We studied the behaviour of AgNPs and matrix interactions within both hydrogel systems. The cell viability of encapsulated human gingival fibroblasts (HGFs) was determined by Prestoblue® assay and live/dead staining. The release of AgNPs was monitored by inductively coupled plasma–mass spectroscopy. The antibacterial properties of the GelMA-AgNP constructs were determined using disc diffusion. Even distribution of AgNPs in GelMA induced a significant decrease in cell viability (p < 0.0001), whereas AgNP aggregates did not induce cytotoxicity in HyStem®-C. AgNPs doses ≥ 0.5 µg/mL in GelMA were significantly toxic to the HGFs (p < 0.0001). The release of AgNPs from GelMA after 48 h was 20% w/w for 0.1 µg/mL and 51% for 100 µg/mL of AgNPs. At ≥5 µg/mL, a significant intra-construct bactericidal effect was observed. The disc diffusion assay shows that GelMA-incorporated AgNPs were found to be effective against both Escherichia coli and Staphylococcus aureus at 50 and 100 µg/mL, respectively. Visible photo-cross-linked GelMA stably incorporated AgNPs to provide an antimicrobial regenerative construct for oral applications.There is a need to develop bifunctional scaffolds that provide antibacterial protection while encouraging host cell attachment/proliferation. This study evaluates HyStem Infections are a common complication of biomaterial placement and often associated with high rates of morbidity due to surgical revision and failure. Thus, a need to fabricate biomaterials with antibacterial properties to hinder the formation of pathogenic biofilms is required. One of the most common modification methods is the incorporation of an antibiotic; however, over recent years, the rate of biomaterial failure caused by antibiotic-resistant bacteria has significantly increased . Silver AgNPs have been studied in conjunction with wound dressings, prosthetic device coatings, topical agents, mouth washes and some dental restorative materials ,6. The iAlpha lipoic-acid is five-membered cyclic disulphide tailing a short hydrocarbon chain on one end and a (COO-) carboxylic group on the other, known to be both antioxidant and anti-inflammatory . While oEscherichia coli DH5α (E. coli) and 12.5 μg/mL for Staphylococcus aureus (S. aureus). This study also reported the formation of AgNP aggregates in tissue culture media leading to poor distribution and sedimentation [Our previous study on 2D culture showed concentration-dependent cytotoxicity where HGFs treated with ≤5 μg/mL had no adverse effects on cell viability, whereas concentrations ≥12.5 μg/mL caused a significant decrease in cell viability . AgNPs hentation .This insPolymeric hydrogels containing AgNPs have attracted great attention for application in the biomedical field, particularly wound dressings and regenerative biomaterials. Gelatin methacryloyl, known as GelMA, is a popular natural polymer-based hydrogel composed of gelatin polymer chains chemically functionalised by methacryloyl (MA) groups . Gelatin®-C, representing different hydrogel systems. HyStem®-C is composed of hyaluronic acid (HA), which is another major constituent of the ECM. According to the manufacturer, denatured collagen was further added this product to enhance cellular adhesion and attachment. Modified forms of HA have been widely studied as wound healing biomaterials and in drug delivery systems [In the present paper, we describe the integration of AgNPs into both GelMA and HyStem systems ,26,27. H systems .Within this context, this research aimed to study the cytotoxicity and antibacterial properties of alpha-lipoic-acid-capped AgNPs by encapsulating both human and bacterial cells in 3D hydrogel scaffolds containing AgNPs in order to (1) prevent AgNP sedimentation and aggregation, (2) to maintain proper AgNP particle distribution and retention of functionality throughout the experiment, and (3) to provide physiological similarities to the native ECM. This study optimised the constructs size and consistency for both hydrogel systems, examined the AgNP/hydrogel interactions and determined both cytotoxicity and antibacterial properties of alpha-lipoic silver nanoparticles in GelMA.3 in a Teflon digestion vessel , followed by gentle heating. The samples were digested at 95 °C for 1 h. During the digestion process, the sample’s volume was reduced to ∼0.2 mL, which was made up again to 3 mL using deionised water. Transmission electron microscopy (TEM) images was obtained using a Philips CM100 BioTWIN TEM  equipped with a LaB6 emitter fitted with a MegaView III Olympus digital camera. The samples were prepared by depositing 10 μL of alpha-lipoic-acid-capped AgNPs onto plasma-glowed, carbon-coated (400 mesh) copper grids. After 60 s, the excess was blotted with filter paper and the sample was left to air dry before viewing. The particle size diameter was measured manually for 100 particles using ImageJ software version 2023, U. S. National Institutes of Health, Bethesda, MD, USA.AgNPs were prepared and characterised as previously described by Cotton et al., 2019 . Concent®-C , was used as per manufacturer instructions. Briefly, the hydrogel components were brought to RT and both Gylcosil and Gelin were reconstituted with 1 mL of degassed water (DG-H2O). Different hydrogel consistencies  were prepared by reconstituting the Extralink with different volumes of the DG-H2O, as per 2, then placed horizontally on a shaker for 2 h (60 cycles per min), until fully dissolved. Within 2 h of reconstituting the solutions with the degassed water, Glycosil and Gelin were mixed in a ratio of 1:1, and slowly pipetted up and down to avoid aeration. The Glycosil/Gelin mix was then placed on a shaker for a further 30 min. The cross-linker was added, and the constructs pipetted directly into a well plate and left undisturbed for 20 min to allow for gelation. AgNPs were added at a concentration of 200 μg/mL or control carrier  to maintain a constant volume.HyStemGelMA was synthesised as previously described by Lim et al., 2019 . Brieflyw/w of GelMA stock solution, 48% PBS, 1% of 50 mM tris dichlororuthenium(II) hexahydrate , and 1% of 500 mM sodium persulphate . Stock solutions of 50 mM Ru (MW = 748.63 g/mol) and 500 mM SPS (MW = 238.1 g/mol) were made in PBS. All solutions were sterilized with a 0.22 μm filter prior to use. Different gel consistencies  were obtained by preparing different concentrations of the stock GelMA solution as per 2.GelMA constructs were prepared by adding 50% g for 5 min and the cell pellet resuspended at 5 million cells/mL of hydrogel as described below.Primary human gingival fibroblasts (HGFs) were obtained with consent from a female patient undergoing routine crown-lengthening surgery at the Faculty of Dentistry, University of Otago. All experiments were performed in accordance with the guidelines of the National Ethics Advisory Committee, New Zealand, and approved by the Human Ethics Committee, University of Otago, reference number H17/112. The HGFs were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% Fetal Bovine Serum , 1% Antibiotic-Antimycotic , 0.5% Gentamicin . Cells were centrifuged at 1000× ®-C constructs were conducted by resuspending the cell pellet at a ratio of 4:1 Glycosil/Gelin mix to Extralink (cross-linker). Constructs of 10 and 25 μL at the three different consistencies of each hydrogel were pipetted into a 96-well plate and incubated at 37 °C and 5% CO2 for 20 min to allow for gelation before adding the media. After gelation, 400 μL of media was gently added at the side of the well to avoid disturbing the construct (n = 3). GelMA/HGF constructs were produced by resuspending the cell pellet in GelMA/PBS and mixing gently, followed by the addition of the cross-linkers Ru and SPS. Constructs of 10 and 25 μL in size were pipetted into a 96-well plate and photo cross-linked for 3 min using visible light (Wavelength = 400–450 nm) at 30 mW/cm2 (n = 3). After cross-linking, 400 μL of media was added gently at the side of the well and the constructs were incubated at 37 °C, 5% CO2. Three cell-free controls of each condition were prepared.Encapsulation of HGF cells within the HyStemn = 3) with stiff HyStem®-C and standard GelMA was determined by incorporating AgNPs at a toxic dose of 200 μg/mL with 5 million HGF cells/mL, as described above. The samples were incubated at 37 °C, 5% CO2, for 20 h, then 40 μL of Prestoblue® cell viability regent  was added to each well. The well plates were incubated again at 37 °C, 5% CO2, and fluorescent measurements recorded the 24 and 48 h time points. Metabolic activity was measured by transferring 50 μL of solution to a 96-well plate, and fluorescence measurements were undertaken using a Synergy 2 Multi-mode microplate reader with an excitation/emission of 535/615 nm.The metabolic activity of cells in the two hydrogels at construct sizes of 10 μL and 25 μL (® and propidium iodide added to each well plate in 400 μL of fresh DMEM/10% FBS. The well plate was incubated for 45 min and then washed (×3) with PBS, prior to being observed under a Nikon-inverted confocal microscopy (Japan). Representative images were used for counting live/dead cells using ImageJ software.Following the cytotoxicity experiments, the same constructs were labelled using cell viability imaging . Constructs were washed with PBS (×3), and 15 μL of both NucBlue®-C and standard GelMA with and without 100 μg/mL of AgNPs was performed using a JEOL JEM-6700F . EDS spectra were obtained from ten randomly chosen areas per sample. Five areas of approximately 5 mm × 5 mm were selected from the electrodense (white) areas representing smaller white areas of NaCl crystals or AgNPs, and five were selected from the greyish-black areas representing the matrix.Samples were prepared as previously outlined and stored overnight at −80 °C before freeze drying for 24 h. Scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) analysis of the carbon-coated stiff HyStem®-C constructs containing 0 or 200 μg/mL AgNPs. Prior to imaging, the media was removed from the wells and replaced with a 100 μL of blue food dye to ensure the gels were visible during processing, particularly the AgNP-free HyStem®-C constructs, which were translucent. After 20 min, the food dye was removed, and the constructs were washed with PBS and then fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 1 h. Constructs were then washed twice for 5 min in 0.1 M sodium cacodylate followed by a third wash in double distilled water for 5 min. The gels were carefully removed from the wells with a spatula and transferred to 1 mL of distilled water in 1.5 mL tubes. Water was removed with a plastic transfer pipette, with a 150-mesh copper grid on the tip, to aid in the retention of the specimens. The specimens