Chrysanthemum Lavandulifolium Research Articles

Cla-miR164-NO APICAL MERISTEM (ClNAM) regulates the inflorescence architecture development of Chrysanthemum lavandulifolium.

Chrysanthemum × morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of Chrysanthemum lavandulifolium, a closely related species of C. × morifolium in the genus. Modification of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compounded corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2, and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influences the architecture of capitulum, but also affects compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.

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.

Comparative Physiological and Transcriptome Analysis of Crossostephium chinense Reveals Its Molecular Mechanisms of Salt Tolerance

Crossostephium chinense is a wild species with strong salt tolerance that has great potential to improve the salt tolerance of cultivated chrysanthemums. Conversely, the unique salt-tolerant molecular mechanisms of Cr. chinense are still unclear. This study performed a comparative physiological and transcriptome analysis of Cr. chinense, Chrysanthemum lavandulifolium, and three hybrids to investigate the salt-tolerant molecular mechanisms of Cr. chinense. The physiological results showed that Cr. chinense maintained higher superoxide dismutase (SOD) activity, alleviating oxidative damage to the membrane. KEGG enrichment analysis showed that plant hormone signaling transduction and the MAPK signaling pathway were mostly enriched in Cr. chinense and hybrids under salt stress. Further weighted gene co-expression network analysis (WGCNA) of DEGs suggested that abscisic acid (ABA) signaling transduction may play a significant role in the salt-tolerant mechanisms of Cr. chinense and hybrids. The tissue-specific expression patterns of the candidate genes related to ABA signaling transduction and the MAPK signaling pathway indicate that genes related to ABA signaling transduction demonstrated significant expression levels under salt stress. This study offers important insights into exploring the underlying salt-tolerant mechanisms of Cr. chinense mediated by ABA signaling transduction and broadens our understanding of the breeding strategies for developing salt-tolerant cultivars utilizing salt-tolerant chrysanthemum germplasms.

Genome-wide identification of the TCP gene family in Chrysanthemum lavandulifolium and its homologs expression patterns during flower development in different Chrysanthemum species.

TCP proteins, part of the transcription factors specific to plants, are recognized for their involvement in various aspects of plant growth and development. Nevertheless, a thorough investigation of TCPs in Chrysanthemum lavandulifolium, a prominent ancestral species of cultivated chrysanthemum and an excellent model material for investigating ray floret (RF) and disc floret (DF) development in Chrysanthemum, remains unexplored yet. Herein, a comprehensive study was performed to analyze the genome-wide distribution of TCPs in C. lavandulifolium. In total, 39 TCPs in C. lavandulifolium were identified, showing uneven distribution on 8 chromosomes. Phylogenetic and gene structural analyses revealed that ClTCPs were grouped into classes I and II. The class II genes were subdivided into two subclades, the CIN and CYC/TB1 subclades, with members of each clade having similar conserved motifs and gene structures. Four CIN subclade genes (ClTCP24, ClTCP25, ClTCP26, and ClTCP27) contained the potential miR319 target sites. Promoter analysis revealed that ClTCPs had numerous cis-regulatory elements associated with phytohormone responses, stress responses, and plant growth/development. The expression patterns of ClTCPs during capitulum development and in two different florets were determined using RNA-seq and qRT-PCR. The expression levels of TCPs varied in six development stages of capitula; 25 out of the 36 TCPs genes were specifically expressed in flowers. Additionally, we identified six key ClCYC2 genes, which belong to the class II TCP subclade, with markedly upregulated expression in RFs compared with DFs, and these genes exhibited similar expression patterns in the two florets of Chrysanthemum species. It is speculated that they may be responsible for RFs and DFs development. Subcellular localization and transactivation activity analyses of six candidate genes demonstrated that all of them were localized in the nucleus, while three exhibited self-activation activities. This research provided a better understanding of TCPs in C. lavandulifolium and laid a foundation for unraveling the mechanism by which important TCPs involved in the capitulum development.

