Epistasis facilitates functional evolution in an ancient transcription factor.

A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.

Tumor formation at ileocecal junction associated with interleukin-1ß upregulation in aryl hydrocarbon receptor-deficient mouse.

Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor. We previously reported spontaneous ileocecal tumorigenesis in AhR-deficient mice after the age of 10 weeks, which originated in the confined area between ileum and cecum. This study aimed to investigate the underlying mechanism that causes tumor development at this particular location. To observe mucosal architecture in detail, tissues of ileocecal region were stained with methylene blue. Gene expression profile in the ileocecal tissue was compared with cecum. Immunohistochemical analysis was performed with ileocecal tissues using antibodies against ileum-specific Reg3ß or cecum-specific Pitx2. In AhR+/+ mice and AhR+/- mice, that do not develop lesions, methylene blue staining revealed the gradually changing shape and arrangement of villi from ileum to cecum. It was also observed in AhR-deficient mice before developing lesions. Microarray-based analysis revealed abundant antimicrobial genes, such as Reg3, in the ileocecal tissue while FGFR2 and Pitx2 were specific to cecum. Immunohistochemical analysis of AhR-deficient mice indicated that lesions originated from the ileocecal junction, a boundary area between different epithelial types. Site-specific gene expression analysis revealed higher expression of IL-1ß at the ileocecal junction compared with the ileum or cecum of 9-11-week-old AhR-deficient mice. These findings indicate that AhR plays a vital function in the ileocecal junction. Regulating AhR activity can potentially manage the stability of ileocecal tissue possessing cancer-prone characteristics. This investigation contributes to understanding homeostasis in different epithelial transitional tissues, frequently associated with pathological states.

Increased prevalence of the COVID-19 associated Neanderthal mutations in the Central European Roma population.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and subsequent COVID-19 has spread world-wide and become pandemic with about 7 million deaths reported so far. Interethnic variability of the disease has been described, but a significant part of the differences remain unexplained and may be attributable to genetic factors. AIM: To analyse genetic factors potentially influencing COVID-19 susceptibility and severity in European Roma minority. SUBJECTS AND METHODS: Two genetic determinants, within OAS-1 (2-prime,5-prime-oligoadenylate synthetase 1, a key protein in the defence against viral infection; it activates RNases that degrade viral RNAs; rs4767027 has been analysed) and LZTFL1 (leucine zipper transcription factor-like 1, expressed in the lung respiratory epithelium; rs35044562 has been analysed) genes were screened in a population-sample of Czech Roma (N = 302) and majority population (N = 2,559). RESULTS: For both polymorphisms, Roma subjects were more likely carriers of at least one risky allele for both rs4767027-C (p < 0.001) and rs35044562-G (p < 0.00001) polymorphism. There were only 5.3% Roma subjects without at least one risky allele in comparison with 10.1% in the majority population (p < 0.01). CONCLUSIONS: It is possible that different genetic background plays an important role in increased prevalence of COVID-19 in the Roma minority.

A Novel Frameshift Variant of the ELF4 Gene in a Patient with Autoinflammatory Disease: Clinical Features, Transcriptomic Profiling and Functional Studies.

We described the diagnosis and treatment of a patient with autoinflammatory disease, named "Deficiency in ELF4, X-linked (DEX)". A novel ELF4 variant was discovered and its pathogenic mechanism was elucidated. The data about clinical, laboratory and endoscopic features, treatment, and follow-up of a patient with DEX were analyzed. Whole exome sequencing and Sanger sequencing were performed to identify potential pathogenic variants. The mRNA and protein levels of ELF4 were analyzed by qPCR and Western blotting, respectively. The association of ELF4 frameshift variant with nonsense-mediated mRNA decay (NMD) in the pathogenesis DEX was examined. Moreover, RNA-seq was performed to identify the key molecular events triggered by ELF4 variant. The relationship between ELF4 and IFN-ß activity was validated using a dual-luciferase reporter assay and a ChIP-qPCR assay. An 11-year-old boy presented with a Behçet's-like phenotype. The laboratory abnormality was the most obvious in elevated inflammatory indicators. Endoscopy revealed multiple ileocecal ulcers. Intestinal histopathology showed inflammatory cell infiltrations. The patient was treated with long-term immunosuppressant and TNF-α blocker (adalimumab), which reaped an excellent response over 16 months of follow-up. Genetic analysis identified a maternal hemizygote frameshift variant (c.1022del, p.Q341Rfs*30) in ELF4 gene in the proband. The novel variant decreased the mRNA level of ELF4 via the NMD pathway. Mechanistically, insufficient expression of ELF4 disturbed the immune system, leading to immunological disorders and pathogen susceptibility, and disrupted ELF4-activating IFN-ß responses. This analysis detailed the clinical characteristics of a Chinese patient with DEX who harbored a novel ELF4 frameshift variant. For the first time, we used patient-derived cells and carried out transcriptomic analysis to delve into the mechanism of ELF4 variant in DEX.