were exposed to serial dehydration using ethanol at 70%, 80%, 95%, and 100% for 5 min each, and then infiltrated with a 50:50 mixture of low viscosity Spurr’s resin and 100% ethanol for 30 min. This was followed by a second infiltration with 100% Spurr’s resin for 1 h. The old resin was then replaced with fresh resin and allowed to infiltrate for another 30 min. Finally, the constructs were removed and placed into silicone moulds, which were filled with resin, covered and cured for 48 h at 60 °C. The cured resin blocks were removed from the silicone moulds and semi-thin sections (~3 µm thick) were prepared. The sections were stained with methyl blue to verify the presence of gel before ultrathin sections of 80 nm were prepared and stained with 0.5% uranyl acetate and 3% lead citrate for contrast (LKB Ultrostain or Leica EM Stain machine). Unstained sections were used for better viewing of AgNPs. The sections were mounted onto formvar-coated copper slot grids and viewed under JEOL JEM-2200FS transmission electron microscope.TEM imaging was performed on standard 10 μL GelMA constructs and 10 μL HyStem®-C and standard GelMA with and without 100 μg/mL of AgNPs were obtained using an FTIR-ATR spectrometer . For each condition (n = 2), 100 scans were acquired in the 4000–400 cm−1 range, with a resolution of 4 cm−1.Samples were prepared as previously outlined and pipetted into 6 mm diameter silicone moulds, then stored overnight at −80 °C before freeze-drying for 24 h. Spectra of stiff HyStemn = 3), and a cell viability assay was preformed and measured as described in To determine the concentration of AgNPs producing cytotoxic effects, 10 μL constructs of standard GelMA were prepared with and without 5 million HGF cells/mL, as described. AgNPs were incorporated at 0, 0.1, 0.5, 1, 5, 10, 50, and 100 μg/mL (n = 3). After 48 h incubation at 37 °C/5% CO2, 250 μL of the media supernatant was transferred to a Teflon digestion vessel and conducted using ICP-MS. A standard curve was generated by preparing media containing AgNPs at 0, 0.0002, 0.002, 0.02, 0.2 and 2 μg/mL.ICP-MS was used to determine the concentration of AgNPs released from the GelMA constructs after 48 h of in vitro incubation in 400 μL of DMEM/10% FBS. Constructs of standard consistency of GelMA (5 wt%) at 10 μL in size were prepared with and without the encapsulation of 5 million HGFs/mL. AgNPs were incorporated at the following concentrations: 0, 0.1, 0.5, 1, 5, 10, 12.5, 25, 50, 100 and 200 μg/mL (n = 3) using ImageJ software.TEM imaging was performed on 10 μL GelMA constructs encapsulating 5 million HGFs/mL and containing 0, 0.1, 100, and 200 μg/mL of AgNPs . The aveEscherichia coli DH5α (E. coli), and Staphylococcus aureus Oxford NCTC6571 (S. aureus) were obtained from the University of Otago culture collection, Dental School and sub-cultured on Tryptic Soy agar plates  with incubation at 37 °C/5% CO2 for 24 h. Sixteen hours prior to experiments, the species were inoculated into 10 mL of tryptic soy broth  and incubated at 37 °C/5% CO2.E. coli and S. aureus into GelMA, a bacterial optical density (OD) of 0.01 per ml of hydrogel was calculated. The amount of broth was subtracted from the total volume of PBS used in the GelMA construct. Then, 10 μL constructs were cross-linked at 450 nm for 3 min in a 48-well plate and incubated at 37 °C/5% CO2 for 20 h.Prior to the encapsulation of E. coli and S. aureus were encapsulated in 10 μL constructs of GelMA with 0, 0.1, 0.5, 1, 5, 10, 50, and 100 μg/mL of AgNPs (n = 3) and no bacterium as a background control. The samples were pipetted into a 48-well plate, placed in a sealable plastic bag to maintain humidity, and incubated at 37 °C/5% CO2. After 20 h of incubation, 400 μL of broth with 40 μL of the Prestoblue® regent were added, and the well plates were incubated for another 3 h before recording at the 24 h time point. Metabolic activity was measured by transferring 50 μL of solution from each well plate to a 96-well plate, and fluorescence measurements were undertaken using a Synergy 2 Multi-mode microplate reader with an excitation/emission of 535/615 nm. MICs was measured as the lowest concentration resulting in bacterial growth inhibition. MBCs was determined by spot-plating 10 μL of the broth onto TSA agar plates, followed by incubation for 24 h, and was recorded as the lowest concentration resulting in completely killing the bacteria.To determine the MICs of AgNPs/GelMA, E. coli and S. aureus was measured and adjusted to 0.1 OD. One ml of the adjusted broth was added to 19 mL of TBS and mixed slowly up and down before pouring in an empty sterile Petri dish, and then left to set. Using a biopsy punch, 3 holes measuring 5 mm in diameter were punched per plate. In total, 10 μL of the standard consistency (5 wt%) of un-cross-linked GelMA with the following concentrations of AgNPs, 0, 25, 50, 100, and 200 μg/mL, was pipetted directly inside the punched hole and cross-linked for 3 min. Afterwards, the plates were incubated for 24 h at 37 °C/5% CO2. Images were taken using a Canon camera and the inhibition zone diameters was measured using Image J software.The bacterial OD of p ≤ 0.05 was considered to be statistically significant.The results were reported as the mean ± standard deviation. The statistical analysis of the results was performed using one-way ANOVA with post hoc analysis using GraphPad Prism. After synthesis of the colloidal suspension of AgNPs, their appearance was dark yellow/brown in colour, which indicated stability, as well as good dispersion of the nanoparticles. The hydrodynamic size of AgNPs measured using DLS was determined to be 1–12 nm in size with a concentration ranging between 1800 and 2900 μg/mL. AgNPs were used within 14 days of being produced. TEM images showed spherical particles with an average diameter of 6 nm A,B.®-C and GelMA with AgNPs , 1548 cm−1 (amide II) and 1241 cm−1 (amide III), which are the characteristic absorption peaks of C=O, N–H, and C–N, respectively [−1 is attributed to C=O stretching of the carboxyl groups within the HA [−1, 1082 cm−1 and 1039 cm−1 are assigned to C-O ester association, C-C groups, and C-OH groups, respectively [−1, attributed to the stretching of the hydrogen-bonded hydroxyl groups. The peaks at 1237 cm−1, 1537 cm−1 and 1630 cm−1 are associated with the C–N stretching of amide III, the N–H stretch of amide II, and C=O stretching of amide I, respectively. The spectrum also displayed the characteristic bands of N-H stretching at 3078 cm−1 (amide B), C-H stretching at 2937 cm−1, and 2878 cm−1 (amide A) and C-H deformation were also detected at 1444 cm−1. Other relevant peaks were identified at: 1161 cm−1 (symmetric C-O-C stretching), 1199 cm−1 (ν (O-C-O), and 1336 cm−1 (CH2). The spectra of the GelMA with AgNPs did not show any new peaks corresponding to the AgNPs, but a shift in certain peaks was identified; this occurred around the 3273 cm−1 region (corresponding to the -OH groups). In addition, amide II and amide I also shifted to lower wavelengths of −1529 cm−1, 1629 cm−1, respectively.  constructs without cells containing 0.1–1 µg/mL AgNPs  constructs made at a standard consistency were used as the preferred model due to an even AgNP distribution/retention and suitable handling properties. Concentrations ≥ 0.5 µg/mL of AgNPs encapsulated with HGFs showed significant cytotoxicity, with no cell growth A. An incmL AgNPs B. HoweveHGF encapsulated with 0 and 0.1 µg/mL AgNPs in 5%wt GelMA and (non-toxic concentration) was imaged using TEM A,B. The E. coli and S. aureus encapsulated in 10 µL standard GelMA  which can potentially cause cellular DNA and tissue damage . AdditioThe AgNPs used in this research have an average diameter size of 6 nm, It has been reported that smaller sized AgNPs possess greater antimicrobial properties in comparison to larger-sized AgNPs , due to Current treatment options for infected wounds and bone defects are limited to the surgical debridement of affected tissue and high doses of antibiotics. The presence of biomaterials in situ further complicates the treatment plan. Once infection is established, biomaterials-mediated infections are resistant to treatment, even with high doses of antibiotics and tend to persist until the prosthetic device is removed . It is a®-C: stiff, standard, and soft . The values obtained from our cytotoxicity experiment are considerably higher than values commonly reported in the literature [Cell viability assay did not show any statistical difference between these two different hydrogel models; however, live/dead stains showed a higher percentage of viable cells in the 10 µL of construct size for stiff HyStem00 µg/mL . The addterature ,52, and terature . Riberioterature . The smaterature , we alsoterature .The exact mechanism of the antibacterial action and cytotoxicity of AgNPs is not fully elucidated. Various theories have been reported regarding how AgNPs exert its toxicity action. It was proposed that AgNPs can penetrate the cell, causing cellular structure damage, thus leading to disruption of cell function, and cell death. Another theory states that the cell death is mediated by the induction of reactive oxygen species (ROS) . ROS canE. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) at 50 and 100 µg/mL, respectively. Gram-negative bacteria were previously reported to be more susceptible to AgNPs than Gram-positive bacteria, owing to the difference in the organization of the peptidoglycan. The lower liability of Gram-positive bacteria can be attributed to the fact that the amount of peptidoglycan is comparatively more in Gram-positive than Gram-negative bacteria with a thinner cell wall of 3 nm compared with 30 nm [S. aureus are less inhibited and Gram-negative E. coli shows substantial inhibition even at lower AgNPs concentrations.The disc diffusion assay shows that GelMA-incorporated AgNPs were found to be effective against both th 30 nm . The chath 30 nm . In addith 30 nm . To concS. aureus and 100 µg/mL for E.coli, when AgNPs of 10 nm were encapsulated in a methylcellulose hydrogel [Panacek et al. reported antimicrobial or MIC values of 50 µg/mL for hydrogel . While ≥We found that the composition of hydrogel can greatly influence the cytotoxicity profile interpretation of AgNPs. Photo-cross-linked GelMA stably incorporated AgNPs offering a non-antibiotic bifunctional scaffold with retained antimicrobial action, while allowing ingress and attachment of mammalian cells for multiple regenerative applications. HGFs were more susceptible to the AgNPs toxicity compared with the bacterial cells. An appropriately designed 3D hydrogel construct could be used as a cytotoxicity model to better mimic the in vivo environment compared with a traditional 2D culture.


Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis remains dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of patients with AML. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a proteolysis-targeting chimera (PROTAC) against the bromodomain and extra-terminal domain (BET) family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment arrested cell cycling and enhanced apoptosis and c-MYC was identified as the downstream target. Collectively, our results indicated that dBET1 had broad anti-cancer effects on AML cell lines with different molecular lesions and provided more benefits to patients with AML. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer characterized by blocked differentiation of myeloid lineage -3. Due tNPM1, FLT3, IDH1/2, KIT, BCL-2, TP53, and MDM2) [AML is highly genetically heterogenous. Cytogenetic analysis revealed major mutations, including chromatin translocation, deletions, insertions, and inversions, among others, in approximately 55% of AML cases. Based on cytogenetic analyses, patients with AML were classified into the favorable, intermediate, and unfavorable groups. More recently, progress in genetics of AML has discovered several recurrent mutations in the remaining 45% of patients with AML, with being prognostic factors  -13. Morend MDM2) , 15. Thein vitro and in vivo [The BET family members  are epigenetic readers that recognize acetylated proteins, including histone and nonhistone proteins  -18. The  in vivo . Further in vivo , 23, 25.Recently, a new technology, proteolysis-targeting chimera (PROTAC), has been developed, consisting of a ligand for a protein of interest, a linker, and an adaptor to recruit an E3 ubiquitin ligase. A PROTAC would bring its target protein in close proximity to a E3 ligase, and initiate polyubiquitination and subsequent proteasome-mediated degradation . There aIn this study, we explored the cytotoxic effects of dBET1 on several AML cell lines , representing different molecular subtypes  of AML. Moreover, we identified the inhibition of cell cycling and enhancing apoptosis as being the downstream effector of the degradation of BRD2, BRD3, and BRD4 by dBET1. Our results shed new light on the molecular mechanism of dBET1’s cytotoxicity and expanded the therapeutic potential of dBET1 to more molecular subtypes of AML.2.NB4, MV4-11, Kasumi, and THP-1 cells were purchased from National Collection of Authenticated Cell Cultures and were grown in culture in RPMI 1640 medium  supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate at 37°C in 5% COhttps://depmap.org/portal/) and shown as a box plot by different tissue/organ. Survival curves of adult patients with AML from TCGA with high/low expression of BRD4/c-MYC were generated from GEPIA2 (http://gepia2.cancer-pku.cn/#index).Public expressions data of BRD4 in 1389 cell lines was downloaded from DepMap . After 48 h of treatment, cell viability was tested with Cell Counting Kit-8 (CCK8) kit (Dojindo Molecular Technologies). Absorbance at 450 nm was applied to estimate the cell viability, which was normalized to DMSO control. Cell viability assay was performed in triplicate, and each assay was repeated at least three times. The half-maximal inhibitory concentration (IC50) of dBET1 was calculated using Graph Prism version 8 .AML cell lines were plated at a density of 2 × 103 cells per well) in six-well plate of soft agar supplemented with variable concentrations of dBET1. After 10 days, cells were first fixed with 100% methanol for 15 min, followed by Giemsa staining for 1 h. Images were acquired using an optical microscope, and the number of colonies were manually counted.NB4, MV4-11, Kasumi, and THP-1 cells were seeded with the same density (4 × 10Short hairpin RNA (shRNA) targeting CRBN (the sequences: CCGGGCCCACGAATAGTTGTCATTTCTCGAGAAATGACAACTATTCG) was cloned into pLKO.1 plasmid , and cDNp value <0.05 and absolute log2 (fold change) > 1. Gene Ontology (GO) and gene set enrichment analysis (GSEA) with Hallmarks gene sets (version 7.1) were conducted using R/Bioconductor package cluster Profiler [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE196693). The data can be accessible with a token upon request.Total RNA was extracted using the RNeasy Mini kit . Library preparation, transcriptome sequencing (Illumina NovaSeq 6000) and clean data filtering were carried out by Novogene Bioinformatics Technology Co., Ltd. . The 150 bp paired-end reads were aligned to hg38 (Ensembl) genome using HISAT2 software (version 2.2.0). Transcriptome assembly and abundance analysis were performed with StringTie software (version 2.1.2). Differentially expressed genes were identified by R/Bioconductor package DESeq2 with adjusted Profiler . HeatmapNB4, MV4-11, Kasumi, and THP-1 cells were treated with dBET1 for 24 h and were then fixed with 70% ethanol and incubated at 4°C for overnight, followed by washing with cold 1× phosphate-buffered saline (PBS) buffer. Fixed cells were then incubated with 1.5 μM propidium iodide (PI)  solution supplemented with RNase A (25 μg/ml) at room temperature for 1 h. Cell cycle distribution was determined using flow cytometry and analyzed using MultiCycle AV DNA analysis software .Cell apoptosis was analyzed as previously described . BrieflyTotal RNA was purified using the RNeasy Mini Kit . First-strand cDNA was generated using a mix of 2 μg of total RNA, 500 ng of random primers , 200U of M-MLV reverse transcriptase , and 20U of RNase inhibitor  in a total volume of 20 μl. qRT-PCR was performed using LightCycler® 480 SYBR Green I Master mix  on a Light cycler 480 Real-Time System  according to the standard protocol. Quantitative mRNA expression of MYC  was calculated using the Ct method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression  as an internal reference.Cells were lysed using RIPA buffer containing protease and phosphatase inhibitor cocktail  for 30 min on ice. The supernatant was collected by centrifugation as total protein and the concentration of total protein was quantified using the Pierce BCA Kit (Thermo Fisher Scientific). Blotting was performed as previously described . Primaryp values were estimated by two-tail t test and <0.05 was regarded as statistically significant . Error bar was indicated by Means ± Standard Deviation (SD).All experiments were performed in triplicates and independently repeated at least three times. Statistical analyses were performed using GraphPad Prism (version 8) . p = 0.044)  .50: 0.1483 μM, NB4 IC50: 0.3357 μM, THP-1 IC50: 0.3551 μM, and MV4-11 IC50: 0.2748 μM)  using Western blotting . BRD4 wa2748 μM) .50 values of dBET1 in these four cell lines: Kasumi (0.1483 μM), followed by MV4-11 (0.2748 μM), NB4 (0.3357 μM), and THP-1 (0.3551 μM)  has cytotoxic effects on AML cell lines, we determined the protein levels of the BRD family in four AML cell lines treated with increasing concentrations of dBET1. Efficient degradation of the BRD family in a dose-dependent manner was observed in all four cell lines, with the most efficiency observed in Kasumi cells, followed by MV4-11, NB4, and THP-1, which conforms to the IC3551 μM) . Besides3551 μM) .To characterize the effects of dBET1 on cell proliferation, we conducted soft agar colony formation assay with incremental dosages of dBET1. The numbers of colonies significantly decreased after treatment with dBET1 in a dosage-dependent manner in the four AML cell lines  . These rTo further define the effects of dBET1 on cell proliferation, we first assessed cell cycle distribution after dBET1 treatment. Treatment with dBET1 significantly decreased the percentage of cells in the S phase compared with that treated with DMSO control in a dosage-dependent manner. In contrast, the percentage of cells in the GO/G1 phase significantly increased after dBET1 treatment. Collectively, these results showed that inhibiting BET family by dBET1 arrested the cell cycle of the four AML cell lines tested. Moreover, we performed flow cytometry using Annexin V and PI to detect apoptosis at 24 h after dBET1 treatment (1 μM and 8 μM) in the four AML cell lines. Treatment with dBET1 induced more apoptotic cells in a dosage-dependent manner (The BRD family plays important roles as a modulator of chromatin remodeling and transcriptional regulation of critical genes in cellular homeostasis and disease progression . Thus, lThe OS rate of AML is poor compared with those of other acute leukemias. Except for APL and CBF AML, the prognosis of AML remains poor, with only approximately 60% of patients progressing to remission and approximately 30% of patients achieving long-term remission , 38. TheBRD4 has emerged as a therapeutic target in AML. In this study, we explored the expression of BRD4 across different cancer cell lines and found that BRD4 was highly expressed in AML cell lines, indicating the dependence of AML cell lines on BRD4 for survival and the usage of BRD4 as a potential therapeutic target. Besides, high BRD4 expression was significantly associated with worse OS of adult patients with AML than low BRD4 expression. These results suggested that high BRD4 expression conferred a survival advantage of AML cell lines and represented as a vulnerability. As expected, inhibiting the BET family proteins using dBET1 demonstrated efficient cytotoxic effects on AML cell lines. Previously, dBET1 has shown cytotoxic effects on MV4-11 cells, without knowing its underlying molecular mechanism . In thisTo further explore the downstream pathways affected by dBET1 treatment, we compared the transcriptomes of AML cell lines with or without dBET1 treatment using RNA-seq. Of note, cell cycle and apoptosis are two enriched pathways because of dBET1 treatment which agrees with the results of the colony formation assay, cell proliferation, cell cycle, and apoptosis analyses. Besides, c-MYC was a well-known downstream target of the BET family, which was also confirmed in this study. Given that c-MYC was an oncogene and played important roles in promoting cell proliferation of cancer cells , the dowThe BCL6 gene was essential for the survival and self-renewal of AML cell lines, especially those with high expression of BCL6 . InteresIn conclusion, we tested the cytotoxic effects of dBET1 on several AML cell lines representing different molecular subtypes of AML and mechanistically explored the downstream effector of dBET1. Our results revealed the therapeutic potential of dBET1 for AML with different molecular lesions.


