Transcription factor CmHSFA4-CmMYBS3 complex enhances salt tolerance in chrysanthemum by repressing CmMYB121 expression.

Excessive soil salinity not only hampers plant growth and development but can also lead to plant death. Previously, we found that heat-shock factor A4 (CmHSFA4) enhances the tolerance of chrysanthemum (Chrysanthemum morifolium) to salt. However, the underlying molecular mechanism remains unclear. In this study, we identified a candidate MYB transcription factor, CmMYB121, which responded to salt stress. We observed that the CmMYB121 transcription is suppressed by CmHSFA4. Moreover, overexpression of CmMYB121 exacerbated chrysanthemum sensitivity to salt stress. CmHSFA4 directly bound to the promoter of CmMYB121 at the heat-shock element. Protein-protein interaction assays identified an interaction between CmHSFA4 and CmMYBS3, a transcriptional repressor, and recruited the corepressor TOPLESS (CmTPL) to inhibit CmMYB121 transcription by impairing the H3 and H4 histone acetylation levels of CmMYB121. Our study demonstrated that a CmHSFA4-CmMYBS3-CmTPL complex modulates CmMYB121 expression, consequently regulating the tolerance of chrysanthemum to salt. The findings shed light on the responses of plants to salt stress.

Divergence of 10 satellite repeats in Artemisia (Asteraceae: Anthemideae) based on sequential fluorescence in situ hybridization analysis: evidence for species identification and evolution.

Artemisia is a large genus encompassing about 400 diverse species, many of which have considerable medicinal and ecological value. However, complex morphological information and variation in ploidy level and nuclear DNA content have presented challenges for evolution studies of this genus. Consequently, taxonomic inconsistencies within the genus persist, hindering the utilization of such large plant resources. Researchers have utilized satellite DNAs to aid in chromosome identification, species classification, and evolutionary studies due to their significant sequence and copy number variation between species and close relatives. In the present study, the RepeatExplorer2 pipeline was utilized to identify 10 satellite DNAs from three species (Artemisia annua, Artemisia vulgaris, Artemisia viridisquama), and fluorescence in situ hybridization confirmed their distribution on chromosomes in 24 species, including 19 Artemisia species with 5 outgroup species from Ajania and Chrysanthemum. Signals of satellite DNAs exhibited substantial differences between species. We obtained one genus-specific satellite from the sequences. Additionally, molecular cytogenetic maps were constructed for Artemisia vulgaris, Artemisia leucophylla, and Artemisia viridisquama. One species (Artemisia verbenacea) showed a FISH distribution pattern suggestive of an allotriploid origin. Heteromorphic FISH signals between homologous chromosomes in Artemisia plants were observed at a high level. Additionally, the relative relationships between species were discussed by comparing ideograms. The results of the present study provide new insights into the accurate identification and taxonomy of the Artemisia genus using molecular cytological methods.

Chrysanthemum (Chrysanthemum morifolium) CmHRE2-like negatively regulates the resistance of chrysanthemum to the aphid (Macrosiphoniella sanborni).

BACKGROUND: The growth and ornamental value of chrysanthemums are frequently hindered by aphid attacks. The ethylene-responsive factor (ERF) gene family is pivotal in responding to biotic stress, including insect stress. However, to date, little is known regarding the involvement of ERF transcription factors (TFs) in the response of chrysanthemum to aphids. RESULTS: In the present study, CmHRE2-like from chrysanthemum (Chrysanthemum morifolium), a transcription activator that localizes mainly to the nucleus, was cloned. Expression is induced by aphid infestation. Overexpression of CmHRE2-like in chrysanthemum mediated its susceptibility to aphids, whereas CmHRE2-like-SRDX dominant repressor transgenic plants enhanced the resistance of chrysanthemum to aphids, suggesting that CmHRE2-like contributes to the susceptibility of chrysanthemum to aphids. The flavonoids in CmHRE2-like-overexpression plants were decreased by 29% and 28% in two different lines, whereas they were increased by 42% and 29% in CmHRE2-like-SRDX dominant repressor transgenic plants. The expression of Chrysanthemum-chalcone-synthase gene(CmCHS), chalcone isomerase gene (CmCHI), and flavonoid 3'-hydroxylase gene(CmF3'H) was downregulated in CmHRE2-like overexpression plants and upregulated in CmHRE2-like-SRDX dominant repressor transgenic plants, suggesting that CmHRE2-like regulates the resistance of chrysanthemum to aphids partially through the regulation of flavonoid biosynthesis. CONCLUSION: CmHRE2-like was a key gene regulating the vulnerability of chrysanthemum to aphids. This study offers fresh perspectives on the molecular mechanisms of chrysanthemum-aphid interactions and may bear practical significance for developing new strategies to manage aphid infestation in chrysanthemums.

Two B-box proteins orchestrate vegetative and reproductive growth in summer chrysanthemum.

