Chrysanthemum Lavandulifolium Research Articles

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.

Isolation and functional analysis of the ClM8-FRUITFULL-like MADS-box gene from Chrysanthemum lavandulifolium

AP1/FUL-like genes encode class-specific MADS box transcription factors in angiosperms and play important roles in the plant floral transition and floral organ identity. Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is one of the original species of Chrysanthemum. Understanding the function of AP1/FUL-like genes in C. lavandulifolium provide molecular-level insight into the flowering mechanism of Chrysanthemum. Here, an AP1/FUL-like gene was isolated from C. lavandulifolium. Sequence and phylogenetic analyses indicated that this gene, ClM8, belongs to the euFUL clade of the AP1/FUL-like genes. We observed different expression levels of ClM8 in C. lavandulifolium roots, stems, fruits and leaves and high expression in the flowers. A subcellular localisation assay showed that ClM8 is localised to the nucleus in vivo. The over-expression of ClM8 in Arabidopsis resulted in altered flowering time and inflorescence structure. These aberrant, transgenic Arabidopsis plants showed early flowering following the production of four to six small rosette leaves, lateral flowers and a composite terminal flower. The terminal flower consisted of two or four pistils surrounded by an abnormal number of sepals, petals and stamens. In addition, the morphology of the style of 35S::ClM8 plants silique differed from wild type, appearing thinner and longer. Further investigation suggested that the expression levels of FLOWERING LOCUS T (FT), LEAFY (LFY) and APETALA1 (AP1) were apparently increased in ClM8-transgenic Arabidopsis. Thus, we speculate that ClM8 promoted flowering in Arabidopsis due to the upregulation of FT. Taken together, our results suggest that ClM8 is involved in flowering induction and floral development that might also play a role in the development of fruits and leaves.

The Multi‐Targeted Effects of Chrysanthemum Herb Extract Against Escherichia coli O157:H7

The Chrysanthemum lavandulifolium extract, which includes chrysoeriol, sudachitin, and acacetin, has excellent antibiotic effects on Escherichia coli O157:H7 (E. coli O157). A notable point is that the antibiotic targets of the herb extract are similar to the targets of commonly used antibiotic drugs, including bacterial cell wall biosynthesis, bacterial protein synthesis, and bacterial DNA replication and repair. In addition, the herbal antibiotic inhibits the etiological factors that contribute to the pathogenic property. The herbal sample was extracted and fractionated and then inoculated through a disk diffusion method to confirm its antibiotic effect against E. coli O157. Total RNA was isolated from the affected bacterial cells, and its expression level was analyzed through a microarray analysis. To confirm the accuracy of the microarray data, a real-time PCR was performed. Three active compounds, chrysoeriol, sudachitin, and acacetin, were identified with a high-performance liquid chromatography-electrospray ionization/mass spectrometry chromatogram, and the disk diffusion study confirmed that chrysoeriol and sudachitin contribute to the antibiotic properties of the herb extract. The results demonstrate that the multi-target efficacy of the herbal sample may indicate the potential for the development of more effective and safer drugs that will act as substitutes for existing antibiotics.

Reference gene selection for RT-qPCR analysis of Chrysanthemum lavandulifolium during its flowering stages

Quantitative real-time RT-PCR (RT-qPCR) is a technology that can be used to analyze the abundance of gene expression. Reference genes, which are assumed to remain at constant levels in different tissues at various developmental stages and photoperiodic treatments, were selected to analyze the expression levels of flowering time genes and floral development genes. Using digital gene expression technology, nine reference genes with moderate expression in the leaves of Chrysanthemum lavandulifolium at the juvenile phase (CK1) and the squaring stage (W1) were selected as the candidate reference genes for further study. A total of 115 biological samples of C. lavandulifolium were analyzed, including different tissues under various developmental stages and leaves with varied photoperiodic treatments. The stability of the nine reference genes was slightly variable across the samples, but MTP, SKIP16 and PGK were the most stable genes overall. In addition, the relative expression level of ClFT in different tissues of plants with the competence to flower was analyzed to verify the reference genes selected in this study. These studies provide a guide for selecting reference genes for analyzing the expression pattern of flowering time genes and floral development genes in C. lavandulifolium.

Transcriptome-wide survey and expression analysis of stress-responsive NAC genes in Chrysanthemum lavandulifolium

The plant-specific NAC (NAM, ATAF, and CUC) transcription factor family plays a vital role in various plant growth and developmental processes as well as in stress resistance. Using RNA sequencing, we found that the ClNAC genes (ClNAC1–44) were the most strongly up-regulated transcription factor family in Chrysanthemum lavandulifolium leaves under salt treatment. We carried out reverse transcriptase polymerase chain reaction to monitor ClNAC genes response against multiple stresses and hormonal treatments including salt, drought, cold, heat, abscisic acid and salicylic acid treatments. The results showed that 35 ClNAC genes were differentially expressed in different organ, and 32 ClNAC genes could respond to at least 2 kinds of treatments. Quantitative real time polymerase chain reaction showed that 10 ClNAC genes belonging to 7 different subfamilies could respond to at least 5 kinds of treatments. Over 50-fold variation in transcriptional levels of ClNAC17 and ClNAC21genes was observed under 6 different types of treatments. In the present study, high-level expression of ClNAC genes under abiotic stresses and hormonal treatments suggests that the NAC transcription factors play important roles in abiotic stress tolerance and adaptation.

