Overexpression of the Chrysanthemum lavandulifolium ROS1 gene promotes flowering in Arabidopsis thaliana by reducing the methylation level of CONSTANS.

DNA demethylation is involved in the regulation of flowering in plants, yet the underlying molecular mechanisms remain largely unexplored. The RELEASE OF SILENCING 1 (ROS1) gene, encoding a DNA demethyltransferase, plays key roles in many developmental processes. In this study, the ROS1 gene was isolated from Chrysanthemum lavandulifolium, where it was strongly expressed in the leaves, buds and flowers. Overexpression of the ClROS1 gene caused an early flowering phenotype in Arabidopsis thaliana. RNA-seq analysis of the transgenic plants revealed that differentially expressed genes (DEGs) were significantly enriched in the circadian rhythm pathway and that the positive regulator of flowering, CONSTANS (CO), was up-regulated. Additionally, whole-genome bisulphite sequencing (WGBS), PCR following methylation-dependent digestion with the enzyme McrBC, and bisulfite sequencing PCR (BSP) confirmed that the methylation level of the AtCO promoter was reduced, specifically in CG context. Overall, our results demonstrated that ClROS1 accelerates flowering by reducing the methylation level of the AtCO promoter. These findings clarify the epigenetic mechanism by which ClROS1-mediated DNA demethylation regulates flowering.

Genome-wide characterization and expression analysis of MYB transcription factors in Chrysanthemum nankingense.

BACKGROUND: Chrysanthemum is a popular ornamental plant worldwide. MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factors play an important role in everything from stress resistance to plant growth and development. However, the MYB family of chrysanthemums has not been the subject of a detailed bioinformatics and expression investigation. RESULTS: In this study, we examined 324 CnMYB transcription factors from Chrysanthemum nankingense genome data, which contained 122 Cn1R-MYB, 183 CnR2R3-MYB, 12 Cn3R-MYB, 2 Cn4R-MYB, and 5 atypical CnMYB. The protein motifs and classification of CnMYB transcription factors were analyzed. Among them, motifs 1, 2, 3, and 4 were found to encode the MYB DNA-binding domain in R2R3-MYB proteins, while in other-MYB proteins, the motifs 1, 2, 3, 4, 5, 6, 7, and 8 encode the MYB DNA-binding domain. Among all CnMYBs, 44 genes were selected due to the presence of CpG islands, while methylation is detected in three genes, including CnMYB9, CnMYB152, and CnMYB219. We analyzed the expression levels of each CnMYB gene in ray floret, disc floret, flower bud, leaf, stem, and root tissues. Based on phylogenetic analysis and gene expression analysis, three genes appeared likely to control cellulose and lignin synthesis in stem tissue, and 16 genes appeared likely to regulate flowering time, anther, pollen development, and flower color. Fifty-one candidate genes that may be involved in stress response were identified through phylogenetic, stress-responseve motif of promoter, and qRT-PCR analyses. According to genes expression levels under stress conditions, six CnMYB genes (CnMYB9, CnMYB172, CnMYB186, CnMYB199, CnMYB219, and CnMYB152) were identified as key stress-responsive genes. CONCLUSIONS: This research provides useful information for further functional analysis of the CnMYB gene family in chrysanthemums, as well as offers candidate genes for further study of cellulose and lignin synthesis, flowering traits, salt and drought stress mechanism.

Comparative analysis of two kinds of garlic seedings: qualities and transcriptional landscape.