Importantly, 16 traits can be good indicators for screening of cold–resistant genotypes of maize. Gene expression analysis showed that GRMZM2G059991, GRMZM2G089982, GRMZM2G088212, GRMZM2G396553, GRMZM2G120578, and GRMZM2G396856 involved in antioxidant enzymes activity and PAs metabolism, and these genes may be used for genetic modification to improve maize cold resistance. Moreover, seven strong cold–resistant genotypes were identified, and they can be used as parents in maize breeding programs to develop new varieties.Maize is a cold-sensitive crop, and it exhibits severe retardation of growth and development when exposed to cold snaps during and right after seedling emergence. Although different agronomic, physiological, and molecular approaches have been tried to overcome the problems related to cold stress in recent years, the mechanisms causing cold resistance in maize are still unclear. Screening and breeding of varieties for cold resistance may be a sustainable option to boost maize production under low-temperature environments. Herein, seedlings of 39 different maize genotypes were treated under both 10 °C low temperature and 22 °C normal temperature conditions for 7 days, to assess the changes in seven growth parameters, two membrane characteristics, two reactive oxygen species (ROS) levels, and four antioxidant enzymes activities. The changes in ten photosynthetic performances, one osmotic substance accumulation, and three polyamines (PAs) metabolisms were also measured. Results indicated that significant differences among genotypes, temperature treatments, and their interactions were found in 29 studied traits, and cold–stressed seedlings were capable to enhance their cold resistance by maintaining high levels of membrane stability index (66.07%); antioxidant enzymes activities including the activity of superoxide dismutase (2.44 Unit g Zea mays), originating from tropical and subtropical regions, is extremely sensitive to cold stress, particularly at the seedling emergence stage  to 37.59% (CAT activity) under the normal temperature of 22 °C and from 8.43% [water use efficiency (WUE)] to 40.13% (CAT activity) under low-temperature stress of 10 °C for the 29 tested traits. We hypothesized that there may be higher broad sense heritability for 22 tested traits including seedling length, root length, root number, seedling fresh weight, root fresh weight, plant biomass, root–shoot ratio, Chl a content, Chl b content, Chl a+b content, net photosynthetic rate (Pn), ribulose Rubisco activity, malondialdehyde (MDA) content, O2•− production rate, H2O2 content, SOD activity, POD activity, CAT activity, APX activity, Pro content, spermidine (Spd) concentration, and Spm concentration since their CVg were above 15% in one or two temperature environments.The genetic differences were confirmed by the genetic variation coefficient (CVng stage . The CVgSeedlings of 39 maize genotypes were subjected to low-temperature stress (10 °C) for 7 days, and the results revealed that seedlings’ growth and development were in general significantly inhibited . Compare2 (Ci) was 7.2% higher in cold–stressed seedlings relative to untreated seedlings  reflect the degree of lipid peroxidation and membrane stability under various environmental stresses ,21. Cold2•− production rate increased by 163.4% in seedlings of 39 maize genotypes compared with the control  correlation between both traits  were conducted under both temperature treatments a–d. Pearh traits a,b. Thish traits c,d. Specij). The results showed that the cold resistance (Uj) of 39 maize genotypes ranged from 0.152 (M1012 genotype) to 0.743 (M0504 genotype), and their coefficient of variation was 34.2% , type II included six moderate cold–resistant genotypes , type III included seven weak cold–resistant genotypes , and type IV included 19 cold–sensitive genotypes , respectively (The cold resistance coefficient (CRC), is a measure of cold stress effects based on the reduction of each trait under cold stress treatments in comparison to un–stress treatments, and thereby was used for the identification of cold–resistant genotypes . The memas 34.2% b. Therefectively b.arginine decarboxylase 1 , spermidine synthase 1 , probable polyamine oxidase 2 , superoxide dismutase 3 , peroxidase 72 , and catalase 1  were examined in maize seedlings of M1012 (cold–sensitive genotype) and M0504 (strong cold–resistant genotype) after treated these seedlings with 10 °C and 22 °C for 7 days. These genes were associated with PAs metabolisms and antioxidant enzymes system and were known to play a role under cold stress. The results showed that the relative expression levels of these six genes were significantly increased in leaves of both maize genotypes seedlings after low temperature treatment (10 °C) (The expression of six candidate cold–induced genes including  (10 °C) .Low temperature is among the major abiotic stresses that limited the productivity, quality, as well as distribution of maize . GeneralIt is well understood that low temperature affected maize seed germination, early leaf development, and seedling growth ; leaf sh2O2 (159.2%) and O2•− (163.4%) levels in leaves of 39 maize genotypes seedlings. Excessive ROS production may be the main factor of membrane lipid peroxidation in the leaves of maize seedlings during chilling damage. Moreover, ROS promoted the degradation of polyunsaturated fatty acids and the production of MDA  in a small tube. Absorption was measured at 470 nm using pH 7.0 phosphoric buffer as a zero point to calculate POD activity. A 0.2 mL aliquot of enzyme extract was added to 2.8 mL H2O2 (0.067 M) for monitoring the decrease of absorbance at 240 nm for 1 min caused by the decomposition of H2O2. CAT activity was calculated subsequently. A 0.1 mL aliquot of enzyme extract was mixed thoroughly with 2.9 mL reaction solution . Then APX activity was measured by observing the decrease in absorbance at 290 nm.A 0.5 g of leaves was homogenized in 1 mL of ice-cold potassium–phosphate buffer  containing potassium chloride (100 mM), ascorbate (1 mM), β-mercaptoethanol (5 mM), and glycerol , kept in an ice bath for 1 h, and then centrifuged at 12,000 rpm  for 30 min at 4 °C. The supernatant was stored at –70 °C for PAs concentration measurements by high-performance liquid chromatography   and temperatures [T]) and their G × T interaction, and to perform PCA, respectively. The violin plot, correlation coefficient, and interactive ring correlation diagrams of all traits were prepared using the Genescloud tool, a free online platform for data analysis  [M–LT and T M–CK were the values of corresponding trait in a genotype under the low temperature and normal temperature treatments, respectively. According to Yan et al. [ij) of the i–th trait for the j–th maize genotype was calculated as follows:LTij and TCKij were the values of the i–th trait for the j–th maize genotype evaluated under low temperature and normal temperature treatment, respectively. Maize cold–resistance in seedling stage was also evaluated by the membership function value [ij was the membership function value of the i–th trait for the j–th maize genotype for cold resistance; CRCi-min and CRCi-max were the minimum and maximum values of the cold resistance coefficient of the i-th trait, respectively, and when the i–th trait was positively or negatively correlated with cold resistance of maize, Equation (7) or Equation (8) was estimated, respectively; jU was the average value of the membership function of measured traits for the j–th maize genotypes for cold resistance. The genetic variation coefficient (CVg) of single trait among all maize genotypes under each treatment was calculated as follows:https://sourceforge.net/projects/mev-tm4/, accessed on 20 May 2022).For all traits under both temperature treatments, we used the IBM-SPSS Statistics v19.0  . Relativry 2022) :RC = (TMis (HCA)  was perfp < 0.01 or p < 0.001) in multiple agronomic and physiological traits, and the extent of changes in the abovementioned traits were genotypes dependent (p < 0.01 or p < 0.001); thereby, different maize genotypes improved cold resistance through different mechanisms according to their genetic constitution. The strong cold-resistant maize genotypes accumulated higher levels of MSI, CAT activity, POD activity, Chl a content, Chl b content, Chl a+b content, Pn, Rubisco activity, Tr, WUE, Put concentration, Spd concentration, Spm concentration, seedling length, seedling fresh weight, and plant biomass, which could be good indicators for screening of cold–resistant genotypes of maize. Gene expression analysis using RT–qPCR also showed that the six candidate genes, i.e., GRMZM2G059991, GRMZM2G089982, GRMZM2G088212, GRMZM2G396553, GRMZM2G120578, and GRMZM2G396856 that involved in antioxidant enzymes activity and PAs metabolism, may be used for genetic modification to improve maize cold resistance. Moreover, the seven genotypes, including M0504, F0305, M02021, M1017, F0801, F1230, and lg104-1, with the strongest resistance to low temperature, can be used as parents in breeding programs to develop new maize varieties.In summary, maize is a cold sensitive crop that shows little capacity to acclimate to low growth temperature. In this study, we found that maize seedlings exposed to 10 °C low temperature stress led to notable changes (


We characterized Müller cell gliosis induced by the activation of transient receptor potential vanilloid-type 4 (TRPV4) and assessed whether statins could modulate the gliosis. The human Müller cell line, MIO-M1, was used to analyze the gliosis caused by glaucomatous stimulation. To induce Müller gliosis in MIO-M1 cells, GSK101 was used to activate TRPV4, and Müller gliosis was evaluated by analyzing vimentin, nestin, and glial fibrillary acidic protein (GFAP) expression. The expression level of TNF-α was determined by ELISA. To evaluate the GSK101 activation of the NF-κB pathway, p65 phosphorylation was measured by Western blotting, and the nuclear translocation of p65 and IκBα phosphorylation were assessed by immunostaining. To assess the effect of statins on MIO-M1 gliosis, cells were pretreated for 24 h with statins before GSK101 treatment. Vimentin, nestin, and GFAP expression were upregulated by GSK101, while statins effectively inhibited them. The expression of TNF-α was increased by GSK101. The phosphorylation and nuclear translocation of p65 and IκBα phosphorylation, which occurs prior to p65 activation, were induced. Statins suppressed the GSK101-mediated phosphorylation of IκBα and p65 translocation. Statins can mitigate gliosis in the human Müller cell line. Because TRPV4 activation in Müller cells reflects glaucoma pathophysiology, statins may have the potential to prevent RGC death. Glaucoma is a progressive neurodegenerative eye disease characterized by irreversible structural damage to the retinal ganglion cells (RGCs) ,2. RGCs RGCs receive physiological and pathological support from glial cells, such as astrocytes and Müller cells. Healthy Müller cells are specialized radial glial cells that span the entire retina and provide metabolic support. However, changes in Müller cells are involved in almost all types of retinal degeneration, such as age-related macular degeneration, diabetic retinopathy, and glaucoma, as well as normal retinal function ,4. When Because the transient receptor potential vanilloid-type 4 (TRPV4) channel contributes to the resting membrane potential of Müller cells, its specific agonist, GSK1016790A (GSK101), was used to activate gliosis ,16. ThenNF-κB (p50/p65), an inducible nuclear transcription factor, regulates inflammatory factors, including TNF-α ,22. NF-κStatins, widely used as hyperlipidemia treatments, not only regulate cholesterol biosynthesis but