Floral transition, the switch from vegetative to reproductive growth, is extremely important for the growth and development of flowering plants. In the summer chrysanthemum, CmBBX8, a member of the subgroup II B-box (BBX) family, positively regulates the transition by physically interacting with CmERF3 to inhibit CmFTL1 expression. In this study, we show that CmBBX5, a B-box subgroup I member comprising two B-boxes and a CCT domain, interacts with CmBBX8. This interaction suppresses the recruitment of CmBBX8 to the CmFTL1 locus without affecting its transcriptional activation activity. CmBBX5 overexpression led to delayed flowering under both LD (long-day) and SD (short-day) conditions, while lines expressing the chimeric repressor gene-silencing (CmBBX5-SRDX) exhibited the opposite phenotype. Subsequent genetic evidence indicated that in regulating flowering, CmBBX5 is partially dependent on CmBBX8. Moreover, during the vegetative growth period, levels of CmBBX5 expression were found to exceed those of CmBBX8. Collectively, our findings indicate that both CmERF3 and CmBBX5 interact with CmBBX8 to dampen the regulation of CmFTL1 via distinct mechanisms, which contribute to preventing the premature flowering of summer chrysanthemum.

Heterografting enhances chrysanthemums resistance to Alternaria alternata via jasmonate-mediated increases in trichomes and terpenoids.

Trichomes, specialized hair-like structures in the epidermal cells of the aboveground parts of plants, protect plants from pests and pathogens and produce valuable metabolites. Chrysanthemum morifolium, used in tea products, has ornamental and medicinal value. However, it is susceptible to Alternaria alternata fungal infection, posing a threat to its production and use, resulting in substantial economic losses. Increasing the density of glandular trichomes enhances disease resistance and improves the production of medicinal metabolites in chrysanthemums. Jasmonate (JA), promotes the formation of glandular trichomes in various plants. However, it remains unclear whether glandular trichome in chrysanthemums are regulated by JA. Grafting, a technique to improve plant resistance to biotic stresses, has been insufficiently explored in its impact on glandular trichomes, terpenoids, and disease resistance. In this study, we demonstrated that grafting with Artemisia vulgaris rootstocks improves the resistance of chrysanthemum scions to A. alternata. Heterografted chrysanthemums exhibited higher trichome density and terpenoid content compared to self-grafted counterparts. Transcriptome analysis highlighted the significant role of CmJAZ1-like in disease resistance in heterografted chrysanthemums. Overexpressing CmJAZ1-like lines exhibited sensitivity to A. alternate, characterized by reduced glandular trichome density and limited terpenoids. Conversely, silencing lines exhibited resistance to A. alternata showcasing increased glandular trichome density and abundant terpenoids. Higher JA content was confirmed in heterografted chrysanthemum scions compared to self-grafted ones. Furthermore, we established that JA promotes the development of glandular trichomes and the synthesis of terpenoids while inducing the degradation of CmJAZ1-like proteins in chrysanthemums. These findings suggest that higher JA increases trichome density and terpenoid content, enhancing resistance to A. alternata by regulating CmJAZ1-like in heterografted chrysanthemums.

A pattern for the early, middle, and late phase of tea chrysanthemum response to Fusarium oxysporum.

Chrysanthemum morifolium is cultivated worldwide and has high ornamental, tea, and medicinal value. With the increasing area of chrysanthemum cultivation and years of continuous cropping, Fusarium wilt disease frequently occurs in various production areas, seriously affecting the quality and yield and causing huge economic losses. However, the molecular response mechanism of Fusarium wilt infection remains unclear, which limits the molecular breeding process for disease resistance in chrysanthemums. In the present study, we analyzed the molecular response mechanisms of 'Huangju,' one of the tea chrysanthemum cultivars severely infested with Fusarium wilt in the field at the early, middle, and late phases of F. oxysporum infestation. 'Huangju' responded to the infestation mainly through galactose metabolism, plant-pathogen interaction, auxin, abscisic acid, and ethylene signalling in the early phase; galactose metabolism, plant-pathogen interaction, auxin, salicylic acid signal, and certain transcription factors (e.g., CmWRKY48) in the middle phase; and galactose metabolism in the late phase. Notably, the galactose metabolism was important in the early, middle, and late phases of 'Huangju' response to F. oxysporum. Meanwhile, the phytohormone auxin was involved in the early and middle responses. Furthermore, silencing of CmWRKY48 in 'Huangju' resulted in resistance to F. oxysporum. Our results revealed a new molecular pattern for chrysanthemum in response to Fusarium wilt in the early, middle, and late phases, providing a foundation for the molecular breeding of chrysanthemum for disease resistance.

Transcriptomic and metabolomic analyses reveal CmMYB308 as a key regulator in the pink flower color variation of 'Dante Purple' chrysanthemum.