BACKGROUND: Facility cultivation is widely applied to meet the increasing demand for high yield and quality, with light intensity and light quality being major limiting factors. However, how changes in the light environment affect development and quality are unclear in garlic. When garlic seedlings are grown, they can also be exposed to blanching culture conditions of darkness or low-light intensity to ameliorate their appearance and modify their bioactive compounds and flavor. RESULTS: In this study, we determined the quality and transcriptomes of 14-day-old garlic and blanched garlic seedlings (green seedlings and blanched seedlings) to explore the mechanisms by which seedlings integrate light signals. The findings revealed that blanched garlic seedlings were taller and heavier in fresh weight compared to green garlic seedlings. In addition, the contents of allicin, cellulose, and soluble sugars were higher in the green seedlings. We also identified 3,872 differentially expressed genes between green and blanched garlic seedlings. The Kyoto Encyclopedia of Genes and Genomes analysis suggested enrichment for plant-pathogen interactions, phytohormone signaling, mitogen-activated protein kinase signaling, and other metabolic processes. In functional annotations, pathways related to the growth and formation of the main compounds included phytohormone signaling, cell wall metabolism, allicin biosynthesis, secondary metabolism and MAPK signaling. Accordingly, we identified multiple types of transcription factor genes involved in plant-pathogen interactions, plant phytohormone signaling, and biosynthesis of secondary metabolites among the differentially expressed genes between green and blanched garlic seedlings. CONCLUSIONS: Blanching culture is one facility cultivation mode that promotes chlorophyll degradation, thus changing the outward appearance of crops, and improves their flavor. The large number of DEGs identified confirmed the difference of the regulatory machinery under two culture system. This study increases our understanding of the regulatory network integrating light and darkness signals in garlic seedlings and provides a useful resource for the genetic manipulation and cultivation of blanched garlic seedlings.

Comparative analysis of the chrysanthemum transcriptome with DNA methylation inhibitors treatment and silencing MET1 lines.

BACKGROUND: As one of the ten most famous flowers in China, the chrysanthemum has rich germplasm with a variety of flowering induction pathways, most of which are photoperiod-induced. After treatment with DNA methylation inhibitors, it was found that DNA methylation plays an important role in flowering regulation, but the mechanism of action remains unclear. Therefore, in this study, curcumin, 5-azaC, their mixed treatment, and MET1-RNAi lines were used for transcriptome sequencing to find out how different treatments affected gene expression in chrysanthemums at different stages of flowering. RESULTS: Genomic DNA methylation levels were measured using HPLC technology. The methylation level of the whole genome in the vegetative growth stage was higher than that in the flowering stage. The methylation level of DNA in the vegetative growth stage was the lowest in the curcumin and mixed treatment, and the methylation level of DNA in the transgenic line, mixed treatment, and curcumin treatment was the lowest in the flowering stage. The flowering rate of mixed treatment and curcumin treatment was the lowest. Analysis of differentially expressed genes in transcriptomes showed that 5-azaC treatment had the most differentially expressed genes, followed by curcumin and transgenic lines, and mixed treatment had the fewest. In addition, 5-azaC treatment resulted in the differential expression of multiple DNA methylation transferases, which led to the differential expression of many genes. Analysis of differentially expressed genes in different treatments revealed that different treatments had gene specificity. However, the down-regulated GO pathway in all 4 treatments was involved in the negative regulation of the reproductive process, and post-embryonic development, and regulation of flower development. Several genes associated with DNA methylation and flowering regulation showed differential expression in response to various treatments. CONCLUSIONS: Both DNA methylase reagent treatment and targeted silencing of the MET1 gene can cause differential expression of the genes. The operation of the exogenous application is simple, but the affected genes are exceedingly diverse and untargeted. Therefore, it is possible to construct populations with DNA methylation phenotypic diversity and to screen genes for DNA methylation regulation.

Development and validation of SSR markers related to flower color based on full-length transcriptome sequencing in Chrysanthemum.

Chrysanthemum (Chrysanthemum moriforlium Ramat.) is one of the most popular flowers worldwide, with very high ornamental and economic values. However, the limitations of available DNA molecular markers and the lack of full genomic sequences hinder the study of genetic diversity and the molecular breeding of chrysanthemum. Here, we developed simple sequence repeat (SSR) from the full-length transcriptome sequences of chrysanthemum cultivar 'Hechengxinghuo'. A total of 11,699 SSRs with mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats were identified, of which eight out of eighteen SSR loci identified based on sixteen transcripts participated in carotenoid metabolism or anthocyanin synthesis were validated as polymorphic SSR markers. These SSRs were used to classify 117 chrysanthemum accessions with different flower colors at the DNA and cDNA levels. The results showed that four SSR markers of carotenoid metabolic pathway divided 117 chrysanthemum accessions into five groups at cDNA level and all purple chrysanthemum accessions were in the group III. Furthermore, the SSR marker CHS-3, LCYE-1 and 3MaT may be related to green color and the PSY-1b marker may be related to yellow color. Overall, our work may be provide a novel method for mining SSR markers associated with specific traits.