also exert pleiotropic effects, such as fibrosis, inflammation, and immune response control ,24,25. SIn this paper, we assessed whether Müller gliosis could be induced by the activation of TRPV4, a mechanosensor that may respond to increased IOP. To do this, we evaluated the expression of gliosis markers, TNF-α expression, and the activation of the NF-κB pathway. Then, we determined whether statins could modulate TRPV4-mediated Müller gliosis.To evaluate Müller gliosis by TRPV4 activation, the expression of the Müller gliosis indicators vimentin, nestin, and GFAP was analyzed by immunostaining. Treatment with the TRPV4 agonist GSK101 upregulated vimentin, nestin, and GFAP expression A–C. PretThen, we asked how the response of Müller gliosis to TRPV4 activation leads to detrimental effects on RGC. The most plausible candidate is TNF-α, because it can induce RGC apoptosis ,10,11,38Because we confirmed that TRPV4 activation induces NF-κB activation and TNF-α secretion, and statins inhibit these events, we determined which step of the NF-κB pathway was inhibited to suppress TNF-α expression and consequently protect RGCs. NF-κB forms homo- or hetero-dimers with DNA-binding proteins. p65 is a major component of these dimers; when NF-κB is activated, p65 is phosphorylated, and the complex translocates to the nucleus. Therefore, we assayed p65 phosphorylation in cytosolic and nuclear fractions with GAPDH and lamin B1 as respective internal controls. We observed the robust induction of p65 phosphorylation in both the cytosolic and nuclear fractions by treatment with GSK101 in a dose-dependent manner . These dBecause statins can suppress TNF-α induction, we tested whether they could suppress GSK101-mediated p65 phosphorylation. Treatment with 25 nM GSK101 consistently induced p65 phosphorylation in cytosolic and nuclear fractions A–C. PretTo confirm that statins suppress the NF-κB pathway, we assessed the phosphorylation of the inhibitory protein IκBα by immunocytochemistry. IκBα functions as an inhibitor through interaction with the NF-κB complex. However, IκBα phosphorylation by stimulatory signals promotes IκBα ubiquitination and degradation, which induces NF-κB complex activation. Consistent with Glaucoma can cause blindness due to progressive, irreversible optic nerve damage. The major risk factor for glaucoma is increased IOP, which is the only adjustable factor to modify glaucoma development and progression. Proposed mechanisms linking RGC injury and IOP elevation include the compressive effect of IOP on the lamina cribrosa , pressurStatins are prescribed for hyperlipidemia treatment; they lower blood cholesterol by competitively inhibiting HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Since statins are structurally similar to HMG-CoA, the active site of the enzyme competes with the native substrate. This competition reduces the rate at which mevalonate is produced, the molecule needed to synthesizes cholesterol. In addition, the inhibition of HMG-CoA by statins hampers the downstream synthesis of isoprenoids, which are crucial lipid attachments for intracellular signaling molecules, such as Rho, Rac1, and CDC42 ,44. The Considerable clinical evidence indicates that statins have beneficial effects on glaucoma patients. De Castro et al.  showed tMüller glia play crucial roles in RGC survival, and Müller gliosis contributes to glaucoma pathogenesis ,52. MüllWe hypothesized that TNF-α production in Müller glia is involved in this mechanism because TRPV4 activation increases the secretion of proinflammatory cytokines, including TNF-α, from glia in other cells . AlthougTNF-α is a well-known proinflammatory cytokine with multiple functions in the immune response, and its expression is induced by transcription factors in the NF-κB pathway p50–p65 complex) ,69,70. I5 complexWe also confirmed that TNF-α induction by GSK101 in MIO-M1 cells is mediated by the NF-κB pathway, which statins inhibit by attenuating IκBα phosphorylation. These findings agree with previous studies ,83,84. S2+ concentration, which increases calpain [This study has some limitations. First, we used a Müller cell line, MIO-M1, rather than primary Müller cells. Therefore, in our next study, we will check whether the same action occurs in primary Müller cells through primary Müller cell culture or in vivo and, furthermore, in animal experiments. Numerous studies in the field of neuroinflammation have shown the potential of statins ,86, but  calpain . There i calpain ; therefoIn conclusion, TRPV4 activation reiterates glaucomatous stimulation by GSK101 in human MIO-M1 cells, increasing TNF-α expression, and statins inhibit this expression by interfering with IκBα phosphorylation. Numerous studies have shown that TNF-α signaling is involved in glaucomatous neurodegeneration and is thus a possible treatment target to improve neuroprotection ,71,72,73TM DMEM medium supplemented with 50 U/mL penicillin, 50 g/mL streptomycin  and 10% fetal bovine serum  [2 in a humidified incubator until the cells reached 80% confluency. The cells were detached by treatment with TrypLE , and the resulting cell suspension was protected with a neutral trypsin solution (Invitrogen-Gibco-Life Science Technology) [MIO-M1, a human Müller glial cell line, was grown in GlutaMAXhnology) . MIO-M1 hnology) .For investigation of the NF-κB pathway, IκBα, p65, and phospho-p65 antibodies were obtained from Cell Signaling Technology . Vimentin, nestin, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. . The Human Quantikine TNF-α ELISA kit was obtained from R&D Systems . The GFAP antibody and RIPA Cell Lysis buffer were purchased from ThermoFisher Scientific . GSK1016790A (GSK101) was purchased from Sigma-Aldrich .MIO-M1 cells were seeded on 12 mm glass coverslips placed in 24-well culture plates in growth medium. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After three washes with phosphate-buffered saline (PBS), the cells were incubated for 1 h at 37 °C with primary antibodies (1:50) in PBS with 1% bovine serum albumin (BSA). After washing three times in PBS for 5 min each, the cells were incubated for 1 h at room temperature with a secondary antibody  (1:100) in PBS. The gliosis of MIO-M1 cells was confirmed through positive staining for vimentin, nestin, and GFAP. Cells were incubated with 1 μg/mL 4′,6-diamidino-2-phenylindole dihydrochloride  to stain the nuclei. The coverslips were mounted with FluorSave , and fluorescent images were captured using an inverted microscope  and analyzed with Zeiss ZEN imaging software (Carl Zeiss Microscopy GmbH).TM NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, cell lysis was demonstrated using the CER-I and CER-II lysis buffers of the NE-PER kit. The tube containing CER-I was centrifuged for 5 min at ~16,000× g in a microcentrifuge and the supernatant (cytosol) was transferred to a new Eppendorf tube. After suspending the insoluble fraction, ice-cold NER was added, and the cells were incubated for 40 min. After adding CER-II lysis buffer, centrifugation took place for 10 min at 16,000× g, the supernatant (nuclei) was transferred to a new Eppendorf tube, and each sample was analyzed by Western blotting.MIO-M1 cells were seeded in 6-well culture plates. Cells were pretreated with statins followed by 25 nM of GSK101, a TRPV4 agonist. Nuclei and cytosols were separated using the PierceEach cytosolic and nuclear sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes , which were blocked in BSA for 1 h. The blots were incubated with antibodies against phospho-IκBα (p-IκBα), GFAP, and GAPDH ; p65 ; and phospho-p65  in blocking solution overnight at 4 °C. The membranes were washed with PBST and then incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The membranes were developed using the Enhanced Chemiluminescence (ECL) detection system . Band densities were quantified using FUJIFILM Science Lab Image Gauge Ver. 4.0 .MIO-M1 cells were cultured in 6-well plates and then treated with GSK101 for 24 h or statins for 1 h followed by GSK101. The MIO-M1 cell culture medium was analyzed for TNF-α using an ELISA kit  according to the manufacturer’s instructions . The abst-tests and one-way ANOVAs were used for statistical comparison. All statistical analyses were performed using an ANOVA test with Prism 6.05 . * p < 0.05 and ** p < 0.001 were considered significant.All values are expressed as means ± standard deviations (SD). The results represent three separate experiments conducted under the same conditions. Unpaired


Beauveria bassiana, a filamentous fungal insect pathogen that serves as the main source of wide-spectrum fungal insecticides. The results demonstrated that these three genes are interrelated at the transcriptional level under normal and heat-shocked conditions. Meanwhile, all the deletion mutants showed significant but differential changes in cell wall integrity, antioxidant activity, hyphal tolerance to carbendazim fungicide, conidial tolerance to 45 °C wet heat and virulence. However, only Δhsp30b showed growth defects on rich and minimal media at 25 °C and Δhsp30a displayed the reduction in conidiophores and conidia. Moreover, the single deletion of hsp30a and hsp30b caused the decreases in hyphal growth at 34 °C and conidial tolerance to UV-B irradiation. Our findings provide a global insight into vital roles of hsp20, hsp30a, and hsp30b in asexual development, environmental adaptation, and fungal virulence of B. bassiana.Small heat shock proteins (sHsps) containing conserved α-crystallin domain play important roles in many cellular processes, but little is known about the functions of sHsps in filamentous entomopathogens. Here, three sHsps of Hsp20, Hsp30a, and Hsp30b were characterized in  Saccharomyces cerevisiae, the sHsp system consists of Hsp26 and Hsp42 [Δhsp26 in S. cerevisiae demonstrated that this gene had no effect on the spore development and germination, cellular growth at various temperatures, thermoresistance during sporulation, and tolerance to ethanol, but is important for the cell morphology under normal and high temperatures [S. cerevisiae Δhsp42 cells showed abnormal morphology [Candida albicans, only one sHsp with α-crystallin domain was identified, namely Hsp21 [hsp21 resulted in various phenotypic changes, including the defects in hyphal formation and cellular growth under conditions of nutrient limitation, the decrease in the resistance to heat shock and kinds of chemical stressors , the delay in germ tube formation under the stress of Hsp90 inhibitor, and reduced pathogenicity, which suggests Hsp21 contributes to thermotolerance, adaptation to oxidative stress, ethanol-induced stress, and antifungal drugs, growth under conditions of nutrient limitation, hyphal formation, and fungal virulence in C. albicans [hsp21 prevented phosphorylation of Cek1 at elevated temperatures and affected the homeostasis of glycerol under osmotic stress, glycogen under oxidative stress, and trehalose under thermal stress [Ustilago maydis, Hsp20, only one sHsp containing α-crystallin domain, positively regulated the fungal virulence and fungal tolerance to hydrogen peroxide [Neurospora crassa, inactivation of hsp30, encoding an α-crystallin-related heat shock protein, showed poor survival under the combined stresses of heat shock and the restriction of glucose and accumulated unphosphorylated glucose at high temperature by reducing the hexokinase activity [Heat shock proteins (Hsps) are a family of stress proteins that are synthesized by cells when exposed to stressful conditions . Accordieratures . Howeverrphology . In Candly Hsp21 . The disperoxide . In Neuractivity ,12. ThesBeauveria bassiana, a classic filamentous entomopathogenic fungus, has been commercially severed as the main source of many eco-friendly