Genome-wide identification and characterization analysis of RWP-RK family genes reveal their role in flowering time of Chrysanthemum lavandulifolium

BackgroundRWP-RKs are plant specific transcription factors, which are widely distributed in plants in the form of polygenic families and play key role in nitrogen absorption and utilization, and are crucial to plant growth and development. However, the genome-wide identification and function of RWP-RK in Compositae plants are widely unknown.ResultsIn this study, 101 RWP-RKs in Chrysanthemum lavandulifolium were identified and tandem repeat was an important way for the expansion of RWP-RKs in Compositae species. 101 RWP-RKs contain 38 NIN-like proteins (NLPs) and 31 RWP- RK domain proteins (RKDs), as well as 32 specific expansion members. qRT-PCR results showed that 7 ClNLPs in leaves were up-regulated at the floral transition stage, 10 ClNLPs were negatively regulated by low nitrate conditions, and 3 of them were up-regulated by optimal nitrate conditions. In addition, the flowering time of Chrysanthemum lavandulifolium was advanced under optimal nitrate conditions, the expression level of Cryptochromes (ClCRYs), phytochrome C (ClPHYC) and the floral integration genes GIGANTEA (ClGI), CONSTANS-LIKE (ClCOL1, ClCOL4, ClCOL5), FLOWERING LOCUS T (ClFT), FLOWERING LOCUS C (ClFLC), SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS 1 (ClSOC1) also were up-regulated. The expression level of ClCRY1a, ClCRY1c, ClCRY2a and ClCRY2c in the vegetative growth stage induced by optimal nitrate reached the expression level induced by short-day in the reproductive growth stage, which supplemented the induction effect of short-day on the transcription level of floral-related genes in advance.ConclusionsIt was speculated that ClNLPs may act on the photoperiodic pathway under optimal nitrate environment, and ultimately regulate the flowering time by up-regulating the transcription level of ClCRYs. These results provide new perspective for exploring the mechanism of nitrate/nitrogen affecting flowering in higher plants.

Genome-wide identification of the MIKCc-type MADS-box gene family in Chrysanthemum lavandulifolium reveals their roles in the capitulum development.

Chrysanthemum ×morifolium is well known throughout the world for its diverse and exquisite flower types. However, due to the complicated genetic background of C. ×morifolium, it is difficult to understand the molecular mechanism of its flower development. And it limits the molecular breeding of improving chrysanthemum flower types. C. ×morifolium has the typical radial capitulum, and many researches showed that the members of the MIKCc-type MADS box gene family play a key role in the formation and development of the capitulum. However, it has been difficult to isolate the important MIKCc and investigate their roles in this process due to the lack of genomic information in chrysanthemum. Here, we identified MIKCc-type MADS box genes at whole genome-wide level in C. lavandulifolium, a diploid species closely related to C. ×morifolium, and investigated their roles in capitulum development by gene expression pattern analysis and protein interaction analysis. A total of 40 ClMIKCc were identified and were phylogenetically grouped into 12 clades. Members of all clades showed different enriched expression patterns during capitulum formation. We speculate that the E-class genes in C. lavandulifolium underwent subfunctionalization because they have a significantly expanded, more diverse expression patterns, and specifically tissue expression than AtSEPs. Meanwhile, we detected the C-class expressed in disc floret corolla, which could be the clue to explore the morphological differences between disc and ray floret corolla. In addition, the potential roles of some MIKCcs in complex inflorescence formation were explored by comparing the number and phylogenetic relationship of MIKCc subfamily members in Asteraceae with different capitulum types. Members of the FLC branch in Asteraceae were found to be possibly related to the differentiation and development of the ray floret.

