SNAI2 enhances HPV­negative cervical cancer cell dormancy by modulating u­PAR expression and the activity of the ERK/p38 signaling pathway in vitro.

The prognosis of patients with human papillomavirus (HPV)­negative cervical cancer is significantly worse than that of patients with HPV­positive cervical cancer. Understanding the mechanisms of this is crucial for preventing disease evolution. In the present study, the GV367­snail family transcriptional repressor 2 (SNAI2) lentiviral vector was constructed and transduced into C­33A cells. Subsequently, the proliferation of tumor cells was detected using the Cell Counting Kit (CCK)­8 method. Flow cytometry was used to analyze the cell cycle progression of tumor cells. The glucose consumption of tumor cells was detected using an oxidase assay, and the senescence of tumor cells was detected using beta­galactosidase staining. The gene expression and the activity of p38 and ERK1/2 were detected using reverse transcription­quantitative PCR and western blotting, respectively. The C­33A­SNAI2 cell line was successfully established. Compared with HeLa and C­33A­Wild cells, the proliferation and percentage of G0/G1­phase cells in the C­33A­SNAI2 group were decreased, as detected by the CCK­8 assay (100±0 vs. 239.1±58.3 vs. 39.7±20.1, P<0.01) and flow cytometry (34.0±7.1% vs. 46.2±10.6% vs. 61.3±5.3%, P<0.05). Compared with the HeLa group, the glucose consumption of the C­33A­Wild and C­33A­SNAI2 groups was significantly decreased (P<0.01). The results of beta­galactosidase staining showed that the proportion of beta­galactosidase­positive cells in the C­33A­SNAI2 group was significantly decreased compared with the C­33A­Wild group (P<0.01). Upregulation of SNAI2 enhanced the increase in p21 expression, and the decrease in CDK1, urokinase plasminogen activator receptor (u­PAR) and cyclin D1 expression in C­33A cells compared with C­33A­Wild cells (P<0.05). In addition, the activities of p38, ERK1/2 and the phosphorylated (p)­ERK1/2/p­p38 ratio were decreased in the C­33A­SNAI2 group compared with the C­33A­Wild and HeLa groups (P<0.05). In conclusion, SNAI2 enhanced HPV­negative cervical cancer C­33A cell dormancy, which was characterized by G0/G1 arrest, by the downregulation of u­PAR expression, and a decrease in the activity of the p­ERK1/2 and p­p38MAPK signaling pathways in vitro. Cancer recurrence and metastases are responsible for most cancer­related deaths. Given that SNAI2 is required for enhancing HPV­negative cervical cancer cell dormancy, regulating this process may promote cervical tumor cells to enter a continuous dormant state, which could be a potential approach for tumor therapy.

A hybrid RNA-protein biosensor for high-throughput screening of adenosylcobalamin biosynthesis.

Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries. Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries. High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level. In this study, we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin, the inverter part was an orthogonal transcriptional repressor to obtain signal inversion, and the output part was a fluorescent protein to be easily detected. The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo. This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization. An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control. Then, using such a biosensor combined with fluorescence-activated cell sorting, we established a high-throughput screening platform for screening adenosylcobalamin overproducers. This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.

Roles of ß-Cell Hypoxia in the Progression of Type 2 Diabetes.

Type 2 diabetes is a chronic disease marked by hyperglycemia; impaired insulin secretion by pancreatic ß-cells is a hallmark of this disease. Recent studies have shown that hypoxia occurs in the ß-cells of patients with type 2 diabetes and hypoxia, in turn, contributes to the insulin secretion defect and ß-cell loss through various mechanisms, including the activation of hypoxia-inducible factors, induction of transcriptional repressors, and activation of AMP-activated protein kinase. This review focuses on advances in our understanding of the contribution of ß-cell hypoxia to the development of ß-cell dysfunction in type 2 diabetes. A better understanding of ß-cell hypoxia might be useful in the development of new strategies for treating type 2 diabetes.

Identification of Mycobacterium tuberculosis transcriptional repressor EthR inhibitors: Shape-based search and machine learning studies.

