m6A modification of lipoyltransferase 1 inhibits bladder cancer progression by activating cuproptosis.

Cuproptosis, a cell death process caused by copper ions, is mediated by protein lipidation related to lipoic acid metabolism. There is a close connection between cuproptosis and the progression and prognosis of various tumors. Here, we identified lipoyltransferase 1 (LIPT1), a key gene related to cuproptosis, was downregulated in bladder cancer (BLCA) and was associated with unfavorable patient prognosis. Restoring the LIPT1 expression in BLCA cells suppressed the proliferation and promoted cuproptosis. Moreover, the consequences of RNA sequencing and Bodipy staining showed that the metabolic pathway mediated by LIPT1 inhibited the accumulation of lipid droplets in cells, disrupted endoplasmic reticulum (ER) homeostasis, and promoted cell apoptosis. Additionally, overexpression of LIPT1 not only repressed the proliferation rate of BLCA cells in vitro but also in vivo. Mechanistically, YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) promoted the degradation of LIPT1 mRNA in a m6A-dependent manner. In summary, these conclusions reveal that LIPT1 promotes cuprotosis and ER stress to inhibit the progression of BLCA, indicating that LIPT1 will provide a powerful treatment direction and drug target for treating BLCA.

Rosiglitazone Promotes Oligodendrocyte Development and Myelin Formation of Repeated Neonatal Sevoflurane Exposure via PPARγ Signaling.

Sevoflurane is one of the most commonly used general anesthetics for children and infants. Recent research indicates that repeated exposure to sevoflurane in neonates induces cognitive and fine motor deficits. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have garnered significant attention as potential therapies for a variety of neurological conditions. In this research, we evaluated whether pretreatment with rosiglitazone in neonatal mice could address myelination defects, cognitive impairment, and fine motor dysfunction via PPARγ. The mice were exposed to 3% sevoflurane for 2 h on postnatal days 6-8 (P6-P8). Behavioral tests were conducted from P29 to P34. Additionally, we evaluated morphological and functional changes related to myelin. Our results showed that rosiglitazone pretreatment significantly ameliorated the cognitive and fine motor impairments of repeated neonatal sevoflurane exposure. In addition, rosiglitazone pretreatment promoted oligodendrocyte precursor cells (OPCs) differentiation and myelination. This suggests that rosiglitazone may be used in clinical settings to enhance the security of neonatal sevoflurane exposure. Furthermore, PPARγ and fatty acid synthase (FASN) may be mediators for rosiglitazone, which alleviates myelination defects, cognitive impairment, and fine motor dysfunction.

H3K18 Lactylation Potentiates Immune Escape of Non-Small Cell Lung Cancer.

The recently discovered epigenetic modification lysine lactylation (Kla) contributes to tumor development and progression in several types of cancer. In addition to the tumor-intrinsic effects, histone lactylation may mediate tumor microenvironment remodeling and immune evasion. Here, we observed elevated pan Kla and H3K18la levels in non-small cell lung cancer (NSCLC) tissues, which was positively correlated with poor patient prognosis. Interruption of glycolysis by 2-DG and oxamate treatment and silencing of LDHA and LDHB reduced H3K18la levels and circumvented immune evasion of NSCLC cells by enhancing CD8+ T cell cytotoxicity. Mechanistically, H3K18la directly activated the transcription of POM121, which enhanced MYC nuclear transport and direct binding to the CD274 promoter to induce PD-L1 expression. In a mouse NSCLC xenograft model, combination therapy with a glycolysis inhibitor and an anti-PD-1 antibody induced intratumoral CD8+ T cell function and exhibited strong anti-tumor efficacy. Overall, this work revealed that H3K18la potentiates the immune escape of NSCLC cells by activating the POM121/MYC/PD-L1 pathway, which offers insight into the role of post-translational modifications in carcinogenesis and provides a rationale for developing an epigenetic-targeted strategy for treating NSCLC.

Deubiquitylase OTUD3 regulates integrated stress response to suppress progression and sorafenib resistance of liver cancer.