KEY MESSAGE: CmMYB308 was identified as a key regulator in chrysanthemum flower color variation from purple to pink by conducting transcriptome and metabolome analysis. CmMYB308 can inhibit anthocyanin biosynthesis by suppressing the expression of CmPAL, CmC4H, and Cm4CL. Flower color variation is a widespread natural occurrence that plays a significant role in floral breeding. We discovered a variation in the flower of the chrysanthemum cultivar 'Dante Purple' (abbreviated as 'DP'), where the flower color shifted from purple to pink. We successfully propagated these pink flowers through tissue culture and designated them as DPM. By conducting transcriptome and metabolome analysis, we identified a reduction in the expression of critical genes involved in anthocyanin biosynthesis-CmPAL, CmC4H, and Cm4CL-in the DPM. This downregulation led to an accumulation of phenylalanine and cinnamic acid within the general phenylpropanoid pathway (GPP), which prevented their conversion into cyanidin and cyanidin 3-glucoside. As a result, the flowers turned pink. Additional transformation and biochemical experiments confirmed that the upregulation of CmMYB308 gene expression in the DPM directly suppressed CmPAL-1 and CmC4H genes, which indirectly affected Cm4CL-3 expression and ultimately inhibited anthocyanin biosynthesis in the DPM. This study offers a preliminary insight into the molecular mechanism underlying chrysanthemum flower color mutation, paving the way for genetic improvements in chrysanthemum flower color breeding.

Intracellular Criegee's mechanism-based synergistic ozone therapy mediated by oleogels for cancer treatment.

Broad cellular components-initiated efficient chemical reactions that occur in malignant cells may contribute to exploring emerging strategies for cancer treatment. Herein, an ozonated oleogel (OG(O)) was developed to achieve cancer ozone therapy (O3-T) based on intracellular Criegee's reaction. By integrating the chemo-drug, the ozone-loaded oleogel (Dox@OG(O)) was prepared as a chemotherapeutic agent for local O3-T, associated with chemotherapy (CT)/radiotherapy (RT)/immunotherapy and wound healing. The in vitro results showed that, Dox@OG(O) could achieve high ozone loading efficiency and ensure its stability. This Oleogel-mediated O3-T could directly destroy tumor cells via intracellular Criegee's reaction occurred on cell membranes, as well as the effects of tumor microenvironment (TME) regulation by the generation of oxygen/reactive oxygen species (ROS) and depletion of glutathione (GSH). Meanwhile, under the stimulation of X-ray, an accelerated free radical's production was observed, further combined with the radio-sensitivity after TME regulation, an effective anti-tumor effect would be achieved. Further on, in vivo results demonstrated that the locally implanted Dox@OG(O) could effectively inhibit the growth of both primary and secondary tumors. Considering these results above, it will serve as inspiration for future studies investigating of O3-T, especially for postoperative skin diseases.

BLADE-ON-PETIOLE interacts with CYCLOIDEA to fine-tune CYCLOIDEA-mediated flower symmetry in monkeyflowers (Mimulus).

Morphological novelties, or key innovations, are instrumental to the diversification of the organisms. In plants, one such innovation is the evolution of zygomorphic flowers, which is thought to promote outcrossing and increase flower morphological diversity. We isolated three allelic mutants from two Mimulus species displaying altered floral symmetry and identified the causal gene as the ortholog of Arabidopsis BLADE-ON-PETIOLE. We found that MlBOP and MlCYC2A physically interact and this BOP-CYC interaction module is highly conserved across the angiosperms. Furthermore, MlBOP self-ubiquitinates and suppresses MlCYC2A self-activation. MlCYC2A, in turn, impedes MlBOP ubiquitination. Thus, this molecular tug-of-war between MlBOP and MlCYC2A fine-tunes the expression of MlCYC2A, contributing to the formation of bilateral symmetry in flowers, a key trait in angiosperm evolution.

Ectopic expression of Chrysanthemum CDM19 in Arabidopsis reveals a novel function in carpel development

BACKGROUND: APETALA3 (AP3) has significant roles in petal and stamen development in accordance with the classical ABC model. RESULTS: The AP3 homolog, CDM19, from Chrysanthemum morifolium cv. Jinba was cloned and sequenced. Sequence and phylogenetic analyses revealed that CDM19 is of DEF/AP3 lineage possessing the characteristic MIKC-type II structure. Expression analysis showed that CDM19 was transcribed in petals and stamens of ray and disc florets with weak expression in the carpels. Ectopic expression of CDM19 in Arabidopsis wild-type background altered carpel development resulting in multi-carpel siliques. CDM19 could only partially rescue the Arabidopsis ap3­­3 mutant. CONCLUSIONS: Our results suggest that CDM19 may partially be involved in petal and stamen development in addition to having novel function in carpel development.

A highly sensitive method for the detection of Chrysanthemum virus B

Background: Chrysanthemum plants are subject to serious viral diseases. The viruses cause severe losses of the quantity and quality of chrysanthemum. The most problematic pathogen of chrysanthemum is typically considered Chrysanthemum virus B (CVB). Thus, a method for the simultaneous detection of CVB is needed. Results: We used gene-specific primers, which were derived from the coat protein gene region of the virus, for reverse transcription to obtain cDNA. Nested amplification polymerase chain reaction (PCR) was employed to detect the viral gene. This method was sensitive enough to detect the virus at up to 10-9 dilution of the cDNA. Conclusion: A highly specific and sensitive nested PCR-based assay has been described for detecting CVB. This new method is highly specific and sensitive for the detection of CVB, which is known to infect chrysanthemum plants in the fields. Further, this protocol has an advantage over traditional methods as it is more cost-effective. This assay is ideal for an early stage diagnosis of the disease.