Genome-Wide Identification and Expression Analysis of HSP70 Gene Family in Chrysanthemum lavandulifolium under Heat Stress

As a molecular chaperone, HSP70 is widely involved in complex activities in plants. Under high temperature, drought, high salt, low temperature, heavy metals, and other stresses, HSP70 is rapidly synthesizes, stabilizes protein and biological macromolecular structures, and improves the stress resistance of plants. In this study, 83 ClHSP70 genes in Chrysanthemum lavandulifolium were identified based on the published Chrysanthemum lavandulifolium genome database. The genes were divided into six clusters based on a phylogenetic analysis, and the gene structures, conserved motifs, and functional domains were relatively conserved. Only two collinear genes were observed, and they formed a pair of duplicating genes. Multiple abiotic stress and phytohormone response elements were observed on the ClHSP70 promoter, such as temperature, drought, methyl jasmonate, abscisic acid, and other stress-related elements, and CpG islands were found on some ClHSP70 promoters, suggesting that they may be related to methylation modifications. Tissue expression analysis showed that the ClHSP70 genes were highly expressed in the roots. In addition, the gene expression changed significantly after 1 h of heat treatment and plays an important role in plant responses to temperature stress. The results of this study provide insights on the ClHSP70 gene family members and a theoretical basis for further research on functional analyses of the ClHSP70 gene family.

ClNAC84 interacts with ClMIP to regulate the cell cycle and reduce the size of Chrysanthemum lavandulifolium organs

The NAC transcription factor is plant-specific proteins and one of the largest families of transcription factors in plants. NAC proteins are involved in various aspects of plant growth and development, but little is known about how NAC proteins regulate the cell cycle. Here, we characterized ClNAC84 from C. lavandulifolium (an NAC transcription factor). ClNAC84 overexpression in C. lavandulifolium resulted in a semi-dwarf phenotype with shorter plant height, smaller leaf size, and smaller flower size than wild-type plants. The number of cells in the S phase during the cell cycle was less in ClNAC84-overexpression transgenic C. lavandulifolium than in wild-type C. lavandulifolium. This indicates that ClNAC84 overexpression can induce cell cycle arrest at the S and G2 phases. To elucidate the ClNAC84 regulatory network, ClMIP protein was shown to interact with ClNAC84 in vitro and in vivo. ClMIP overexpression in C. lavandulifolium also resulted in dwarfism and decreased cell numbers, and the expression level of ClKRP5 was higher in transgenic C. lavandulifolium than in wild-type plants. We also found that ClMIP can bind to the promoter of ClKRP5. Our data indicate that the interaction between ClNAC84 and ClMIP may promote ClKRP5 expression and inhibit S and G2 phases of the cell cycling.

Co-option of a carotenoid cleavage dioxygenase gene (CCD4a) into the floral symmetry gene regulatory network contributes to the polymorphic floral shape-color combinations in Chrysanthemum sensu lato.

Morphological novelties, including formation of trait combinations, may result from de novo gene origination and/or co-option of existing genes into other developmental contexts. A variety of shape-color combinations of capitular florets occur in Chrysanthemum and its allies. We hypothesized that co-option of a carotenoid cleavage dioxygenase gene into the floral symmetry gene network would generate a white zygomorphic ray floret. We tested this hypothesis in an evolutionary context using species in Chrysanthemum sensu lato, a monophyletic group with diverse floral shape-color combinations, based on morphological investigation, interspecific crossing, molecular interaction and transgenic experiments. Our results showed that white color was significantly associated with floret zygomorphy. Specific expression of the carotenoid cleavage dioxygenase gene CCD4a in marginal florets resulted in white color. Crossing experiments between Chrysanthemum lavandulifolium and Ajania pacifica indicated that expression of CCD4a is trans-regulated. The floral symmetry regulator CYC2g can activate expression of CCD4a with a dependence on TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING (TCP) binding element 8 on the CCD4a promoter. Based on all experimental findings, we propose that gene co-option of carotenoid degradation into floral symmetry regulation, and the subsequent dysfunction or loss of either CYC2g or CCD4a, may have led to evolution of capitular shape-color patterning in Chrysanthemum sensu lato.

Characterization of WRKY Gene Family in Whole-Genome and Exploration of Flowering Improvement Genes in Chrysanthemum lavandulifolium.