Tuberculosis has been a challenge to the world since prehistoric times, and with the advent of drug-resistant strains, it has become more challenging to treat this infection. Ethionamide (ETH), a second-line drug, acts as a prodrug and targets mycolic acid synthesis by targeting the enoyl-acyl carrier protein reductase (InhA) enzyme. Mycobacterium tuberculosis (Mtb) EthR is an ethA gene repressor required to activate prodrug ETH. Recent studies suggest targeting the EthR could lead to newer drug molecules that would help better activate the ETH or complement this process. In this report, we have attempted and successfully identified three new molecules from the drug repurposing library that can target EthR protein and function as ETH boosters. These molecules were obtained after rigorous filtering of the database for their physicochemical, toxicological properties and safety. The molecular docking, molecular dynamics simulations and binding energy studies yielded three compounds, Ethyl (2-amino-4-((4-fluorobenzyl)amino)phenyl)carbamate) (L1), 2-((2,2-Difluorobenzo [d] [1,3]dioxol-5-yl)amino)-2-oxoethyl (E)-3-(5-bromofuran-2-yl)acrylate (L2), and N-(2,3-Dihydrobenzo [b] [1,4]dioxin-6-yl)-4-(2-((4-fluorophenyl)amino)-2-oxoethoxy)-3-methoxy benzamide (L3) are potential EthR inhibitors. We applied machine learning methods to evaluate these molecules for toxicity and synthesisability, suggesting safety and ease of synthesis for these molecules. These molecules are known for other pharmacological activities and can be repurposed faster as adjuvant therapy for tuberculosis.

Systematic Engineering of Genistein Biosynthetic Pathway through Genetic Regulators and Combinatorial Enzyme Screening.

Microbial production of genistein, an isoflavonoid primarily found in soybeans, is gaining prominence in the food industry due to its significant nutritional and health benefits. However, challenges arise in redesigning strains due to intricate regulatory nodes between cell growth and genistein production and in systematically exploring core enzymes involving genistein biosynthesis. To address this, this study devised a strategy that simultaneously and precisely rewires flux at both acetyl-CoA and malonyl-CoA nodes toward genistein synthesis. In particular, naringenin, the primary precursor of genistein, was accumulated 2.6 times more than the unoptimized strain through transcriptional repressor-based genetic regulators. Building upon this, a combination of isoflavone synthase and cytochrome P450 reductase with the remarkable conversion of naringenin to genistein was screened from enzyme homologue libraries. The integrated metabolic engineering strategy yields the highest reported production (98 mg/L of genistein) to date, providing a framework for the biosynthesis of diverse flavonoids, including genistein.

Canalizing cell fate by transcriptional repression.

Precision in the establishment and maintenance of cellular identities is crucial for the development of multicellular organisms and requires tight regulation of gene expression. While extensive research has focused on understanding cell type-specific gene activation, the complex mechanisms underlying the transcriptional repression of alternative fates are not fully understood. Here, we provide an overview of the repressive mechanisms involved in cell fate regulation. We discuss the molecular machinery responsible for suppressing alternative fates and highlight the crucial role of sequence-specific transcription factors (TFs) in this process. Depletion of these TFs can result in unwanted gene expression and increased cellular plasticity. We suggest that these TFs recruit cell type-specific repressive complexes to their cis-regulatory elements, enabling them to modulate chromatin accessibility in a context-dependent manner. This modulation effectively suppresses master regulators of alternative fate programs and their downstream targets. The modularity and dynamic behavior of these repressive complexes enables a limited number of repressors to canalize and maintain major and minor cell fate decisions at different stages of development.

MicroRNA-124 plays an inhibitory role in cutaneous squamous cell carcinoma cells via targeting SNAI2, an immunotherapy determinant.