The integrated stress response (ISR) is activated in response to intrinsic and extrinsic stimuli, playing a role in tumor progression and drug resistance. The regulatory role and mechanism of ISR in liver cancer, however, remain largely unexplored. Here, we demonstrate that OTU domain-containing protein 3 (OTUD3) is a deubiquitylase of eukaryotic initiation factor 2α (eIF2α), antagonizing ISR and suppressing liver cancer. OTUD3 decreases interactions between eIF2α and the kinase EIF2ΑK3 by removing K27-linked polyubiquitylation on eIF2α. OTUD3 deficiency in mice leads to enhanced ISR and accelerated progression of N-nitrosodiethylamine-induced hepatocellular carcinoma. Additionally, decreased OTUD3 expression associated with elevated eIF2α phosphorylation correlates with the progression of human liver cancer. Moreover, ISR activation due to decreased OTUD3 expression renders liver cancer cells resistant to sorafenib, while the combined use of the ISR inhibitor ISRIB significantly improves their sensitivity to sorafenib. Collectively, these findings illuminate the regulatory mechanism of ISR in liver cancer and provide a potential strategy to counteract sorafenib resistance.

m6A-Mediated Induction of 7-Dehydrocholesterol Reductase Stimulates Cholesterol Synthesis and cAMP Signaling to Promote Bladder Cancer Metastasis.

Dysregulation of cholesterol homeostasis occurs in multiple types of tumors and promotes cancer progression. Investigating the specific processes that induce abnormal cholesterol metabolism could identify therapeutic targets to improve cancer treatment. In this investigation, we observed upregulation of 7-dehydrocholesterol reductase (DHCR7), a vital enzyme involved in the synthesis of cholesterol, within bladder cancer (BC) tissues in comparison to normal tissues, which was correlated with increased BC metastasis. Increased expression of DHCR7 in BC was attributed to decreased mRNA degradation mediated by YTHDF2. Loss or inhibition of DHCR7 reduced BC cell invasion in vitro and metastasis in vivo. Mechanistically, DHCR7 promoted BC metastasis by activating the cAMP/PKA/FAK pathway. Specifically, DHCR7 increased cAMP levels by elevating cholesterol content in lipid rafts, thereby facilitating the transduction of signaling pathways mediated by cAMP receptors. DHCR7 additionally enhanced the cAMP signaling pathway by reducing the concentration of 7-DHC and promoting the transcription of the G protein-coupled receptor GIPR. Overall, these findings demonstrate that DHCR7 plays an important role in BC invasion and metastasis by modulating cholesterol synthesis and cAMP signaling. Furthermore, inhibition of DHCR7 shows promise as a viable therapeutic strategy for suppressing BC invasion and metastasis.

Functional Analysis of Stress Resistance of Bacillus cereus SCL10 Strain Based on Whole-Genome Sequencing.

A Gram-positive, rod-shaped, aerobic, motile, and spore-forming bacterium, designated SCL10, was isolated from Acaudina molpadioides exposure to Co-60 radiation. In this study, whole-genome sequencing was performed to identify the strain as Bacillus cereus and functional characterization, with a focus on stress resistance. The genome of the B. cereus SCL10 strain was sequenced and assembled, revealing a size of 4,979,182 bp and 5167 coding genes. The genes involved in biological functions were annotated by using the GO, COG, KEGG, NR, and Swiss-Prot databases. The results showed that genes related to alkyl hydroperoxide reductase (ahpC, ahpF), DNA-binding proteins from starved cells (dps), spore and biofilm formation (spoVG, spo0A, gerP), cold shock-like protein (cspC, cspE), ATP-dependent chaperone (clpB), and photolyase, small, acid-soluble spore protein (SASP) and DNA repair protein (recA, radD) could explain the stress resistance. These findings suggest that antioxidant activity, sporulation, biofilm formation, and DNA protection may be considered as the main resistance mechanisms under exposure to radiation in the B. cereus SCL10 strain.

Cu(II)-Catalyzed Hydroboration Reactions of 1,1-Disubstituted α,ß-Unsaturated Ketones, Esters, and Amides in Pure Water.