Comparative Expression Analysis of DFL, a LFY/FLO Homologue Gene in <i>Dendranthema lavandulifolium</i> Responds to Photoperiodic Induction

he expression of the floral regulators DFL, a LFY/FLO homologue from Chrysanthemum lavandulifolium was examined during short day light treatments. Quantitative real-time RT-PCR experiments showed that DFL was expressed in the vegetative apices and throughout the shoot apex following photoperiodic induction. After 5 days of induction, DFL expression was increased markly and expressed in highest levels after 20 days induction. Expression of DFL in the shoot apex at the time of floral determination indicating that DFL gene is involved in the first steps of the transition from vegetative to reproductive development.

Development of method for preparing high molecular weight DNA from chrysanthemum

We developed an improved method for preparing high molecular weight DNA (HMW-DNA) from leaves of Chrysanthemum lavandulifolium that has high content of phenolic compounds. Compared with three previous methods for preparing HMW-DNA, only with the improved method a considerable yield of HMW-DNA more than 2Mb was achieved. The improved method utilizes a modified buffer system including 1% (v/v) β-mercaptoethanol, 4mM spermidine and 6% (w/v) polyvinylpyrrolidone (PVP)-40 and a centrifugal speed of 1600×g to extract and suspend HMW-DNA. With the improved method, phenolic compounds were successfully removed and antioxidants protected HMW-DNA from oxidation and degradation. No difference on the yield of DNA was observed with different lysis times. The HMW-DNA can be partially digested to 23–80kb in size, and is suitable for artificial chromosome library construction.

Expression Analysis of Nudix Hydrolase Genes in Chrysanthemum lavandulifolium

Nudix hydrolases are associated with detoxification processes in plants under abiotic stress and regulate disease resistance signal transduction pathways. Investigating expression patterns and phyletic evolution of eight Nudix hydrolase gene family members in Chrysanthemum lavandulifolium will provide insights into detoxification pathways. In this study, full-length open reading frames of eight Nudix hydrolase genes (ClNUDX1-8) were obtained using a C. lavandulifolium expressed sequence tag (EST) database, EST splicing, and rapid-amplification of cDNA ends. Sequence similarity analysis of the eight ClNUDXs using MEGA4.0 software revealed that these genes belonged to seven subfamilies of the plant Nudix hydrolase gene family. Reverse transcription-polymerase chain reaction analysis demonstrated that five genes exhibited organ-specific expression; these included ClNUDX1, ClNUDX2, ClNUDX3, CINUDX7, and ClNUDX8. Among those, ClNUDX1, ClNUDX3, ClNUDX7, and ClNUDX8 were induced by salt, drought, heat, and cold stresses. ClNUDX2 was only influenced by salt and drought. Transcription levels of both ClNUDX1 and ClNUDX2 were also regulated by abscisic acid and salicylic acid. The promoter of ClNUDX1, CN1P, was cloned. Transient expression analysis indicated that CN1P was capable of driving expression of the GUS reporter gene. Thus, it was concluded that different members of Nudix hydrolase genes in C. lavandulifolium demonstrated varied expression patterns in response to different abiotic stresses. These findings further elucidated knowledge on the response mechanisms to abiotic stresses in C. lavandulifolium.

CDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C.×morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3h, 12h, 24h and 48h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.

Visualization of genomic relationships in allotetraploid hybrids between Chrysanthemum lavandulifolium X C. chanetii by fluorescence in situ hybridization and genomic in situ hybridization

Artificial cross between Chrysanthemum lavandulifolium (2n=18 diploid) and C. chanetii (2n=36, tetraploid) made succesfully interspecific F1 hybrid (2n=36 tetraploid instead of 2n=27 tripolid) for the first time. The F1 hybrid had 12 signals of 45S rDNA and and four signals of 5S rDNA by FISH. The chromosome complement of the F1 hybrid showed each set of the both parental chromosomes by GISH by using the biotin labeled probe of C. lavandulifolium mixed with approximately 20X blocking DNA of C. chanetii. Eighteen yellow-green colored chromosomes were hybridized with the probe of C. lavandulifolium, that could be involved with C. lavandulifolium, while the other 18 orange-red colored chromosomes were not GISH-hybridized chromosomes with the probe, that could be involved with C. chanetii. The extra nine chromosome set in the hybrids might be resulted due to duplication of the haploid chromosome set of C. lavandulifolium at/after fertilization.

New sesquiterpene lactones and acetylenes from chrysanthemum lavandulifolium

The aerial parts of Chrysanthemum lavandulifolium Mak. (Asteraceae) yielded three new acetylenes of the spiroacetal type 1-3, as well as two new germacranolides 4-5 and several known lactones.