mycoinsecticides [B. bassiana [B. bassiana due to differential roles in the maintenance of cell wall integrity, antioxidant activity and cellular cation homeostasis [B. bassiana possesses 28 Hsps consisting of 3 sHsps, 6 Hsp40s, 1 Hsp60, 15 Hsp70s, 1 Hsp90, and 2 Hsp100s [cticides . The actcticides . Therefobassiana ,16. In aeostasis . These l Hsp100s . HoweverB. bassiana genome [S. cerevisiae Hsp26 and Hsp42. Hsp20, Hsp30a, and Hsp30b consist of 432, 197, and 232 amino acids with molecular masses of 47.04, 21.78, and 25.72 kDa and isoelectric points of 6.45, 6.05, and 5.49, respectively, and were predicted to locate in the cytoplasm. Comparisons of the amino acid sequences and a phylogenetic analysis revealed that B. bassiana Hsp20, Hsp30a, and Hsp30b share 25.66–47.41% sequence identity with one another and 22.88–93.91% sequence identity with 23 other Hsp20s or Hsp30s in fungi and Hsp20 was subdivided into the ScHsp26 clade whereas Hsp30a and Hsp30b were subdivided into the ScHsp42 clade , Hsp30a (EJP62362), and Hsp30b (EJP69022), containing the α-crystallin domain were found in a genome  with the42 clade .hsp20, hsp30a and hsp30b were identified and confirmed by PCR  analyses  under normal and heat-shocked conditions. In n the WT A. In Δhsn the WT A. In Δhsn the WT A. These 50s; min) for conidial tolerance to a wet-heat stress at 45 °C in Δhsp20, Δhsp30a, and Δhsp30b were shortened by 9.68%, 22.57%, and 23.38%, respectively  than that of the WT  of the concentration required to inhibit colony growth by 50% in Δhsp20, Δhsp30a, and Δhsp30b were lowered by 20.36%, 54.83%, and 54.61%, respectively, compared with the estimate of WT -labeled mannose residues increased by 47.39%, 15.09%, and 20.00% on the conidial surfaces of Δhsp20, Δhsp30a, and Δhsp30b, respectively, compared with the WT , Δhsp30a and Δhsp30b exhibited 44.53% and 36.40% reduction in conidial tolerance to UV-B irradiation, respectively, compared with the WT estimate  in many filamentous fungi such as naporthe . S. cerenditions A. Meanwhf hsp30b A. The alnditions A. AdditiΔhsp30b displayed a moderate defect in hyphal growth and only Δhsp30a showed the reduction in conidial yields  and its mutants were cultivated in SDAY consisting of 4% glucose, 1% peptone, 1% yeast extract, and 1.5% agar at 25 °C in a light/dark cycle of 12:12 h for normal growth and conidia germination. All the above strains were also grown in CZA containing 3% sucrose, 0.3% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.05% MgSO4, and 0.001% FeSO4 plus 1.5% agar or 1/4 SDAY (amended with 1/4 of each SDAY nutrient) for their responses to nutritional and chemical stresses at the same regime as SDAY. Escherichia coli DH5α  was cultured in Luria-Bertani (LB) medium at 37 °C for plasmid propagation. Additionally, all chemicals used in this study were analytical grade and purchased from Sigma  unless indicated otherwise.The WT strain of B. bassiana Bb2860 [S. cerevisiae. The identified genes  were amplified from Bb2860 via PCR with designed primer pairs (http://blast.ncbi.nlm.nih.gov/blast.cgi (accessed on 20 March 2022)), followed by phylogenetic analysis with sHsp containing α-crystallin domain from other fungi in the NCBI protein database using a neighbor-joining method in MEGA 7.0 software . The molecular size and theoretical isoelectric point of the deduced proteins were predicted at https://web.expasy.org/protparam/ (accessed on 12 April 2022) and their subcellular localization was predicted at https://wolfpsort.hgc.jp/ (accessed on 13 April 2022).Three α-crystallin domain-containing sHsps were found in the genome of a Bb2860  using Hser pairs  and sequHsp20, hsp30a, and hsp30b were deleted by homologous replacement as described previously [bar cut with appropriate restriction enzymes , yielding three deleted plasmids of p0380-5′hsp20-bar-3′hsp20, p0380-5′hsp30a-bar-3′hsp30a, and p0380-5′hsp30b-bar-3′hsp30b for the deletion of hsp20, hsp30a, and hsp30b, respectively. The full-length coding sequences of hsp20, hsp30a, and hsp30b with its flanking regions were amplified from the genome of WT and ligated into the plasmid of p0380-sur-gateway to exchange for the gateway fragment under the action of Gateway®® BP Clonase™ II Enzyme Mix (Invitrogen), forming complemented plasmids of p0380-sur-hsp20, p0380-sur-hsp30a, and p0380-sur-hsp30b for targeted gene complementation. All paired primers used for gene deletion and complementation are listed in Agrobacterium-mediated transformation [bar resistance to ammonium glufosinate (200 μg/mL) and the sur resistance to chlorimuron ethyl (10 μg/mL), respectively, followed by the identification via PCR and qRT-PCR with paired primers (eviously . Brieflyormation . Putativ primers . Positiv7 conidia mL−1 (the same used below unless specified) was initially spread on SDAY with cellophane and incubated at 25 °C and 12:12 h for 3 days. For a heat shock treatment, the 3-days-old hyphal cultures were exposed to 40 °C for 1 h. The total RNA was extracted from the cultures with RNAiso™ Plus Reagent  and reversely transcribed into cDNAs with PrimeScript®® RT reagent kit (TaKaRa), respectively. The transcriptional levels of each gene were determined by qRT-PCR with paired primers (®® Premix Ex TaqTM (TaKaRa). The transcript of fungal γ-actin gene was used as an internal standard. The relative transcript levels of each gene in the deleted and complementary mutants were calculated based on the 2−ΔΔCt method with respect to the WT standard [An aliquot of 100 μL of a conidial suspension of 10 primers  under thstandard .6 conidia mL−1 was dropped on the plates (9 cm diameter) of SDAY, CZA, and modified CZA media with different carbon or nitrogen sources. The modified CZA media originated from the standard CZA by replacing sucrose with glucose, galactose, glycerol, fructose, trehalose, maltose, or acetate (NaAc) as the sole carbon source, replacing NaNO3 with NaNO2 or NH4Cl as the sole nitrogen source, and removing sucrose (carbon starvation) and NaNO3 (nitrogen starvation), respectively. After 8-day incubation at 25 °C, the diameter of each colony was recorded using two measurements taken perpendicular to each other across the center of colony. Additionally, SDAY plates with 1 μL of conidial suspension were also incubated at 34 °C and 12:12 h for 8 days, followed by measuring colony diameters as a growth index of each strain at the high temperature. All the experiments were conducted in triplicate.A suspension of 1 μL of 102 plate culture. All the experiments were conducted in triplicate.For quantification of the capacity for conidiation, 100 μL of conidial suspension was spread evenly on SDAY plate (9 cm diameter) and cultured at 25 °C for 7 days. From day 4 onwards, three 5 mm-diameter plugs were randomly taken from each plate daily using a cork borer and immersed into 1 mL of 0.02% Tween 80. After the vibration and removal of hyphal debris by filtration, the number of conidia was measured using a hemocytometer and converted to the number of conidia per cm50. All the experiments were conducted in triplicate.To assess the quality and viability of conidia from SDAY plates, 50 μL of conidial suspension was uniformly spread on GM plates (6 cm diameter) consisting of 2% sucrose, 0.5% peptone, and 1.5% agar. Percentage germination was measured every 2 h under the microscopy. The time required for 50% conidial germination of each strain was calculated by model fitting analysis and was defined as GT2O2 (20–80 mM), carbendazim (0.4–2 μg/mL), and NaCl (0.4–2 M). All plates were cultured at 25 °C for 6 d. The diameter of each colony was measured and an EC50 for each chemical to inhibit 50% of the colony growth was calculated by modeling analyses of relative growth indices of each strain over the chemical gradient. All the experiments were conducted in triplicate.100 μL of conidial suspension was spread on cellophane overlaid SDAY plates and incubated at 25 °C. After 3 days, hyphal discs (5 mm diameter) were cut off from the culture and attached to the plates (9 cm diameter) of 1/4 SDAY alone (control) or 1/4 SDAY supplemented with a concentration of Congo red (0.5–3 mg/mL), menadione (2–8 mM), H2O2, 1.2 M NaCl. After 24 h incubation at 25 °C, the relative germination was calculated as the ratio of percent germination under each stress over that in the control. Conidial thermotolerance and UV-B resistance were quantified as LT50 after exposure to 45 °C wet heat for 0–120 min and LD50 after exposure to UV-B irradiation at 0–0.8 J cm−2, as described previously [G. mellonella larvae by normal cuticle infection, as described previously [100 μL of conidial suspension of each strain was evenly spread on GM plates alone (control) and containing 1.0 mg/mL Congo red, 0.2 mM menadione, or 4 mM Heviously . Conidiaeviously . All the4 labeled conidia were quantified on the flow cytometer FC 500 MCL  using an argon laser at the excitation/emission wavelengths of 488/530 nm. Finally, cell walls of aerial conidia were examined via transmission electron microscopy as described previously [Several methods were used to reveal the differences in cell wall integrity in the WT and mutants. First, the fragility of the cell wall was assessed using a method of cell wall degradation . Hyphal −1) of SODs and CATs were assayed as described previously [For antioxidant enzyme activities, the total activity  and subjected to a one-factor (strain) analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test. Results were statistically significant when B. bassiana and are transcriptionally interrelated under normal and heat-shocked conditions. Moreover, hsp20, hsp30a, and hsp30b played important but differential roles in sustaining cell wall integrity, antioxidant activity, fungal tolerances to high temperature and carbendazim fungicide, and fungal virulence in B. bassiana. Additionally, hsp30a positively regulated fungal conidiation and conidial tolerance to UV-B irradiation, and hsp30b positively contributed to hyphal growth in rich and minimal media and conidial germination in response to UV-B irradiation. These findings unveil possible means of improving field persistence and efficacy of a fungal formulation by manipulating the sHsp genes in a candidate strain.In summary, three sHsps containing conserved α-crystallin domain exist in the genome of