Here, a Cu2(OH)2CO3-catalyzed hydroboration reaction of 1,1-disubstituted α,ß-unsaturated compounds has been developed. The reaction was carried out using water as a solvent at room temperature except for N-monosubstituted α,ß-unsaturated amides. This method is applicable to diverse 1,1-disubstituted α,ß-unsaturated ketones, esters, and amides, showing excellent reactivity (up to 98% yield). Gram-scale experiments and functional group transformations further demonstrated the practicality of this method.

Cooperation and competition between denitrification and chromate reduction in a hydrogen-based membrane biofilm reactor.

Competition and cooperation between denitrification and Cr(VI) reduction in a H2-based membrane biofilm reactor (H2-MBfR) were documented over 55 days of continuous operation. When nitrate (5 mg N/L) and chromate (0.5 mg Cr/L) were fed together, the H2-MBfR maintained approximately 100 % nitrate removal and 60 % chromate Cr(VI) removal, which means that nitrate outcompeted Cr(VI) for electrons from H2 oxidation. Removing nitrate from the influent led to an immediate increase in Cr(VI) removal (to 92 %), but Cr(VI) removal gradually deteriorated, with the removal ratio dropping to 14 % after five days. Cr(VI) removal resumed once nitrate was again added to the influent. 16S rDNA analyses showed that bacteria able to carry out H2-based denitrification and Cr(VI) reduction were in similar abundances throughout the experiment, but gene expression for Cr(VI)-reduction and export shifted. Functional genes encoding for energy-consuming chromate export (encoded by ChrA) as a means of bacterial resistance to toxicity were more abundant than genes encoding for the energy producing Cr(VI) respiration via the chromate reductase ChrR-NdFr. Thus, Cr(VI) transport and resistance to Cr(VI) toxicity depended on H2-based denitrification to supply energy. With Cr(VI) being exported from the cells, Cr(VI) reduction to Cr(III) was sustained. Thus, cooperation among H2-based denitrification, Cr(VI) export, and Cr(VI) reduction led to sustained Cr(VI) removal in the presence of nitrate, even though Cr(VI) reduction was at a competitive disadvantage for utilizing electrons from H2 oxidation.

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.

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.

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.

Cellular senescence and metabolic reprogramming model based on bulk/single-cell RNA sequencing reveals PTGER4 as a therapeutic target for ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the prevailing histological subtype of renal cell carcinoma and has unique metabolic reprogramming during its occurrence and development. Cell senescence is one of the newly identified tumor characteristics. However, there is a dearth of methodical and all-encompassing investigations regarding the correlation between the broad-ranging alterations in metabolic processes associated with aging and ccRCC. We utilized a range of analytical methodologies, such as protein‒protein interaction network analysis and least absolute shrinkage and selection operator (LASSO) regression analysis, to form and validate a risk score model known as the senescence-metabolism-related risk model (SeMRM). Our study demonstrated that SeMRM could more precisely predict the OS of ccRCC patients than the clinical prognostic markers in use. By utilizing two distinct datasets of ccRCC, ICGC-KIRC (the International Cancer Genome Consortium) and GSE29609, as well as a single-cell dataset (GSE156632) and real patient clinical information, and further confirmed the relationship between the senescence-metabolism-related risk score (SeMRS) and ccRCC patient progression. It is worth noting that patients who were classified into different subgroups based on the SeMRS exhibited notable variations in metabolic activity, immune microenvironment, immune cell type transformation, mutant landscape, and drug responsiveness. We also demonstrated that PTGER4, a key gene in SeMRM, regulated ccRCC cell proliferation, lipid levels and the cell cycle in vivo and in vitro. Together, the utilization of SeMRM has the potential to function as a dependable clinical characteristic to increase the accuracy of prognostic assessment for patients diagnosed with ccRCC, thereby facilitating the selection of suitable treatment strategies.

Multi-omic profiling of clear cell renal cell carcinoma identifies metabolic reprogramming associated with disease progression.