BET inhibition reforms the immune microenvironment and alleviates T cell dysfunction in chronic lymphocytic leukemia.

Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eµ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.

Reprofiling synthetic glucocorticoid-induced leucine zipper fusion peptide as a novel and effective hair growth promoter.

Hair is a biofilament with unique multi-dimensional values. In human, in addition to physiologic impacts, hair loss and hair related disorders can affect characteristic features, emotions, and social behaviors. Despite significant advancement, there is a dire need to explore alternative novel therapies with higher efficacy, less side effects and lower cost to promote hair growth to treat hair deficiency. Glucocorticoid-induced leucine zipper (GILZ) is a protein rapidly induced by glucocorticoids. Studies from our group and many others have suggested that a synthetic form of GILZ, TAT-GILZ, a fusion peptide of trans-activator of transcription and GILZ, can function as a potent regulator of inflammatory responses, re-establishing and maintaining the homeostasis. In this study, we investigate whether TAT-GILZ could promote and contribute to hair growth. For our pre-clinical model, we used 9-12 week-old male BALB/c and nude (athymic, nu/J) mice. We applied TAT-GILZ and/or TAT (vehicle) intradermally to depilated/hairless mice. Direct observation, histological examination, and Immunofluorescence imaging were used to assess the effects and compare different treatments. In addition, we tested two current treatment for hair loss/growth, finasteride and minoxidil, for optimal evaluation of TAT-GILZ in a comparative fashion. Our results showed, for the first time, that synthetic TAT-GILZ peptide accelerated hair growth on depilated dorsal skin of BALB/c and induced hair on the skin of athymic mice where hair growth was not expected. In addition, TAT-GILZ was able to enhance hair follicle stem cells and re-established the homeostasis by increasing counter inflammatory signals including higher regulatory T cells and glucocorticoid receptors. In conclusion, our novel findings suggest that reprofiling synthetic TAT-GILZ peptide could promote hair growth by increasing hair follicle stem cells and re-establishing homeostasis.

Transcriptome-wide identification of ARF gene family in medicinal plant Polygonatum kingianum and expression analysis of PkARF members in different tissues.

BACKGROUND: Polygonatum kingianum holds significant importance in Traditional Chinese Medicine due to its medicinal properties, characterized by its diverse chemical constituents including polysaccharides, terpenoids, flavonoids, phenols, and phenylpropanoids. The Auxin Response Factor (ARF) is a pivotal transcription factor known for its regulatory role in both primary and secondary metabolite synthesis. However, our understanding of the ARF gene family in P. kingianum remains limited. METHODS AND RESULTS: We employed RNA-Seq to sequence three distinct tissues (leaf, root, and stem) of P. kingianum. The analysis revealed a total of 31,558 differentially expressed genes (DEGs), with 43 species of transcription factors annotated among them. Analyses via gene ontology and the Kyoto Encyclopedia of Genes and Genomes demonstrated that these DEGs were predominantly enriched in metabolic pathways and secondary metabolite biosynthesis. The proposed temporal expression analysis categorized the DEGs into nine clusters, suggesting the same expression trends that may be coordinated in multiple biological processes across the three tissues. Additionally, we conducted screening and expression pattern analysis of the ARF gene family, identifying 12 significantly expressed PkARF genes in P. kingianum roots. This discovery lays the groundwork for investigations into the role of PkARF genes in root growth, development, and secondary metabolism regulation. CONCLUSION: The obtained data and insights serve as a focal point for further research studies, centred on genetic manipulation of growth and secondary metabolism in P. kingianum. Furthermore, these findings contribute to the understanding of functional genomics in P. kingianum, offering valuable genetic resources.