Chrysanthemum is a well-known ornamental plant with numerous uses. WRKY is a large family of transcription factors known for a variety of functions ranging from stress resistance to plant growth and development. Due to the limited research on the WRKY family in chrysanthemums, we examined them for the first time in Chrysanthemum lavandulifolium. A total of 138 ClWRKY genes were identified, which were classified into three groups. Group III in C. lavandulifolium contains 53 members, which is larger than group III of Arabidopsis. The number of introns varied from one to nine in the ClWRKY gene family. The “WRKYGQK” motif is conserved in 118 members, while other members showed slight variations. AuR and GRE responsive cis-acting elements were located in the promoter region of WRKY members, which are important for plant development and flowering induction. In addition, the W box was present in most genes; the recognition site for the WRKY gene may play a role in autoregulation and cross-regulation. The expression of the most variable 19 genes in terms of different parameters was observed at different stages. Among them, 10 genes were selected due to the presence of CpG islands, while nine genes were selected based on their close association with important Arabidopsis genes related to floral traits. ClWRKY36 and ClWRKY45 exhibit differential expression at flowering stages in the capitulum, while methylation is detected in three genes, including ClWRKY31, ClWRKY100, and ClWRKY129. Our results provide a basis for further exploration of WRKY members to find their functions in plant growth and development, especially in flowering traits.

Detection and analysis of the volatile components in the essential oils of <i>Chrysanthemum</i> and <i>Opisthopappus</i> species and their hybrid progeny

<italic>Chrysanthemum</italic> and <italic>Opisthopappus</italic> are genera that include perennial herbaceous floral species, including excellent varieties with strong fragrances resulting from long-term artificial selection. Thus, they are ornamentally and economically important flower resources. In this study, a water distillation method was used to extract essential oils from the inflorescences of <italic>Chrysanthemum</italic> and <italic>Opisthopappus</italic> wild resources and hybrid progeny with high essential oil contents (<italic>Chrysanthemum</italic> <italic>morifolium</italic> 'xiangjin', <italic>C. morifolium</italic> 'xiangyun', <italic>C. morifolium</italic> 'xinjiboju', <italic>Opisthopappus taihangensis</italic>, <italic>Opisthopappus longilobus</italic>, <italic>Chrysanthemum lavandulifolium</italic>, and <italic>C. morifolium</italic> 'minghuangju'). The essential oil extraction rates were as follows: 1.17‰, 2‰, 1.67‰, 2.17‰, 0.43‰, 1‰, and 1.17‰. On the basis of HS-SPME-GC-MS (Headspace solid phase microextraction-gas chromatography-mass spectrometry), 225 volatile compounds were detected in the seven analyzed essential oil samples. Each essential oil had a relative volatile component content exceeding 0.3. The three most abundant compounds were olefins (46 types), alcohols (34 types), and esters (18 types). The volatile components with relatively high contents included thymol, D-camphor, pinene, eucalyptol, 2-terpineol, terpineol, trans-caryophyllene, and <italic>β</italic>-elemene. These volatile compounds have strong biological activities and are useful components of medicines and daily-use products. An evaluation of their antibacterial effects demonstrated that the essential oils of <italic>C.</italic> 'xiangjin', <italic>C.</italic> 'xiangyun', <italic>C.</italic> 'xinjiboju', <italic>O. taihangensis</italic>, <italic>O. longilobus</italic>, and <italic>C.</italic> 'minghuangju' inhibited the growth of <italic>Escherichia coli</italic>. The <italic>C. lavandulifolium</italic> essential oil inhibited the growth of <italic>Pectobacterium carotovorum</italic>. The results of this study will provide researchers with an important theoretical basis for the development and application of <italic>Chrysanthemum</italic> and <italic>Opisthopappus</italic> essential oils.