Comparison of chrysanthemum flowers grown under hydroponic and soil-based systems: yield and transcriptome analysis.

BACKGROUND: Flowers of Chrysanthemum × morifolium Ramat. are used as tea in traditional Chinese cuisine. However, with increasing population and urbanization, water and land availability have become limiting for chrysanthemum tea production. Hydroponic culture enables effective, rapid nutrient exchange, while requiring no soil and less water than soil cultivation. Hydroponic culture can reduce pesticide residues in food and improve the quantity or size of fruits, flowers, and leaves, and the levels of active compounds important for nutrition and health. To date, studies to improve the yield and active compounds of chrysanthemum have focused on soil culture. Moreover, the molecular effects of hydroponic and soil culture on chrysanthemum tea development remain understudied. RESULTS: Here, we studied the effects of soil and hydroponic culture on yield and total flavonoid and chlorogenic acid contents in chrysanthemum flowers (C. morifolium 'wuyuanhuang'). Yield and the total flavonoids and chlorogenic acid contents of chrysanthemum flowers were higher in the hydroponic culture system than in the soil system. Transcriptome profiling using RNA-seq revealed 3858 differentially expressed genes (DEGs) between chrysanthemum flowers grown in soil and hydroponic conditions. Gene Ontology (GO) enrichment annotation revealed that these differentially transcribed genes are mainly involved in "cytoplasmic part", "biosynthetic process", "organic substance biosynthetic process", "cell wall organization or biogenesis" and other processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment in "metabolic pathways", "biosynthesis of secondary metabolites", "ribosome", "carbon metabolism", "plant hormone signal transduction" and other metabolic processes. In functional annotations, pathways related to yield and formation of the main active compounds included phytohormone signaling, secondary metabolism, and cell wall metabolism. Enrichment analysis of transcription factors also showed that under the hydroponic system, bHLH, MYB, NAC, and ERF protein families were involved in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. CONCLUSIONS: Hydroponic culture is a simple and effective way to cultivate chrysanthemum for tea production. A transcriptome analysis of chrysanthemum flowers grown in soil and hydroponic conditions. The large number of DEGs identified confirmed the difference of the regulatory machinery under two culture system.

Transcriptome analysis of the molecular mechanism of Chrysanthemum flower color change under short-day photoperiods.

Chrysanthemum [Dendranthema morifolium Tzvel.] is an ornamental plant grown under long-term artificial cultivation conditions. In production, early Chrysanthemum blossoms are often promoted by artificial short-day treatment. However, we found that the flower colour of Chrysanthemum blossoms induced by artificial short-day treatment was lighter than those induced by the natural photoperiod. To explore the intrinsic mechanism of colour fading in flowers, we performed full-length transcriptome sequencing of Chrysanthemum morifolium cv. 'Jinbeidahong' using single-molecule real-time sequencing and RNA-sequencing under natural daylight (ND) and short daylight (SD) conditions. The clustered transcriptome sequences were assigned to various databases, such as NCBI, Swiss-Prot, Gene Ontology and so on. The comparative results of digital gene expression analysis revealed that there were differentially expressed transcripts (DETs) in the four stages under ND and SD conditions. In addition, the expression patterns of anthocyanin biosynthesis structural genes were verified by quantitative real-time PCR. The major regulators of the light signalling ELONGATED HYPOCOTYL5 genes were markedly upregulated under ND conditions. The patterns of anthocyanin accumulation were consistent with the expression patterns of CHI1 and 3GT1. The results showed that the anthocyanin synthesis is tightly regulated by the photoperiod, which will be useful for molecular breeding of Chrysanthemum.