Chrysanthemum morifolium Ramat.), inhibited by long-day lighting, can be reversed with a short period of low supplemental blue light (S-BL). Both flowering and the reactive oxygen species (ROS) scavenging processes are primarily driven by sugars created by photosynthetic carbon assimilation. In addition, the antioxidant ability potentially affects flowering in photoperiod- and/or circadian rhythm-dependent manners. This indicates that there is an interactive relationship among blue (B) light, photosynthetic efficiency, sugar accumulation, and antioxidant ability in flowering regulation. Here, 4 h of 30 μmol·m−2·s−1 photosynthetic photon flux density (PPFD) S-BL was applied at the end of a 13-h long-day period (LD13 + 4B) at different intervals during 60 days of experimental duration. The five experimental groups were named according to the actual number of days of S-BL and their intervals: applied once every day, “60 days-(LD13 + 4B) (100.0%)”; once every other day, “30 days-(LD13 + 4B) (50.0%)”; once every three days, “15 days-(LD13 + 4B) (25.0%)”; once every five days, “10 days-(LD13 + 4B) (16.7%)”; and once every seven days, “7 days-(LD13 + 4B) (11.7%)”. Two non-S-BL control groups were also included: 60 10-h short days (60 days-SD10) and 13-h long days (60 days-LD13). At the harvest stage, varying degrees of flowering were observed except in “60 days-LD13” and “7 days-(LD13 + 4B) (11.7%)”. The number of flowers increased and the flower buds appeared earlier as the proportion of S-BL days increased in LD13 conditions, although the “60 days-SD10” gave the earliest flowering. The proportion of initial, pivotal, and optimal flowering was 16.7% (“10 days-(LD13 + 4B)”), 50.0% (“30 days-(LD13 + 4B)”), and 100.0% (“60 days-(LD13 + 4B)”), respectively. Meanwhile, a series of physiological parameters such as the production of enzymatic or non-enzymatic antioxidants, chlorophyll content, photosynthetic efficiency, enzyme activities, and carbohydrate accumulation were significantly improved by “30 days-(LD13 + 4B) (50.0%)” as a turning point until the peaks appeared in “60 days-(LD13 + 4B) (100.0%)”, as well as the expression of florigenic or anti-florigenic and some antioxidant-synthetic genes. Furthermore, the results of principal component analysis (PCA) indicated that S-BL days positively regulated flowering, photosynthesis, carbohydrate accumulation, and antioxidant production. In aggregate, the pivotal and optimal proportions of S-BL days to reconcile the relationship among flowering, photosynthetic carbon assimilation, and antioxidant ability were 50.0% and 100.0%, respectively. However, there are still significant gaps to be filled in order to determine the specific involvement of blue light and antioxidant abilities in flowering regulation.The flowering of chrysanthemum ( Chrysanthemum morifolium Ramat.) is a qualitative short-day plant (SDP), where a sufficiently long night over a certain critical level is absolutely required for flowering. The flowering characteristic that depends on day-length variation is “photoperiodism” [The commercially and ornamentally important plant, chrysanthemum . The absorbance peaks of phytochromes are in red  and far red  light, and to a lesser extent in blue  light. Blue light is also absorbed by cryptochromes. Cryptochromes, especially those found in Brassicaceae, have a role in the suppression of stem elongation [a Heynh. . Moreovea Heynh. ,12,13,14The light quality affects the photosynthesis, and therefore the sugar accumulation. In addition to providing energy to plants, sugars also function as signaling molecules within them, thus regulating growth and development . CapsicuFlowering is primarily driven by sugars created from photosynthetic carbon assimilation . Plant fIn the current research, an innovation of supplemental LED blue light (S-BL) was provided for the horticulture and floriculture industry. The S-BL is innovatively applied at different intervals in a long-day period to understand the efficient proportions of S-BL days to the whole experimental duration in chrysanthemum flowering regulation. According to our results, (1) putting forward more possible options of proportions of S-BL days in initial, pivotal, and optimal flowering, respectively, (2) providing effective and optimal proportions of S-BL days to reconcile the relationship among flowering, sugar accumulation, and antioxidant properties, (3) and supporting flexibility of blue light application based on actual demands, will reduce the cultivation cost, save energy, and comply with the purpose of sustainable development. Our study could inspire researchers and growers to choose the appropriate LED lighting options for flowering control, phytochemical contents, nutritional values, etc.Chrysanthemum morifolium Ramat.) “Gaya Glory”, a qualitative SDP, was used as the experimental material. Flowers Breeding Research Institute, Gyeongnam Agricultural Research & Extension Services, Changwon, Gyeongnam, Korea, provided rooted cuttings with 8 ± 2 leaves per plant, which were separately transplanted into 10 cm plastic pots with a commercial medium , one plant per pot. After planting, plants were moved to this closed–type plant factory and acclimated to 23/18 °C (light/dark), 70 ± 10% RH, and 270 ± 5 μmol·m−2·s−1 PPFD supplied with F48T12-CW-VHO fluorescent lamps . A pressurized gas tank supplied CO2 to complement plant photosynthesis on time, and an electrolyte CO2 sensor  monitored online maintained a concentration of 350 ± 50 parts per million (PPM). The air circulation system in this location was designed with the fans uniformly spaced so that the conditioned air could blow horizontally into the developing rooms through several regularly spaced openings. The plants were treated with photoperiodic light treatments after one week of acclimation (with 16-h LD). Daily irrigation with a multipurpose nutrient solution  [The pot experiment was carried out at Gyeongsang National University in Jinju, Korea, in a closed-type plant factory . In this work, the ornamental species chrysanthemum (H = 6.5)  was carr−2·s−1 PPFD provided by white (W) MEF50120 LEDs  with a wide spectrum ranging from 400 to 720 nm and a distinct peak at 435 nm in blue (−2·s−1 PPFD of S-BL [−2·s−1 PPFD) at the end of LD13 (LD13 + 4B)  C at diffThe plant growth parameters, such as plant height, number of branches, leaves, and flowers per plant, leaf area per leaf, and plant dry weight, were collected after 60 days of photoperiodic light treatments. The days to visible flower buds in each treatment were determined by counting the number of days from initiating the light treatment to the date when the first flower bud appeared. The number of flowers per plant contained both blooming flowers and visible flower buds at the harvest stage. The leaves with a length > 1 cm were counted to determine the total number of leaves per plant. The top fourth true leaves were selected to measure the fully expanded leaf area using a leaf area meter . After careful cleaning, divided samples of shoots were oven-dried  at 85 °C for 5~7 days until a constant mass was reached to determine the dry weight. Additionally, the harvested samples were immediately placed in liquid nitrogen and then stocked in a −80 °C refrigerator for the subsequent physiological investigations.v/v) acetone overnight at 4 °C until the leaf samples were completely discolored. A UV spectrophotometer  was used for colorimetry at A663 nm and A646 nm. The pigment contents were calculated from the following equations: chlorophyll a (Chl a) = 12.25 × A663 − 2.79 × A646; chlorophyll b (Chl b) = 21.50 × A646 − 5.10 × A663. For each experiment, 3 technical and 6 biological replicates were performed.The leaf chlorophyll (Chl) content was determined according to Lichtenthaler and Buschmann . After 6Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) of the top fourth completely expanded adult leaf for each plant at harvest time. The average of six measurements taken on each leaf was utilized. To maintain the same stable circumstances, these parameters were monitored in the closed-type plant factory from 9:00 to 11:00 a.m.A leaf porometer  was used to measure the net photosynthetic rate (−2·s−1 PPFD) was applied to obtain the maximum fluorescence (Fm) and minimum fluorescence (F0). The leaf was then light-adapted for 5 min with continuous actinic light  with saturating pulses every 25 s, and the maximum light-adapted fluorescence (Fm′) and steady-state fluorescence (Fs) were measured. The maximal PSII quantum yield (Fv/Fm) was calculated as Fv/Fm = (Fm − F0)/Fm [F0′), the actinic light was switched off and a far-red pulse was delivered. Fv′/Fm′ = (Fm′ − Fs)/Fm′ was used to compute the photochemical efficiency (Fv′/Fm′) of PSII. Furthermore, the photochemical quenching coefficient (qP) was determined to be qP = (Fm′ − Fs)/(Fm′ − F0′) [A photosystem  was used to quantify the leaf chlorophyll fluorescence. As previously stated, the top fourth fully expanded adult leaf from each plant was chosen for these measures. After dark-adapting the leaves for 30 min with a leaf clip, a 0.6 s saturating light pulse (3450 μmol·m− F0)/Fm . To prod′ − F0′) . For eacv/v) triton-X, pH 7.0), 0.1 g of the powder was homogenized. The resultant mixture was then centrifuged  to extract the supernatant, which was then utilized for the total protein estimation and enzyme activity assays. The total protein assessments were led by [630 nm, A485 nm, and A590 nm, respectively. For each experiment, 3 technical and 6 biological replicates were performed.Weighed measurements of 0.3 g of −80 °C stocked leaf samples were collected at 10:00 p.m. after 60 days of the photoperiodic light treatments and the anthrone colorimetric technique was used to determine the amounts of starch and soluble sugars ,41. Stoce led by . A UV spv/v) KOH solution was added to this combination, which was then put in a 100 °C water bath for 10 min before being progressively cooled to room temperature. After applying a 200 μL 0.15% (v/v) anthrone-sulfuric acid solution, the mixture was incubated at 40 °C for 20 min while the enhancement of A620 nm was measured. The phosphoenolpyruvate carboxykinase (PEPC) was assayed in a 1 mL reaction mixture consisting of 50 mM Tris-HCl (pH 8.0), 5 mM MnCl2, 2 mM DTT, 10 mM NaHCO3, 0.2 mM NADH, 5-unit NAD-MDH, and a 160 μL enzyme extract. To begin the process, 2.5 mM phosphoenolpyruvate was added (PEP). In a 1.5 mL reaction mixture comprising of 100 mM imidazole-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 0.05% (w/v) BSA, 2 mM DTT, 150 μM NADH, 1-unit LDH, 2 mM ADP, and a 150 μL enzyme extract, the phosphoenolpyruvate phosphatase (PEPP) was measured. The reaction was started with 2 mM PEP, and the change in A412 nm was measured. The total activity of Ribulose 1,5-diphosphate carboxylase/oxygenase (RuBisCO) was determined by injecting 100 μL of the supernatant into 400 μL of an assay mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 10 mM MgCl2, 0.1 mM EDTA, and 20 mM NaH14CO3 (2.0 GBq mmol−1) at 30 °C. After a 5-min activation time, the reaction was started by adding RuBP to 0.5 mmol L−1 and was stopped after 30 s with 100 μL of 6 mol L−1 HCl. Moreover, the activities of soluble starch synthase (SSS), adenosine diphosphate glucose pyro-phosphorylase (ADPGPPase), and uridine diphosphate glucose pyro-phosphorylase (UDGPPase) were measured according to the protocol described by Doehlert et al. [The preceding step’s total protein solution was utilized to examine the enzymatic activities, which were quantified using a UV spectrophotometer . Three technical and six biological replicates were conducted for each enzymatic measurement. At 34 °C for 1 h, sucrose synthase (SS) and sucrose phosphate synthase (SPS) were measured in a 1-mL reaction mixture comprising of a 500 μL enzyme extract. A 300 μL 30% . Castillo et al. [470 nm was calculated using an extinction value of 26.6 mM−1 cm−1. Becana et al. [560 nm. According to Nakano and Asada [290 nm (extinction coefficient 2.9 mM−1 cm−1). Three technical and six biological duplicates were conducted for each measurement.The activities of enzymes that scavenge reactive oxygen species (ROS) were also investigated. According to Aebi , the cato et al.  presentea et al.  investignd Asada , the actTo obtain carotenoids, 1 g of frozen leaf samples was milled with 1 g of quartz and 15 mL of acetone containing 1% BHT  . The mix3COONa). Using a UV spectrophotometer, the absorbance of the mixtures was measured at 535 and 700 nm in comparison to pure water . The value (Abs535 − Abs700)pH1.0 − (Abs535 − Abs700)pH4.5 corresponds to the absorbance due to the anthocyanin. Results were expressed as “mg” of kuromanin equivalents (KuE) per gram of frozen weight.In search of anthocyanins, 1 g of frozen leaf samples was crushed along with 1 g of quartz and 15 mL of 1% HCl in methanol . After s3). The mixture was mixed for 1 h at 4 °C before being centrifuged for 15 min at 15,000 rpm. The concentration of reduced ascorbic acid was determined using the association of vitamin chemical 2,6-dichloroindophenol (DCIP) technique [For the compound ascorbic acid, 4 g of frozen leaves were crushed along with 1 g of quartz and 80 mL of the