MicroRNAs (miRs) play multiple roles during cutaneous squamous cell carcinoma (CSCC) progression. Previous studies suggest miR-124 could inhibit cancer development in CSCC. METHODS: Obtained 63 pairs of CSCC and adjacent tissues for analysis. Cultured HaCaT and two CSCC cell lines (A431 and SCL-1) in DMEM (10 % FBS). Transfected cells using Lipofectamine 2000 with various miR-124 mimics, inhibitors, or Snail family transcriptional repressor 2 (SNAI2) expression plasmid. Performed a series of assays, including real-time quantitative PCR, Western blot, CCK8, wound healing, transwell, and luciferase reporter gene assay, to examine the effects of miR-124 on CSCC cells. RESULTS: An evident downregulation of miR-124 in CSCC tissues, which was related to advanced disease stage and nodal metastasis. Overexpressing miR-124 could reduce the proliferation, migration, and invasion abilities of CSCC cells. It was verified that miR-124 targets the SNAI2 in CSCC cells. Moreover, ectopic expression of SNAI2 rescued the suppressive effects on CSCC cells induced by miR-124 overexpression. Furthermore, miR-124 increased cell sensitivity to cisplatin. Besides, SNAI2 is a critical factor in the immune-related aspects of CSCC and its modulation may influence the response to immunotherapy. CONCLUSION: We demonstrate that miR-124 inhibits CSCC progression through downregulating SNAI2, and thus it may be a molecular candidate for treating CSCC in the clinic.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

A Ratiometric Gene-Switch System for miRNA Sensing and Gene Regulation.

microRNAs (miRNAs) are a class of non-coding, small RNAs that play an important role in diverse biological processes and diseases. By regulating the expression of eukaryotic genes post-transcriptionally in a sequence-specific manner, miRNAs are widely used to design synthetic RNA switches. However, most of the RNA switches are often dependent on the corresponding ligand molecules, whose specificity and concentration would affect the efficiency of synthetic RNA circuits. Here, a fused transcriptional repressor Gal4BD-Rluc based gene-switch system Gal-miR for miRNA visualization and gene regulation is described. By placing a luciferase downstream gene under the control of endogenous miRNA machinery, the Gal-miR system makes the conversion of miRNA-mediated gene silencing into a ratiometric bioluminescent signal, which quantitatively reflected miRNA-206 activity during myogenic differentiation. Moreover, it demonstrates that this gene-switch system can effectively inhibit breast cancer cell viability, migration and invasion under the control of specific miRNAs by replacing the downstream gene with melittin functional gene. The study proposes a powerful modular genetic design for achieving precise control of transgene expression in a miRNA responsive way, as well as visualizing the dynamics of miRNA activity.

Ssn6 Interacts with Polar Tube Protein 2 and Transcriptional Repressor for RNA Polymerase II: Insight into Its Involvement in the Biological Process of Microsporidium Nosema bombycis.

Nosema bombycis is a representative species of Microsporidia, and is the pathogen that causes pebrine disease in silkworms. In the process of infection, the polar tube of N. bombycis is injected into the host cells. During proliferation, N. bombycis recruits the mitochondria of host cells. The general transcriptional corepressor Ssn6 contains six tetratricopeptide repeats (TPR) and undertakes various important functions. In this study, we isolated and characterized Nbssn6 of the microsporidium N. bombycis. The Nbssn6 gene contains a complete ORF of 1182 bp in length that encodes a 393 amino acid polypeptide. Indirect immunofluorescence assay showed that the Ssn6 protein was mainly distributed in the cytoplasm and nucleus at the proliferative phase of N. bombycis. We revealed the interaction of Nbssn6 with polar tube protein 2 (Nbptp2) and the transcriptional repressor for RNA polymerase II (Nbtrrp2) by Co-IP and yeast two-hybrid assays. Results from RNA interference further confirmed that the transcriptional level of Nbptp2 and Nbtrrp2 was regulated by Nbssn6. These results suggest that Nbssn6 impacts the infection and proliferation of N. bombycis via interacting with the polar tube protein and transcriptional repressor for RNA polymerase II.

A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei.

BACKGROUND: The well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering. RESULTS: To identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter. CONCLUSIONS: XTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei.

Characterization of a novel ArsR regulates divergent ars operon in Ensifer adhaerens strain ST2.