DNA demethylation during Chrysanthemum floral transition following short-day treatment

Background: Analytical techniques such as methylation-sensitive amplification polymorphism and high-performance liquid chromatography were used to detect variation in DNA methylation of mature Chrysanthemum leaves during the floral transition induced by short-day (SD) treatment. Results: For both early- and late-flowering cultivars, the time from the date of planting to the appearance of the capitulum bud and early blooming were significantly shorter than those of the control. The capitulum development of the early-flowering cultivar was significantly accelerated compared to the control, unlike the late-flowering cultivar. The DNA methylation percentage of leaves was significantly altered during flower development. For the early-flowering cultivar, DNA methylation was 42.2-51.3% before the capitulum bud appeared and 30.5-44.5% after. The respective DNA methylation percentages for the late-flowering cultivar were 43.5-56% and 37.2-44.9%. Conclusions: The DNA methylation percentage of Chrysanthemum leaves decreased significantly during floral development. The decline in DNA methylation was elevated in the early-flowering cultivar compared with the late-flowering cultivar.

Gene chip analysis of Arabidopsis thaliana genomic DNA methylation and gene expression in response to carbendazim.

OBJECTIVES: To examine the effects of carbendazim on Arabidopsis genomic DNA methylation and gene expression. RESULTS: Carbendazim caused widespread changes in gene loci methylation and gene expression. With 0.1 mM (D2) and 0.2 mM (D3) carbendazim, there were, respectively, 1522 and 2278 demethylated sites and 1541 and 2790 methylated sites. A total of 279 and 505 genes were up-regulated by more than 300 % and 175 and 609 genes were down-regulated by 67 % in D2 and D3 treatments, respectively, compared with the control. Conjoint analysis showed that 20 and 39 demethylated genes were up-regulated >300 % and 21 and 24 methylated genes were down-regulated <67 % in D2 and D3, respectively. CONCLUSIONS: Carbendazim causes methylation or demethylation of certain genes and changes the expression of these genes. These findings provide a theoretical basis for novel epigenetics-based methods to detect organic food and a new interpretation for the degradation of crop varieties.

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Background In recent years, nickel (Ni) has been widely applied in industrial and agricultural production and has become a kind of environmental pollution. In this study, the effect of nickel chloride (NiCl2) with different concentrations on Arabidopsis genomic stability and DNA methylation has been demonstrated. The nucleolus variation and 18S rDNA methylation after NiCl2 treatment have been analyzed. Results The results are as follows: (1) The NiCl2 could result in heritable genomic methylation variations. The genomic DNA methylation variations have been detected by methylation-sensitive amplified polymorphism (MSAP) molecular markers, and the result showed that after NiCl2 treatment, there was methylation variation in T0 generation seedlings, and partial site changes maintained in T1 generation, which suggested that the effects of NiCl2 on DNA methylation could be heritable in offspring. (2) NiCl2 brought deformity and damage to nucleolar structure in Arabidopsis root tip cells, and the damage was positively correlated with the NiCl2 concentration. 3. In the nucleolus, there was an increased cytosine methylation in 18S rDNA. The plant nucleolus variation and 18S rDNA methylation may be used as an examination indicator for Ni pollution in soil or plant. Conclusions NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

Effect of cryopreservation on the efficiency of exogenous gene, genetic transformation and expression level of Arabidopsis thaliana

Background: Cryopreservation refers to the storage of a living organism at ultra-low-temperature for long-term preservation of plant germplasm. The effect of cryopreservation on the efficiency of exogenous gene genetic transformation and expression level were studied herein. In this work, transgenic Arabidopsis thaliana were successfully conserved in vitro by cryopreservation methods. Results: The effects of osmotic stress due to cryoprotectants during pretreatment and of storage at -196ºC on the stability, the efficiency of genetic transformation and the expression level of exogenous gene were analyzed in Arabidopsis. The results showed that there had not any significant increasing in the efficiency of genetic transformation after cryopreservation, and our observation was not in agreement with earlier reports. Transgenic Arabidopsis lines over-expressing ATOST1 gene were used for the real-time PCR analysis, and the result indicated that the expression of the ATOST1 gene was up-regulated about 2.4-fold in the transgenic seedlings tissues retrieved from cryopreservation than those non-cryopreserved counterparts. Conclusions： Cryopreservation could improve the expression of exogenous gene, however, could not promote the genetic transformation obviously.