MBD protein recognizes flower control genes regulated by DNA methylation in &lt;i&gt;Chrysanthemum lavandulifolium&lt;/i&gt;

Dynamic changes in DNA methylation regulate the expression of genes and play important roles especially in the flowering processes of higher plants. Methyl-CpG-binding domain protein could specifically recognize hypermethylated regions in the genome, thus MBD sequencing technology and CpG islands analysis of the sequences were used to identify candidate genes that were regulated by DNA methylation, in particular the flowering induction stage of <em>Chrysanthemum lavandulifolium</em>. MBD-seq identified 89 candidate genes which included 49 genes exhibiting changes in DNA methylation status during floral induction. Based on CpG islands analysis of the sequences, 27 candidate genes were selected that may be regulated by DNA methylation. The expression levels of 30 candidate genes and nine key genes were determined by RT-PCR and qRT-PCR during floral induction (7D), four genes (<em>ClFT</em>, <em>ClMET</em>, <em>DFL</em> and <em>ClWRKY21</em>) were similarly up-regulated. Methylation-specific PCR analysis also indicated that there were changes in the DNA methylation status in the <em>DFL</em> and <em>ClWRKY21</em>. The changes in the DNA methylation status during the induction phase of flowering may lead to changes in gene expression. In this study, a set of genes were identified that are proposed to be involved in floral induction and two key genes were identified (<em>DFL, ClWRKY21</em>) that were regulated by DNA methylation during the flowering process of <em>C. lavandulifolium</em>.

Chrysanthemum lavandulifolium homolog ClMAD1 modulates the floral transition during temperature shift

Flower initiation and development in chrysanthemum requires accurate cell division and expansion, which are regulated by a complex network involved with many cell cycle regulators. Mitotic arrest deficiency 1 (MAD1) is a key component of spindle assembly checkpoint, which was reported to regulate floral transition in Arabidopsis through the interaction with Suppressor of FRIGIDA 4 (SUF4) to modulate Flowering Locus C (FLC) transcription. So far, no MAD1, SUF4 and FLC homologous genes, and related studies were reported in chrysanthemum. Here we first isolated and clone ClMAD1 and ClSUF4 homologs in Chrysanthemum lavandulifolium. Both ClMAD1 and ClSUF4 localized in nuclei, and their expression varied in different tissues. Overexpression of ClMAD1 in Arabidopsis promoted endopolyploidization in leaves, and restored cell cycle and flowering phenotypes of mad1 mutants to different extents. Overexpression of ClSUF4 in Arabidopsis resulted in significantly late flowering, which was correlated with enhanced transcription of FLC and reduced transcription of FT gene. ClMAD1 was predicted to have three coiled-coil (CC) domains, which is one more than that in Arabidopsis. Similar to AtSUF4, ClSUF4 was predicted to have two Zn-C2H2 domains. Protein-protein interaction assays revealed that ClSUF4 could interact with AtMAD1, and the third coiled-coil domain of ClMAD1 not the full-length protein. All these data suggest that both ClMAD1 and ClSUF4 are functional homologs. We further isolated their downstream signaling component ClFLC-like (ClFLCl) genes. ClFLCl had high expression in leaves at vegetative growth stage, and significantly reduced after the bolting suggesting a negative role in the floral transition. Long-term cold treatment significantly repressed the transcription of ClFLCl genes. Temperature shift promoted floral transition of chrysanthemum, which is correlated with the fluctuation of ClMAD1, ClSUF4 and ClFLCl transcription. Taken together, we reported a novel machinery on the regulation of floral transition in chrysanthemum that ClMAD1 interacts with ClSUF4 to modulate ClFLCl-mediated floral transition during the temperature shift.