Transcriptome analysis and functional validation reveal the novel role of LhCYCL in axillary bud development in hybrid Liriodendron.

Shoot branching significantly influences yield and timber quality in woody plants, with hybrid Liriodendron being particularly valuable due to its rapid growth. However, understanding of the mechanisms governing shoot branching in hybrid Liriodendron remains limited. In this study, we systematically examined axillary bud development using morphological and anatomical approaches and selected four distinct developmental stages for an extensive transcriptome analysis. A total of 9,449 differentially expressed genes have been identified, many of which are involved in plant hormone signal transduction pathways. Additionally, we identified several transcription factors downregulated during early axillary bud development, including a noteworthy gene annotated as CYC-like from the TCP TF family, which emerged as a strong candidate for modulating axillary bud development. Quantitative real-time polymerase chain reaction results confirmed the highest expression levels of LhCYCL in hybrid Liriodendron axillary buds, while histochemical ß-glucuronidase staining suggested its potential role in Arabidopsis thaliana leaf axil development. Ectopic expression of LhCYCL in A. thaliana led to an increase of branches and a decrease of plant height, accompanied by altered expression of genes involved in the plant hormone signaling pathways. This indicates the involvement of LhCYCL in regulating shoot branching through plant hormone signaling pathways. In summary, our results emphasize the pivotal role played by LhCYCL in shoot branching, offering insights into the function of the CYC-like gene and establishing a robust foundation for further investigations into the molecular mechanisms governing axillary bud development in hybrid Liriodendron.

Causal gene regulatory analysis with RNA velocity reveals an interplay between slow and fast transcription factors.

Single-cell expression dynamics, from differentiation trajectories or RNA velocity, have the potential to reveal causal links between transcription factors (TFs) and their target genes in gene regulatory networks (GRNs). However, existing methods either overlook these expression dynamics or necessitate that cells be ordered along a linear pseudotemporal axis, which is incompatible with branching trajectories. We introduce Velorama, an approach to causal GRN inference that represents single-cell differentiation dynamics as a directed acyclic graph of cells, constructed from pseudotime or RNA velocity measurements. Additionally, Velorama enables the estimation of the speed at which TFs influence target genes. Applying Velorama, we uncover evidence that the speed of a TF's interactions is tied to its regulatory function. For human corticogenesis, we find that slow TFs are linked to gliomas, while fast TFs are associated with neuropsychiatric diseases. We expect Velorama to become a critical part of the RNA velocity toolkit for investigating the causal drivers of differentiation and disease.

Knocking-down long non-coding RNA LINC01094 prohibits chondrocyte apoptosis via regulating microRNA-577/metal-regulatory transcription factor 1 axis.

PURPOSE: The abnormal function and survival of chondrocytes result in articular cartilage failure, which may accelerate the onset and development of osteoarthritis (OA). This study is aimed to investigate the role of LINC01094 in chondrocyte apoptosis. METHODS: The viability and apoptosis of lipopolysaccharide (LPS)-induced chondrocytes were evaluated through CCK-8 assay and flow cytometry analysis, respectively. The expression levels of LINC01094, miR-577 and MTF1 were detected by qRT-PCR. Dual luciferase reporter tests were implemented for the verification of targeted relationships among them. Western blotting was employed to measure the levels of pro-apoptotic proteins (Caspase3 and Caspase9). RESULTS: The viability of LPS-induced chondrocytes was overtly promoted by loss of LINC01094 or miR-577 upregulation, but could be repressed via MTF1 overexpression. The opposite results were observed in apoptosis rate and the levels of Caspase3 and Caspase9. LINC01094 directly bound to miR-577, while MTF1 was verified to be modulated by miR-577. Both LINC01094 and MTF1 were at high levels, whereas miR-577 was at low level in OA synovial fluid and LPS-induced chondrocytes. Furthermore, the highly expressed miR-577 abolished the influences of MTF1 overexpression on LPS-induced chondrocytes. CONCLUSIONS: Silencing of LINC01094 represses the apoptosis of chondrocytes through upregulating miR-577 expression and downregulating MTF1 levels, providing a preliminary insight for the treatment of OA in the future.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Structural basis for the regulation of plant transcription factor WRKY33 by the VQ protein SIB1.

