium and Artemisia vulgaris exhibiting enhanced resistance to chrysanthemum aphid (Macrosiphoniella sanbourni).
Planta
PUBLISHED: 07-22-2009
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Aphids represent the most destructive of chrysanthemum pests to cultivation. Reliable variety sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. An intergeneric hybrid between Dendranthema morifolium (chrysanthemum) variety Zhongshanjingui and Artemisia vulgaris (mugwort) Variegata was attempted. Most of the hybrid embryos aborted at an early developmental stage. Embryo rescue allowed the generation of hybrid plants, whose hybridity was confirmed by a combination of morphological, cytological and GISH analysis. The hybrids were vigorous, flowered normally, and their flower and leaf shape resembled those of the chrysanthemum more than those of the mugwort parent. The hybrids showed much higher resistance to chrysanthemum aphid (Macrosiphoniella sanbourni) than maternal chrysanthemum by inoculation test. The leaves of the hybrid developed a higher density of trichomes and secretory glands compared to the maternal chrysanthemum. GC-MS analysis revealed that approximately 51% of the essential oil in the hybrid leaves were monoterpenoids and sesquiterpenoids, while the proportion in the chrysanthemum was approximately 37%, and in the mugwort was approximately 90%. It is inferred that higher aphid resistance in the hybrid mainly owed to the leaf micromorphology and bioactive essential oil content.
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Downregulation of apurinic/apyrimidinic endonuclease 1/redox factor-1 enhances the sensitivity of human pancreatic cancer cells to radiotherapy in vitro.
Cancer Biother. Radiopharm.
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Abstract Background: Radiotherapy is an important treatment for the patients with advanced pancreatic cancer. Emerging studies determined apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) might associate with the resistance of human pancreatic cancer cells to radiotherapy. Aims: To investigate whether downregulation of APE1/Ref-1 expression by ribonucleic acid interference would increase the sensitivity of chromic-P32 phosphate to pancreatic cancer cells. Methods: The plasmids containing APE-specific and unspecific short hairpin were transfected into Patu-8898 cells. Stable cell clones were selected by G418. The mRNA expression of APE1/Ref-1 was detected by semiquantitative reverse transcription-polymerase chain reaction and the protein expression of APE1/Ref-1 was detected by Western blot analysis; cell proliferation was studied by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colony formation assay; apoptosis was detected by flow cytometry. Results: After 24 hours irradiation, APE1/Ref-1 mRNA and protein expression were upregulated, in a concentration-dependent manner. Suppression of APE1/Ref-1 by siRNA increased the pancreatic cancer cells hypersensitive to (32)P-CP. In the combination of (32)P-CP and siRNA group, MTT assay showed that the cell inhibition increased to (74.33%±9.02%), the surviving fraction in the colony formation assay was only 25.00%, and the apoptosis rate was up to (16.77%±0.98%). Conclusions: Knockdown APE1/Ref-1 gene expression may significantly sensitize the Patu-8988 cells to radiotherapy, which may be a useful target for modifying radiation resistance of pancreatic cancer cells to irradiation.
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Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization.
Mol. Biol. Rep.
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The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known.
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Combination of multiple resistance traits from wild relative species in Chrysanthemum via trigeneric hybridization.
PLoS ONE
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With the objective of combining multiple resistant traits from wild relative species in florists chrysanthemums, trigeneric hybridization was conducted by crossing two intergeneric F(1) hybrids Chrysanthemum grandiflorum × Artemisia vulgaris and Chrysanthemum crassum × Crossostephium chinense.
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Identification and expression of an APETALA2-like gene from Nelumbo nucifera.
Appl. Biochem. Biotechnol.
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Arabidopsis transcription factor APETALA2 (AP2) controls multiple aspects of plant growth and development, including seed development, stem cell maintenance, and specification of floral organ identity. Based on sequence similar of Arabidopsis AP2 and its homologues genes from other plant species, degenerate RT-PCR and rapid amplification of cDNA ends assay were used to clone AP2 genes from lotus (Nelumbo nucifera). A 2,048-bp cDNA fragment was obtained, which contains a 1,536-bp open reading frame encoding a protein of 511 amino acids. The protein contains two AP2 domains that are conserved in AP2 proteins from other plant species, thus was named as N. nucifera APETALA2 (NnAP2). Quantitative RT-PCR revealed that NnAP2 gene was expressed in flowers, roots, leaves, and stems of N. nucifera, with flowers which have the highest transcript levels. Further analysis showed that in all five lotus cultivars examined, including "Zhongguogudailian," "Yaoniangyujiao," "Jinxia," "Hongtailian," and "Yiliangqianban," petals always have the highest expression levels when compared with the other four flower organs, though the number of petals in these cultivars ranged from simple to thousands. However, NnAP2 expression level in four nonsimple petal flower cultivars was higher than that in the simple petal flower cultivar Zhongguogudailian, indicating that NnAP2 may play a role in specification of petal identity during the evolutionary process of the ancient species N. nucifera.
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The constitutive expression of Chrysanthemum dichrum ICE1 in Chrysanthemum grandiflorum improves the level of low temperature, salinity and drought tolerance.
Plant Cell Rep.
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The quality and productivity of chrysanthemum are severely compromised by various abiotic stresses. Here, we describe the isolation of CdICE1 from Chrysanthemum dichrum using RACE PCR, which shared identical nucleotide of ICE1 ORF from Chrysanthemum grandiflorum variety Jinba. CdICE1 contains a conserved bHLH domain, a nuclear localization domain, a S-rich motif and a ACT domain. The constitutive expression of CdICE1 in C. grandiflorum improved the tolerance of C. grandiflorum to low temperature/freezing, drought and salinity. When the transgene was inserted in the antisense direction, the expression of the endogenous ICE1 gene was down-regulated, and the level of the plants sensitivity to abiotic stress increased. The level of expression of CgDREBa and CgDREBb, activities of superoxide dismutase and peroxidase and the proline content were enhanced in the sense transgenic lines, and lowered in the antisense ones under stresses. In conclusion, CdICE1 represents a promising candidate for a biotechnological approach to improve the level of crop abiotic stress tolerance.
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The AP2-like gene NsAP2 from water lily is involved in floral organogenesis and plant height.
J. Plant Physiol.
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APETALA2 (AP2) genes are ancient and widely distributed among the seed plants, and play an important role during the plant life cycle, acting as key regulators of many developmental processes. In this study, an AP2 homologue, NsAP2, was characterized from water lily (Nymphaea sp. cv. Yellow Prince) and is believed to be rather primitive in the evolution of the angiosperms. In situ RNA hybridization showed that NsAP2 transcript was present in all regions of the floral primordium, but had the highest level in the emerging floral organ primordium. After the differentiation of floral organs, NsAP2 was strongly expressed in sepals and petals, while low levels were found in stamens and carpels. The NsAP2 protein was suggested to be localized in the cell nucleus by onion transient expression experiment. Overexpression of NsAP2 in Arabidopsis led to more petal numbers, and Arabidopsis plants expressing NsAP2 exhibited higher plant height, which may be a result of down-regulated expression of GA2ox2 and GA2ox7. Our results indicated that the NsAP2 protein may function in flower organogenesis in water lily, and it is a promising gene for plant height improvement.
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