Volatiles inheriting from Crossostephium chinense act as repellent weapons against aphids in Chrysanthemum lavandulifolium cultivars

Chrysanthemum lavandulifolium is a kind of medicinal plant with large industrial scale and widely cultivated in southern China. Chrysanthemum aphids (Macrosiphoniella sanborni) seriously restricted the development of C. lavandulifolium industry. In this study, in order to breed aphid resistant cultivars, we introduced a potential aphid resistant germplasm Crossostephium chinense to participate in breeding, and the role of volatiles in this process was analyzed. Nine true hybrids were obtained by distant hybridization combined with embryo rescue. The aphid resistance and behavioral effects of their volatiles on aphids were tested. All hybrids behaved stronger resistance and repellency than C. lavandulifolium. The repellency of H1, H2 and H11 was significantly higher than other hybrids. Forty-seven major volatiles were detected in the parent and hybrid leaves by using solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC–MS). H1, H2 and H11 can be separated from other plant materials by PCA analysis, 15 volatiles such as 1, 2-Oxidolinalool were identified as their characteristic difference compounds. Methacrolein was demonstrated to be a repellent compound to aphids by using standard chemical. These findings may lead to novel control strategies against M. sanborni of C. lavandulifolium and its relative species.

Dysfunction of CYC2g is responsible for the evolutionary shift from radiate to disciform flowerheads in the Chrysanthemum group (Asteraceae: Anthemideae).

Evolutionary shifts among radiate, disciform and discoid flowerheads have occurred repeatedly in a number of major lineages across the Asteraceae phylogeny; such transitions may also appear within evolutionarily young groups. Although several studies have demonstrated that CYC2 genes partake in regulating floral morphogenesis in Asteraceae, the evolution of capitulum forms within a recently diverging lineage has remained poorly understood. Here, we study the molecular regulation of the shift from a radiate to a disciform capitulum within the Chrysanthemum group. This is a recently radiating group mainly comprising two genera, Chrysanthemum and Ajania, that are phylogenetically intermingled but distinct in flowerhead morphology: Chrysanthemumspp. with radiate capitula and Ajaniaspp. with disciform capitula. We found that the morphogenesis of zygomorphy in the marginal floret in Ajania was disrupted soon after floral primordium emergence; CYC2g, one of the CYC2 copies that was expressed prominently in the ray floret of Chrysanthemum was not expressed in flowerheads of Ajania. Weakening the expression of ClCYC2g in Chrysanthemum lavandulifolium led to the gradual transition of a ray flower toward the disc-like form. Molecular evolutionary analyses indicated that the disciform capitulum might have evolved only once, approximately 8Mya, arising from dysfunction of the CYC2g orthologs. A 20-nt deletion, including a putative TATA-box of the Ajania-type CYC2g promoter, appeared to inhibit the expression of the gene. Considering the divergent habitats of Chrysanthemum and Ajania, we propose that the shift from radiate to disciform capitulum must have been related to changes in pollination strategies under selective pressure.

Methylation-sensitive amplification polymorphism analysis reveals the DNA methylation dynamics during capitulum development in Chrysanthemum lavandulifolium

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

AfLFY, a LEAFY homolog in Argyranthemum frutescens, controls flowering time and leaf development

Flowering is important for plant propagation and survival, and it is also closely related to human life. Identifying the molecular mechanisms underlying flower development is essential for plant improvement and breeding. Flower development is a complex physiological process that is regulated by multiple genes. LFY genes play important roles in the floral meristem transition and act as crucial integrators in regulating the floral gene network. Argyranthemum frutescens is an ornamental species cultivated for floral displays, yet little is known about molecular mechanisms driving its flower development. In this study, the LEAFY gene homologue, AfLFY, was identified and cloned from A. frutescens, and its role and expression patterns were characterized. Two distinct copies of AfLFY were found in the A. frutescens genome and both sequences contained a 1248 bp open reading frame that encoded 415 amino acids. The putative protein sequences have a typical LFY family domain. In addition, AfLFY was expressed at the highest levels in young leaves of the vegetative stage and in the shoot apical bud meristem of the reproductive stage. Phylogenetic analysis showed that AfLFY was most closely related to DFL from Chrysanthemum lavandulifolium. Subcellular localization studies revealed that AfLFY localized to the nucleus. Heterologous expression of AfLFY in transgenic tobacco plants shortened its period of vegetative growth, converted the lateral meristems into terminal flowers and promoted precocious flowering. In addition, transgenic plants exhibited obvious morphological changes in leaf shape. qRT-PCR analysis indicated that the expression levels genes related to flowering, FT, SOC1, and AP1 were significantly upregulated in AfLFY transgenic plants. Our findings suggested that the AfLFY gene plays a vital role in promoting flowering and leaf development in A. frutescens. These results laid a foundation for us to understand the mechanism of AfLFY in regulation flowering, and the results will be helpful in improving A. frutescens through molecular breeding.