extraction solution (20 g/L HPOechnique . In brie2) (5% w/v) was first incubated at room temperature for 6 min. After that, 150 μL of AlCl3·6H2O (10% w/v) was added and incubated at room temperature for 6 min. 800 μL of NaOH (10% w/v) solution was added and incubated at room temperature for 15 min. The extract was replaced with distilled water for the blank. A UV spectrophotometer  was used to measure the absorbance at 510 nm. The total flavonoid was expressed in “mg” Quercetin equivalent (QE) per gram of frozen weight.For total flavonoids, 1 g of frozen leaf samples was crushed with 1 g of quartz and 10 mL of 100% acetone. The mixture was mixed for 1 h at 70 °C before being centrifuged for 15 min at 17,000 rpm. The supernatant was discarded, and the material was extracted again using the same process but only incubating for 15 min. The supernatants were pooled and diluted as needed for the analyses. Perva-Uzunali et al.  provided2CO3 (20% w/v) was added and incubated for 2 h at room temperature in the dark. A UV spectrophotometer  was used to measure the absorbance at 765 nm. Instead of aliquot extract, distilled water was used to make a blank. The total phenolics were expressed in “mg” of gallic acid equivalent (GAE) per gram of frozen weight.To determine the total phenolics, the procedures for aliquoting the leaf extract based on well-established references ,59,60 we® Reverse Transcriptase  was used to synthesize cDNA from 1 μg of total RNA, in accordance with the manufacturer’s instructions. The cDNA was diluted 10-fold, and 5 μL of it was utilized in a 15-μL quantitative RT-PCR (qRT-PCR) experiment using SYBR Premix Ex Taq™ II , which was carried out on a Roche Light Cycler 96 real-time fluorescence quantitative PCR apparatus . The relative expression levels of each gene were determined using the 2−∆∆Ct method [Arabidopsis were denoted as “Cm + gene” The data were averaged and standardized against the expression of the reference genes CmACTIN and CmEF1 (elongation factor 1α). The primer sequences and PCR conditions used in the analyses are listed in The total RNA was extracted using the RNeasy Plant Mini Kit  and processed with RNase-free DNase  as directed by the manufacturer. A PrimeScriptt method . In our For the flowering-, photosynthesis-, carbohydrate-, or antioxidant-related parameters, the principal component analysis (PCA) was conducted on the three replicates, using the MINITAB 16.2.1 statistical software, aiming to extract trends when multiple qualitative variables were used, formulating new variables correlated to the original ones . The PCAp) ≤ 0.05 with a statistical program . The differences between each treatment were tested by Student’s t-test (p) ≤ 0.05. Moreover, the experimental assays used to obtain all results were repeated six times and presented as the mean ± standard error.All plants in our study were picked at random. Excel 2016 and the DPS software package  were used to process, plot, and statistically analyze the data. Significant differences among the treatments were assessed by an analysis of variance (ANOVA), followed by the Duncan’s multiple range test at a probability  supplied at different intervals in the LD13 period significantly affected the photoperiodic flowering, morphology, and growth of “Gaya Glory” plants . FirstlySecondly, we also investigated how the growth and morphology of chrysanthemums responded to the different number of days of S-BL in the photoperiodic light treatments. As shown in The chlorophyll (Chl) content of chrysanthemum leaves was considerably affected by the different proportions of S-BL days . In thisPn value increased as the number of days of S-BL increased. The maximum Pn value of chrysanthemum leaves appeared in “60 days-(LD13 + 4B)”. This increase in the net photosynthetic rate indicates that the number of days of blue light application is positively related to the leaf development and chlorophyll content in chrysanthemum plants, and further improves the photosynthetic efficiency. “60 days-(LD13 + 4B)” induced the greatest values of Tr, Gs, and Ci, and the lowest values were observed in the two control treatments “60 days-LD13 and SD10”.Fv’/Fm’ or qP was observed: “60 days-(LD13 + 4B)” > “30 days-(LD13 + 4B)” > “15 days-(LD13 + 4B)” = “10 days-(LD13 + 4B)” > “7 days-(LD13 + 4B)” = “60 days-LD13” > “60 days-SD10”. However, there were insignificant differences in the Fv/Fm among all treatments due to the un-stressful environments, were only “60 days-(LD13 + 4B)” and “30 days-(LD13 + 4B)” showed slightly increased Fv/Fm values  and three well-characterized floral meristem identity genes APETALA1 (CDM111), FRUITFULL (CmAFL1), and LEAFY (CmFL) were selected and analyzed by qRT-PCR in leaves and shoot apices, respectively (CmTFL1 was obviously higher in the non-flowered treatments “7 days-(LD13 + 4B)” and “60 days-LD13”, and showed the opposite proportional to the flowering capacity. However, these 3 floral meristem identity genes CDM111, CmAFL1, and CmFL were expressed inversely, the higher expression was observed in flowering-promoted treatments “60 days-(LD13 + 4B)” and “30 days-(LD13 + 4B)”, while significantly lower in non-flowered treatments “7 days-(LD13 + 4B)” and “60 days-LD13”.To study the tissue-specific expression patterns of flowering-related genes in “Gaya Glory” in response to the proportion of S-BL days, the anti-florigenic ectively A–D. AfteCmPAL) and the dihydroflavonol 4-reductase (DFR) gene (CmDFR) in the Chrysanthemum morifolium ” treatment always resulted in the best expression of these two antioxidant synthesis-related genes, both in the leaves and the petals.The antioxidant ability of “Gaya Glory” plants strongly responded to the various proportions of S-BL days . We alsorifolium E,F. The The PCA of all analyzed parameters highlighted that the first two principal components (PCs) were related with eigenvalues higher than 1 and explained 94.1% of the total variance, with PC1 and PC2 accounting for 89.3% and 4.8%, respectively. The type of S-BL treatment contributed to the clear separation on PC1 . “60 dayBlue light has been shown to benefit the Chl content in numerous species, by advancing chloroplasts for an upgraded light capture capacity, thus improving the photosynthetic productivity ,74. “30 2 content, which boosts the photosynthetic parameter values such as the net photosynthetic rate [Gs, Tr, Ci, etc. improved; 50.0% (“30 days-(LD13 + 4B)”) was a turning point in boosting those features, and the Pn improved significantly over this proportion of S-BL days  [According to Shimazaki et al., stomata effectively opened in response to blue light . Stomatatic rate . In our -BL days . Chl flute (ETR) . As showAdditionally, plants cultivated with blue light have higher contents of proteins, total soluble sugars, sucrose, starch, and greater dry weight, but shorter shoots compared to plants grown with white light ,86. Our CmTFL1 gene boosted secondary branching in Arabidopsis and axillary buds in Chrysanthemum, indicating that the high expression level of CmTFL1 in the stem stimulated the establishment of lateral meristems. Similar effects have been observed in other species having homologous TFL1 genes. The LpTFL1 gene in Lolium perenne L. not only restored the tfl1 mutant’s phenotype but also generated a large number of secondary branches and leaves with improved vegetative growth [Arabidopsis AtTFL1, Prunus serotine PsTFL1, and Lotus japonicas LjCEN1 genes all increased the number of branches and leaves [TFL1 gene has a conserved role in controlling branching and leafing. Moreover, the constitutive expression of CsTFL1 was significantly delayed in flower production in Chrysanthemum seticuspe under SD circumstances. CmTFL1 function was confirmed using five transgenic lines, and CmTFL1 functionally influenced flower development in Chrysanthemum morifolium [JcTFL1b-RNAi Jatropha showed consistent early blooming [CmTFL1 expression was typically higher in LD conditions and particularly high in treatments with a greater number of branches and leaves, especially in the non-flowered treatments “7 days-(LD13 + 4B)” and “60 days-LD13” , a facultative SDP, may blossom even in non-inductive LD circumstances. Two florigenic genes (Hd3a and RFT1) of rice are activated depending on the length of the day, and the RFT1 protein is proposed as the LD Florien [CmFTL1 in chrysanthemum may operate as an LD florigenic gene, comparable to RFT1 in rice. Under photoperiod-unfavorable conditions, it may be anticipated that residual CmFTL3 and enhanced CmFTL1 eventually cause flowering, which was also proved by our pervious study exploring the LD florigenic-like gene CmFTL1 involved in photoperiodic flowering in SDP chrysanthemum [Furthermore, three chrysanthemum orthologues of lowering . The CsF the SAM . Overexp Florien . CmFTL1 anthemum . That miw/v) sucrose may activate LFY (LEAFY) expression and stimulate plant blooming via the LFY factor [CDM111, CmAFL1, and CmFL)  B–D. Furtnd CmFL) A,B.In general, enzymatic and non-enzymatic antioxidant defense mechanisms interact together synergistically to keep ROS levels balanced and avoid oxidative stresses. It has been claimed that the light quality, particularly blue light, an efficient activator in stimulating antioxidant systems, has a considerable influence on the antioxidant capacity in many plant species ,104. FurThe non-enzymatic antioxidant capacity of chrysanthemum leaves cultivated under different proportions of S-BL days was also investigated. Carotenoids, tocopherol, flavonoids, ascorbate, and phenolic compounds are antioxidants found in plants that play key roles in protection against photo-oxidative damage ,22. The CmPAL and CmDFR, were tested. Phenylalanine ammonia-lyase (PAL) is the 1st rate-limiting enzyme of the phenylalanine metabolic pathway which can be involved in plant immune response, and is a key enzyme in the process of plant stress responses [PAL may be related to the synthesis of anthocyanins [Chrysanthemum morifolium trend increased with the proportion of S-BL days as did the pattern in the antioxidant capacity. Still, the “60 days-(LD13 + 4B)” treatment always resulted in the best expression of these two antioxidant synthesis-related genes in both leaves and petals  indicated that the proportion of S-BL days positively regulated flowering, photosynthesis, carbohydrate accumulation, and antioxidant production. When the proportion of days of S-BL was over 50.0% (“30 days-LD13 + 4B)”), these parameters were significantly improved . Accordi + 4B”), In conclusion, the proportion of S-BL days positively regulated flowering, photosynthesis, carbohydrate accumulation, and antioxidant production. According to our current findings, (1) the number of flowers increased and the flower buds appeared earlier as the proportion of S-BL days increased; (2) the proportion of initial, pivotal, and optimal flowering is 16.7% (“10 days-(LD13 + 4B)”), 50.0% (“30 days-(LD13 + 4B)”), and 100.0% (“60 days-(LD13 + 4B)”), respectively; (3) the 50.0% (“30 days-(LD13 + 4B)”) appears to be a turning point for the change in the physiological traits until peaks appeared in 100.0% (“60 days-(LD13 + 4B)”). In aggregate, the pivotal and optimal proportions of S-BL days to reconcile the relationship among flowering, photosynthetic carbon assimilation, and antioxidant ability is 50.0% (“30 days-(LD13 + 4B)”) and 100.0% (“60 days-(LD13 + 4B)”), respectively. We hope our study could inspire researchers and growers to choose the appropriate LED lighting options for flowering control, phytochemical contents, nutritional value, etc. This study also provides support for the use of blue light in horticulture. Studies continue to elucidate the precise role of blue light and antioxidant abilities in regulating flowering.