Clear cell renal cell carcinoma (ccRCC) is a complex disease with remarkable immune and metabolic heterogeneity. Here we perform genomic, transcriptomic, proteomic, metabolomic and spatial transcriptomic and metabolomic analyses on 100 patients with ccRCC from the Tongji Hospital RCC (TJ-RCC) cohort. Our analysis identifies four ccRCC subtypes including De-clear cell differentiated (DCCD)-ccRCC, a subtype with distinctive metabolic features. DCCD cancer cells are characterized by fewer lipid droplets, reduced metabolic activity, enhanced nutrient uptake capability and a high proliferation rate, leading to poor prognosis. Using single-cell and spatial trajectory analysis, we demonstrate that DCCD is a common mode of ccRCC progression. Even among stage I patients, DCCD is associated with worse outcomes and higher recurrence rate, suggesting that it cannot be cured by nephrectomy alone. Our study also suggests a treatment strategy based on subtype-specific immune cell infiltration that could guide the clinical management of ccRCC.

Metabolic reprogramming based on RNA sequencing of gemcitabine-resistant cells reveals the FASN gene as a therapeutic for bladder cancer.

Bladder cancer (BLCA) is the most frequent malignant tumor of the genitourinary system. Postoperative chemotherapy drug perfusion and chemotherapy are important means for the treatment of BLCA. However, once drug resistance occurs, BLCA develops rapidly after recurrence. BLCA cells rely on unique metabolic rewriting to maintain their growth and proliferation. However, the relationship between the metabolic pattern changes and drug resistance in BLCA is unclear. At present, this problem lacks systematic research. In our research, we identified and analyzed resistance- and metabolism-related differentially expressed genes (RM-DEGs) based on RNA sequencing of a gemcitabine-resistant BLCA cell line and metabolic-related genes (MRGs). Then, we established a drug resistance- and metabolism-related model (RM-RM) through regression analysis to predict the overall survival of BLCA. We also confirmed that RM-RM had a significant correlation with tumor metabolism, gene mutations, tumor microenvironment, and adverse drug reactions. Patients with a high drug resistance- and metabolism-related risk score (RM-RS) showed more active lipid synthesis than those with a low RM-RS. Further in vitro and in vivo studies were implemented using Fatty Acid Synthase (FASN), a representative gene, which promotes gemcitabine resistance, and its inhibitor (TVB-3166) that can reverse this resistance effect.

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.

Constructing gradient reflection and scattering porous framework in composite aerogels for enhanced microwave absorption.

Developing high-performance microwave absorption (MA) materials becomes an urgent concern in the field of electromagnetic protection. Constructing porous framework is an efficient approach to MA owing to the abilities of adjusting impedance matching and providing more reflection and scattering paths for electromagnetic waves. Herein, a cellulose nanofibril (CNF)/honeycomb-like carbon-shell encapsulated FeCoNi@C/carbon nanotube (CNT) composite aerogel was fabricated via a facile freeze-drying method. The super-lightweight composites showed a distinctive gradient structure for reflection and scattering inside aerogel pores, micrometer small pores, and nano-fillers on the pore walls. The composite aerogel showed an ideal minimum reflection loss (RLmin) of -43.6 dB and remarkable adjustable effective absorption bandwidth (EAB) of 12.18 GHz due to good impedance matching, unique gradient porous structure, and synergies of multiple loss mechanisms. Therefore, this work will provide a viable strategy to improve the MA capability of absorbers by taking full advantage of constructing gradient reflection and scattering porous structure.

CmNAC25 targets CmMYB6 to positively regulate anthocyanin biosynthesis during the post-flowering stage in chrysanthemum.