Morphological variation of Chrysanthemum lavandulifolium induced by 5-azaC treatment

To explore the possible role of DNA methylation in plant development. Different concentrations of the DNA methyltransferase inhibitor 5-azacytidine were used on different components of Chrysanthemum lavandulifolium (stem and hypocotyl). The effects of 5-azaC on plantlet growth and flower development were investigated. Results showed that low concentrations of 5-azaC (20 μmol L−1–100 μmol L−1) inhibited plantlet growth (from the stem segments), and high concentrations of 5-azaC (150 μmol L−1–200 μmol L−1) promoted plantlet growth. This inhibition is similar in the regeneration of hypocotyls, except that the concentration range of 5-azaC is different. Low concentration of 5-azaC (10 μmol L−1–20 μmol L−1) inhibit generation or growth of callus-generated plantlets, high concentration of 5-azaC (150 μmol L−1–200 μmol L−1) promoted the growth of hypocotyl regeneration seedlings. In thus, the inhibitory effect of plantlet was similar in hypocotyl regeneration. Therefore, different explants have different sensitivities to methyltransferase inhibitor (5-azaC) treatment. Progeny plants treated with stems of C. lavandulifolium under the short days condition, the control plants (about 15 leaves) did not bloom, plants undergoing high concentration treatments flowered in some strains (flowering rate 80% in treatment 6), and the pitch of all unflowering plants was dwarfed. It seems that 5-azaC can break the C. lavandulifolium adolescent period limit, potentially by inducing the photoperiodic in advance or through another mechanism, after 5-azaC processing may alters key gene methylation, resulting in flowering. In the hypocotyl-treated progeny after multiple passages, all treatments had flowering and non-flowering lines under short-day conditions, and the flowering rate was between 39.29% and 78.43%. We also obtained a continuous flowering line was obtained (2-8-2), and this trait exhibited stable by asexual reproduction. In addition, abundant variation in leaf morphology, flower shape and stem color variation were observed in hypocotyl regeneration plants. All the evidence suggests that the methyltransferase inhibitor (5-azaC) may affect the DNA methylation level of many key genes regulating plant growth and development. This study provides phenotypic data and materials for further exploring the role of DNA methylation in the growth and flower induction of C. lavandulifolium.

Transcriptome Analysis of Chrysanthemum lavandulifolium Response to Salt Stress and Overexpression a K+ Transport ClAKT Gene-enhanced Salt Tolerance in Transgenic Arabidopsis

Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.

The expression and interactions of ABCE-class and CYC2-like genes in the capitulum development of Chrysanthemum lavandulifolium and C. × morifolium

The head-like inflorescence in C. × morifolium was considered as a pseudianthia that bears numerous types of floral organs. ABCE-class and CYC2-like genes have been proven to play a key role in the identity of floral organs. To further understand their roles in capitulum development, expression pattern and protein interactions of ABCE-class and CYC2-like genes were detected using quantitative PCR and yeast-two hybrid. A-class genes, DenFUL and ClAP2, particularly accumulated in ray floret corollas and formed heterodimers with CYC2-like proteins. CYC2-like genes are particularly expressed in ray floret corollas, and these proteins form homodimers and heterodimers. The present study indicated that A-class genes in chrysanthemums interacted with CYC2-like genes and might be involved in the identity of reproductive organs and ray florets, especially the differentiation of disc and ray floret corollas. Moreover, the floral organ identity model in Chrysanthemums was proposed based on insights from the gene expression and protein interactions of ABCE-class and CYC2-like genes.