The WRKY transcription factors play essential roles in a variety of plant signaling pathways associated with biotic and abiotic stress response. The transcriptional activity of many WRKY members are regulated by a class of intrinsically disordered VQ proteins. While it is known that VQ proteins interact with the WRKY DNA-binding domains (DBDs), also termed as the WRKY domains, structural information regarding VQ-WRKY interaction is lacking and the regulation mechanism remains unknown. Herein we report a solution NMR study of the interaction between Arabidopsis WRKY33 and its regulatory VQ protein partner SIB1. We uncover a SIB1 minimal sequence neccessary for forming a stable complex with WRKY33 DBD, which comprises not only the consensus "FxxhVQxhTG" VQ motif but also its preceding region. We demonstrate that the ßN-strand and the extended ßN-ß1 loop of WRKY33 DBD form the SIB1 docking site, and build a structural model of the complex based on the NMR paramagnetic relaxation enhancement and mutagenesis data. Based on this model, we further identify a cluster of positively-charged residues in the N-terminal region of SIB1 to be essential for the formation of a SIB1-WRKY33-DNA ternary complex. These results provide a framework for the mechanism of SIB1-enhanced WRKY33 transcriptional activity.

Expressing banana transcription factor MaERFVII3 in Arabidopsis confers enhanced waterlogging tolerance and root growth.

Background: Waterlogging poses a significant threat to plant growth and yield worldwide. Identifying the genes responsible for mitigating waterlogging stress is crucial. Ethylene-responsive factors (ERFs) are transcriptional regulators that respond to various biotic and abiotic stresses in plants. However, their roles and involvement in responding to waterlogging stress remain largely unexplored. Hence, this study aimed to elucidate the role of ERFs in enhancing banana plant resilience to waterlogging. Methods: We hypothesized that introducing a group VII ERF transcription factor in Arabidopsis could enhance waterlogging stress tolerance. To test this hypothesis, we isolated MaERFVII3 from banana roots, where it exhibited a significant induction in response to waterlogging stress. The isolated MaERFVII3 was introduced into Arabidopsis plants for functional gene studies. Results: Compared with wild-type plants, the MaERFVII3-expressing Arabidopsis showed increased survival and biomass under waterlogging stress. Furthermore, the abundance of transcripts related to waterlogging and hypoxia response showed an elevation in transgenic plants but a decrease in wild-type and empty vector plants when exposed to waterlogging stress. Our results demonstrate the significant contribution of MaERFVII3 to waterlogging tolerance in Arabidopsis, providing baseline data for further exploration and potentially contributing to crop improvement programs.

Transcriptome analysis reveals important regulatory factors for condensed tannins synthesis in acorn.

Condensed tannins are widely present in the fruits and seeds of plants and effectively prevent them from being eaten by animals before maturity due to their astringent taste. In addition, condensed tannins are a natural compound with strong antioxidant properties and significant antibacterial effects. Four samples of mature and near-mature Quercus fabri acorns, with the highest and lowest condensed tannin content, were used for genome-based transcriptome sequencing. The KEGG enrichment analysis revealed that the differentially expressed genes (DEGs) were highly enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism. Given that the phenylpropanoid biosynthesis pathway is a crucial step in the synthesis of condensed tannins, we screened for significantly differentially expressed transcription factors and structural genes from the transcriptome data of this pathway and found that the expression levels of four MADS-box, PAL, and 4CL genes were significantly increased in acorns with high condensed tannin content. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiment further validated this result. In addition, yeast one-hybrid assay confirmed that three MADS-box transcription factors could bind the promoter of the 4CL gene, thereby regulating gene expression levels. This study utilized transcriptome sequencing to discover new important regulatory factors that can regulate the synthesis of acorn condensed tannins, providing new evidence for MADS-box transcription factors to regulate the synthesis of secondary metabolites in fruits.

The Brachypodium distachyon DREB transcription factor BdDREB-39 confers oxidative stress tolerance in transgenic tobacco.