Microbes evolved resistance determinates for coping with arsenic toxicity are commonly regulated by a variety of transcriptional repressors (ArsRs). Ensifer adhaerens strain ST2 was previously shown tolerance to environmental organoarsenical methylarsenite (MAs(III)), which has been proposed to be a primordial antibiotic. In E. adhaerens strain ST2 chromosomal ars operon, two MAs(III) resistance genes, arsZ, encoding MAs(III) oxidase, and arsK, encoding MAs(III) efflux transporter, are controlled by a novel ArsR transcriptional repressor, EaArsR. It has two conserved cysteine pairs, Cys91-92 and Cys108-109. Electrophoretic mobility shift assays (EMSAs) demonstrate that EaArsR binds to two inverted-repeat sequences within the ars promoter between arsR and arsZ to repress ars operon transcription and that DNA binding is relieved upon binding of As(III) and MAs(III). Mutation of either Cys91 or Cys92 to serine (or both) abolished these mutants binding to the ars promoter. In contrast, both C108S and C109S mutants kept responsiveness to As(III) and MAs(III). These results suggest that cysteine pair Cys91-Cys92 and either Cys108 or Cys109 contribute to form arsenic binding site. Homology modeling of EaArsR indicates the binding site consisted of Cys91-Cys92 pair from one monomer and Cys108-Cys109 pair from the other monomer, which displays the diverse evolution of arsenic binding site in the ArsR metalloregulators.

Methylation of ESR1 promoter induced by SNAI2-DNMT3B complex promotes epithelial-mesenchymal transition and correlates with poor prognosis in ERα-positive breast cancers.

Estrogen receptor α (ERα) serves as an essential therapeutic predictor for breast cancer (BC) patients and is regulated by epigenetic modification. Abnormal methylation of cytosine phosphoric acid guanine islands in the estrogen receptor 1 (ESR1) gene promoter could silence or decrease ERα expression. In ERα-negative BC, we previously found snail family transcriptional repressor 2 (SNAI2), a zinc-finger transcriptional factor, recruited lysine-specific demethylase 1 to the promoter to transcriptionally suppress ERα expression by demethylating histone H3 lysine 4 dimethylation (H3K4me2). However, the role of SNAI2 in ERα-positive BC remains elusive. In this study, we observed a positive correlation between SNAI2 and ESR1 methylation, and SNAI2 promoted ESR1 methylation by recruiting DNA methyltransferase 3 beta (DNMT3B) rather than DNA methyltransferase 1 (DNMT1) in ERα-positive BC cells. Subsequent enrichment analysis illustrated that ESR1 methylation is strongly correlated with cell adhesion and junction. Knocking down DNMT3B could partially reverse SNAI2 overexpression-induced cell proliferation, migration, and invasion. Moreover, high DNMT3B expression predicted poor relapse-free survival and overall survival in ERα-positive BC patients. In conclusion, this study demonstrated the novel mechanisms of the ESR1 methylation mediated with the SNAI2/DNMT3B complex and enhanced awareness of ESR1 methylation's role in promoting epithelial-mesenchymal transition in BC.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Hypoxia causes pancreatic ß-cell dysfunction and impairs insulin secretion by activating the transcriptional repressor BHLHE40.

Hypoxia can occur in pancreatic ß-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on ß-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human ß-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or ß-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic ß-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in ß-cells that inhibit insulin secretion by suppressing MAFA expression.

Identification and Functional Evaluation of Alternative Splice Variants of Dax1 in Mouse Embryonic Stem Cells.

Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.

Comprehensively prognostic and immunological analysis of snail family transcriptional repressor 2 in pan-cancer and identification in pancreatic carcinoma.