BACKGROUND: Anthocyanin is a class of important secondary metabolites that determines colorful petals in chrysanthemum, a famous cut flower. 'Arctic Queen' is a white chrysanthemum cultivar that does not accumulate anthocyanin during the flowering stage. During the post-flowering stage, the petals of 'Arctic Queen' accumulate anthocyanin and turn red. However, the molecular mechanism underlying this flower color change remains unclear. RESULTS: In this study, by using transcriptome analysis, we identified CmNAC25 as a candidate gene promoting anthocyanin accumulation in the post-flowering stage of 'Arctic Queen'. CmNAC25 is directly bound to the promoter of CmMYB6, a core member of the MBW protein complex that promotes anthocyanin biosynthesis in chrysanthemum, to activate its expression. CmNAC25 also directly activates the promoter of CmDFR, which encodes the key enzyme in anthocyanin biosynthesis. CmNAC25 was highly expressed during the post-flowering stage, while the expression level of CmMYB#7, a known R3 MYB transcription factor interfering with the formation of the CmMYB6-CmbHLH2 complex, significantly decreased. Genetic transformation of both chrysanthemum and Nicotiana tabacum verified that CmNAC25 was a positive regulator of anthocyanin biosynthesis. Another two cultivars that turned red during the post-flowering stages also demonstrated a similar mechanism. CONCLUSIONS: Altogether, our data revealed that CmNAC25 positively regulates anthocyanin biosynthesis in chrysanthemum petals during the post-flowering stages by directly activating CmMYB6 and CmDFR. Our results thus revealed a crucial role of CmNAC25 in regulating flower color change during petal senescence and provided a target gene for molecular design breeding of flower color in chrysanthemum.

Fatty Acid Oxidation Mediated by Malonyl-CoA Decarboxylase Represses Renal Cell Carcinoma Progression.

Fatty acid metabolism reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Increased lipid storage supports ccRCC progression, highlighting the importance of understanding the molecular mechanisms driving altered fatty acid synthesis in tumors. Here, we identified that malonyl-CoA decarboxylase (MLYCD), a key regulator of fatty acid anabolism, was downregulated in ccRCC, and low expression correlated with poor prognosis in patients. Restoring MLYCD expression in ccRCC cells decreased the content of malonyl CoA, which blocked de novo fatty acid synthesis and promoted fatty acid translocation into mitochondria for oxidation. Inhibition of lipid droplet accumulation induced by MLYCD-mediated fatty acid oxidation disrupted endoplasmic reticulum and mitochondrial homeostasis, increased reactive oxygen species levels, and induced ferroptosis. Moreover, overexpressing MLYCD reduced tumor growth and reversed resistance to sunitinib in vitro and in vivo. Mechanistically, HIF2α inhibited MLYCD translation by upregulating expression of eIF4G3 microexons. Together, this study demonstrates that fatty acid catabolism mediated by MLYCD disrupts lipid homeostasis to repress ccRCC progression. Activating MLYCD-mediated fatty acid metabolism could be a promising therapeutic strategy for treating ccRCC. SIGNIFICANCE: MLYCD deficiency facilitates fatty acid synthesis and lipid droplet accumulation to drive progression of renal cell carcinoma, indicating inducing MYLCD as a potential approach to reprogram fatty acid metabolism in kidney cancer.

The RAV transcription factor TEMPRANILLO1 involved in ethylene-mediated delay of chrysanthemum flowering.

TEMPRANILLO1 (TEM1) is a transcription factor belonging to related to ABI3 and VP1 family, which is also known as ethylene response DNA-binding factor 1 and functions as a repressor of flowering in Arabidopsis. Here, a putative homolog of AtTEM1 was isolated and characterized from chrysanthemum, designated as CmTEM1. Exogenous application of ethephon leads to an upregulation in the expression of CmTEM1. Knockdown of CmTEM1 promotes floral initiation, while overexpression of CmTEM1 retards floral transition. Further phenotypic observations suggested that CmTEM1 involves in the ethylene-mediated inhibition of flowering. Transcriptomic analysis established that expression of the flowering integrator CmAFL1, a member of the APETALA1/FRUITFULL subfamily, was downregulated significantly in CmTEM1-overexpressing transgenic plants compared with wild-type plants but was verified to be upregulated in amiR-CmTEM1 lines by quantitative RT-PCR. In addition, CmTEM1 is capable of binding to the promoter of the CmAFL1 gene to inhibit its transcription. Moreover, the genetic evidence supported the notion that CmTEM1 partially inhibits floral transition by targeting CmAFL1. In conclusion, these findings demonstrate that CmTEM1 acts as a regulator of ethylene-mediated delayed flowering in chrysanthemum, partly through its interaction with CmAFL1.