Key message BdDREB-39 is a DREB/CBF transcription factor, localized in the nucleus with transactivation activity, and BdDREB-39-overexpressing transgenic yeasts and tobacco enhanced the tolerance to oxidative stress.Abstract The DREB/CBF transcription factors are generally recognized to play an important factor in plant growth, development and response to various abiotic stresses. However, the mechanism of DREB/CBFs in oxidative stress response is largely unknown. This study isolated a DREB/CBF gene BdDREB-39 from Brachypodium distachyon (B. distachyon). Multiple sequence alignment and phylogenetic analysis showed that BdDREB-39 was closely related to the DREB proteins of oats, barley, wheat and rye and therefore its study can provide a reference for the excavation and genetic improvement of BdDREB-39 or its homologs in its closely related species. The transcript levels of BdDREB-39 were significantly up-regulated under H2O2 stress. BdDREB-39 was localised in the nucleus and functioned as a transcriptional activator. Overexpression of BdDREB-39 enhanced H2O2 tolerance in yeast. Transgenic tobaccos with BdDREB-39 had higher germination rates, longer root, better growth status, lesser reactive oxygen species (ROS) and malondialdehyde (MDA), and higher superoxide dismutase (SOD) and peroxidase (POD) activities than wild type (WT). The expression levels of ROS-related and stress-related genes were improved by BdDREB-39. In summary, these results revealed that BdDREB-39 can improve the viability of tobacco by regulating the expression of ROS and stress-related genes, allowing transgenic tobacco to accumulate lower levels of ROS and reducing the damage caused by ROS to cells. The BdDREB-39 gene has the potential for developing plant varieties tolerant to stress.

Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzymes activities in maize (Zea mays L.).

KEY MESSAGE: The Dof22 gene encoding a deoxyribonucleic acid binding with one finger in maize, which is associated with its drought tolerance. The identification of drought stress regulatory genes is essential for the genetic improvement of maize yield. Deoxyribonucleic acid binding with one finger (Dof), a plant-specific transcription factor family, is involved in signal transduction, morphogenesis, and environmental stress responses. In present study, by weighted correlation network analysis (WGCNA) and gene co-expression network analysis, 15 putative Dof genes were identified from maize that respond to drought and rewatering. A real-time fluorescence quantitative PCR showed that these 15 genes were strongly induced by drought and ABA treatment, and among them ZmDof22 was highly induced by drought and ABA treatment. Its expression level increased by nearly 200 times after drought stress and more than 50 times after ABA treatment. After the normal conditions were restored, the expression levels were nearly 100 times and 40 times of those before treatment, respectively. The Gal4-LexA/UAS system and transcriptional activation analysis indicate that ZmDof22 is a transcriptional activator regulating drought tolerance and recovery ability in maize. Further, overexpressed transgenic and mutant plants of ZmDof22 by CRISPR/Cas9, indicates that the ZmDof22, improves maize drought tolerance by promoting stomatal closure, reduces water loss, and enhances antioxidant enzyme activity by participating in the ABA pathways. Taken together, our findings laid a foundation for further functional studies of the ZmDof gene family and provided insights into the role of the ZmDof22 regulatory network in controlling drought tolerance and recovery ability of maize.

Genome-wide analysis of NAC transcription factors and exploration of candidate genes regulating selenium metabolism in Broussonetia papyrifera.