Background: Snail family transcriptional repressor 2 (SNAI2) is a transcription factor that induces epithelial to mesenchymal transition in neoplastic epithelial cells. It is closely related to the progression of various malignancies. However, the significance of SNAI2 in human pan-cancer is still largely unknown. Methods: The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Cancer Cell Line Encyclopedia (CCLE) databases were taken to examine the SNAI2 expression pattern in tissues and cancer cells. The link between SNAI2 gene expression levels and prognosis, as well as immune cell infiltration, was investigated using the Kaplan-Meier technique and Spearman correlation analysis. We also explored the expression and distribution of SNAI2 in various tumor tissues and cells by the THPA (Human Protein Atlas) database. We further investigated the relationship between SNAI2 expression levels and immunotherapy response in various clinical immunotherapy cohorts. Finally, the immunoblot was used to quantify the SNAI2 expression levels, and the proliferative and invasive ability of pancreatic cancer cells was determined by colony formation and transwell assays. Results: We discovered heterogeneity in SNAI2 expression in different tumor tissues and cancer cell lines by exploring public datasets. The genomic alteration of SNAI2 existed in most cancers. Also, SNAI2 exhibits prognosis predictive ability in various cancers. SNAI2 was significantly correlated with immune-activated hallmarks, cancer immune cell infiltrations, and immunoregulators. It's worth noting that SNAI2 expression is significantly related to the effectiveness of clinical immunotherapy. SNAI2 expression was also found to have a high correlation with the DNA mismatch repair (MMR) genes and DNA methylation in many cancers. Finally, the knockdown of SNAI2 significantly weakened the proliferative and invasive ability of pancreatic cancer cells. Conclusion: These findings suggested that SNAI2 could be used as a biomarker in human pan-cancer to detect immune infiltration and poor prognosis, which provides a new idea for cancer treatment.

CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis).

Anthocyanin and catechin production in tea (Camellia sinensis) leaves can positively affect tea quality; however, their regulatory mechanisms are not fully understood. Here we report that, while the CsMYB75- or CsMYB86-directed MYB-bHLH-WD40 (MBW) complexes differentially activate anthocyanin or catechin biosynthesis in tea leaves, respectively, CsMYBL2a and CsMYBL2b homologs negatively modified the light- and temperature-induced anthocyanin and catechin production in both Arabidopsis and tea plants. The MBW complexes activated both anthocyanin synthesis genes and the downstream repressor genes CsMYBL2a and CsMYBL2b. Overexpression of CsMYBL2b, but not CsMYBL2a, repressed Arabidopsis leaf anthocyanin accumulation and seed coat proanthocyanin production. CsMYBL2b strongly and CsMYBL2a weakly repressed the activating effects of CsMYB75/CsMYB86 on CsDFR and CsANS, due to their different EAR and TLLLFR domains and interactions with CsTT8/CsGL3, interfering with the functions of activating MBW complexes. CsMYBL2b and CsMYBL2a in tea leaves play different roles in fine-tuning CsMYB75/CsMYB86-MBW activation of biosynthesis of anthocyanins and catechins, respectively. The CsbZIP1-CsmiR858a-CsMYBL2 module mediated the UV-B- or cold-activated CsMYB75/CsMYB86 regulation of anthocyanin/catechin biosynthesis by repressing CsMYBL2a and CsMYBL2b. Similarly, the CsCOP1-CsbZIP1-CsPIF3 module, and BR signaling as well, mediated the high temperature repression of anthocyanin and catechin biosynthesis through differentially upregulating CsMYBL2b and CsMYBL2a, respectively. The present study provides new insights into the complex regulatory networks in environmental stress-modified flavonoid production in tea plant leaves.

Transcriptional repressor AGL79 positively regulates flowering time in Arabidopsis.

The MADS-box gene family is widely distributed in higher plants and the members of the angiosperm-specific APETALA1/FRUITFULL (AP1/FUL) subfamily plays important roles in the regulation of plant reproductive development. Recent studies revealed that the AP1/FUL subfamily member Dt2, VEGETATIVE1/PsFRUITFULc (VEG1/PsFULc) and MtFRUITFULc (MtFULc) are essential for the stem growth, branching and inflorescence development in legume species soybean (Glycine max), pea (Pisum sativum) and Medicago truncatula. However, the biological function of their homologue in Arabidopsis thaliana, AGAMOUS-LIKE 79 (AGL79), has not been well elucidated. In this study, we investigated the developmental roles of Arabidopsis AGL79 by CRISPR/Cas9-mutagenesis and molecular and physiological analyses. We found that AGL79 mainly acts as a transcriptional repressor and positively regulates Arabidopsis flowering time. We further revealed that AGL79 interacts with SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and represses the expression of TERMINAL FLOWER 1 (TFL1). Our results demonstrated the AGL79-mediated flowering regulation in Arabidopsis and added an additional layer of complexity to the understanding of flowering time regulation in dicot plants.