MAIN CONCLUSION: Genome-wide identification revealed 79 BpNAC genes belonging to 16 subfamilies, and their gene structures and evolutionary relationships were characterized. Expression analysis highlighted their importance in plant selenium stress responses. Paper mulberry (Broussonetia papyrifera), a deciduous arboreal plant of the Moraceae family, is distinguished by its leaves, which are abundant in proteins, polysaccharides, and flavonoids, positioning it as a novel feedstock. NAC transcription factors, exclusive to plant species, are crucial in regulating growth, development, and response to biotic and abiotic stress. However, extensive characterization of the NAC family within paper mulberry is lacking. In this study, 79 BpNAC genes were identified from the paper mulberry genome, with an uneven distribution across 13 chromosomes. A comprehensive, genome-wide analysis of BpNACs was performed, including investigating gene structures, promoter regions, and chromosomal locations. Phylogenetic tree analysis, alongside comparisons with Arabidopsis thaliana NACs, allowed for categorizing these genes into 16 subfamilies in alignment with gene structure and motif conservation. Collinearity analysis suggested a significant homologous relationship between the NAC genes of paper mulberry and those in Morus notabilis, Ficus hispida, Antiaris toxicaria, and Cannabis sativa. Integrating transcriptome data and Se content revealed that 12 BpNAC genes were associated with selenium biosynthesis. Subsequent RT-qPCR analysis corroborated the correlation between BpNAC59, BpNAC62 with sodium selenate, and BpNAC55 with sodium selenite. Subcellular localization experiments revealed the nuclear functions of BpNAC59 and BpNAC62. This study highlights the potential BpNAC transcription factors involved in selenium metabolism, providing a foundation for strategically breeding selenium-fortified paper mulberry.

Sequence and epigenetic variations of R2R3-MYB transcription factors determine the diversity of taproot skin and flesh colors in different cultivated types of radish (Raphanus sativus L.).

KEY MESSAGE: This study found that three paralogous R2R3-MYB transcription factors exhibit functional divergence among different subspecies and cultivated types in radish. Cultivated radish taproots exhibit a wide range of color variations due to unique anthocyanin accumulation patterns in various tissues. This study investigated the universal principles of taproot color regulation that developed during domestication of different subspecies and cultivated types. The key candidate genes RsMYB1 and RsMYB2, which control anthocyanin accumulation in radish taproots, were identified using bulked segregant analysis in two genetic populations. We introduced the RsMYB1-RsF3'H-RsMYB1Met genetic model to elucidate the complex and unstable genetic regulation of taproot flesh color in Xinlimei radish. Furthermore, we analyzed the expression patterns of three R2R3-MYB transcription factors in lines with different taproot colors and investigated the relationship between RsMYB haplotypes and anthocyanin accumulation in a natural population of 56 germplasms. The results revealed that three paralogous RsMYBs underwent functional divergence during radish domestication, with RsMYB1 regulating the red flesh of Xinlimei radish, and RsMYB2 and RsMYB3 regulating the red skin of East Asian big long radish (R. sativus var. hortensis) and European small radish (R. sativus var. sativus), respectively. Moreover, RsMYB1-H1, RsMYB2-H10, and RsMYB3-H6 were identified as the primary haplotypes exerting regulatory functions on anthocyanin synthesis. These findings provide an understanding of the genetic mechanisms regulating anthocyanin synthesis in radish and offer a potential strategy for early prediction of color variations in breeding programs.

Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.

YAP prevents senescence of dermal fibroblast and inhibits melanogenesis via paracrine effect of DKK1.

Senile skin hyperpigmentation displays remarkable histopathological features of dermal aging. The crosstalk between melanocytes and dermal fibroblasts plays crucial roles in aging-related pigmentation. While senescent fibroblasts can upregulate pro-melanogenic factors, the role of anti-melanogenic factors, such as dickkopf1 (DKK1), and the upstream regulatory mechanism during aging remain obscure. This study investigated the roles of yes-associated protein (YAP) and DKK1 in the regulation of dermal fibroblast senescence and melanogenesis. Our findings demonstrated decreased YAP activity and DKK1 levels in intrinsic and extrinsic senescent fibroblasts. YAP depletion induced fibroblast senescence and downregulated the expression and secretion of DKK1, whereas YAP overexpression partially reversed the effect. The transcriptional regulation of DKK1 by YAP was supported by dual-luciferase reporter and chromatin immunoprecipitation assays. Moreover, YAP depletion in fibroblasts upregulated Wnt/ß-catenin in melanocytes and stimulated melanogenesis, which was partially rescued by the re-supplementation of DKK1. Conversely, overexpression of YAP in senescent fibroblasts decreased Wnt/ß-catenin levels in melanocytes and inhibited melanogenesis. Additionally, reduced levels of YAP and DKK1 were verified in the dermis of solar lentigines. These findings suggest that, during skin aging, epidermal pigmentation may be influenced by YAP in the dermal microenvironment via the paracrine effect of DKK1.