[Astragali Radix-Curcumae Rhizoma inhibits colon cancer progression and enhances 5-FU efficacy by regulating hypoxia-inducible factors and tumor stem cells].

The animal and cell models were used in this study to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in inhibiting colon cancer progression and enhancing the efficacy of 5-fluorouracil(5-FU) by regulating hypoxia-inducible factors and tumor stem cells. The animal model was established by subcutaneous transplantation of colon cancer HCT116 cells in nude mice, and 24 successfully modeled mice were randomized into model, 5-FU, HQEZ, and 5-FU+HQEZ groups. The tumor volume was measured every two days. Western blot was employed to measure the protein levels of epidermal growth factor receptor(EGFR), dihydropyrimidine dehydrogenase(DPYD), and thymidylate synthase(TYMS), the key targets of the hypoxic core region, as well as the hypoxia-inducible factors HIF-1α and HIF-2α and the cancer stem cell surface marker CD133 and SRY-box transcription factor 2(SOX2). The results of animal experiments showed that HQEZ slowed down the tumor growth and significantly increased the tumor inhibition rate of 5-FU. Compared with the model group, HQEZ significantly down-regulated the protein levels of EGFR and DPYD, and 5-FU+HQEZ significantly down-regulated the protein levels of EGFR and TYMS in tumors. Compared with the model group, HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, SOX2, and CD133 in the hypoxic core region. Compared with the 5-FU group, 5-FU+HQEZ lowered the protein levels of HIF-1α, HIF-2α, and SOX2. The cell experiments showed that the protein le-vels of HIF-1α and HIF-2α in HCT116 cells elevated significantly after low oxygen treatment. Compared with 5-FU(1.38 µmol·L~(-1)) alone, HQEZ(40 mg·mL~(-1)) and 5-FU+HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, and TYMS. In conclusion, HQEZ can inhibit the expression of hypoxia-responsive molecules in colon cancer cells and reduce the properties of cancer stem cells, thereby enhancing the therapeutic effect of 5-FU on colon cancer.

A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Genome-wide characterization of the bHLH gene family in Gynostemma pentaphyllum reveals its potential role in the regulation of gypenoside biosynthesis.

BACKGROUND: Gynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated. RESULTS: This study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees. CONCLUSIONS: In conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

Near-infrared light and PIF4 promote plant antiviral defense by enhancing RNA interference.

The molecular mechanism underlying phototherapy and light treatment, which utilize various wavelength spectra of light, including near-infrared (NIR), to cure human and plant diseases, is obscure. Here we revealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-activated RNA interference (RNAi) in plants. PIF4, a central transcription factor involved in light signaling, accumulates to high levels under NIR light in plants. PIF4 directly induces the transcription of two essential components of RNAi, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and ARGONAUTE 1 (AGO1), which play important roles in resistance to both DNA and RNA viruses. Moreover, the pathogenic determinant ßC1 protein, which is evolutionarily conserved and encoded by betasatellites, interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization. These findings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the exploration of NIR antiviral treatment.

Tartary Buckwheat (Fagopyrum tataricum) FtTT8 Inhibits Anthocyanin Biosynthesis and Promotes Proanthocyanidin Biosynthesis.

Tartary buckwheat (Fagopyrum tataricum) is an important plant, utilized for both medicine and food. It has become a current research hotspot due to its rich content of flavonoids, which are beneficial for human health. Anthocyanins (ATs) and proanthocyanidins (PAs) are the two main kinds of flavonoid compounds in Tartary buckwheat, which participate in the pigmentation of some tissue as well as rendering resistance to many biotic and abiotic stresses. Additionally, Tartary buckwheat anthocyanins and PAs have many health benefits for humans and the plant itself. However, little is known about the regulation mechanism of the biosynthesis of anthocyanin and PA in Tartary buckwheat. In the present study, a bHLH transcription factor (TF) FtTT8 was characterized to be homologous with AtTT8 and phylogenetically close to bHLH proteins from other plant species. Subcellular location and yeast two-hybrid assays suggested that FtTT8 locates in the nucleus and plays a role as a transcription factor. Complementation analysis in Arabidopsis tt8 mutant showed that FtTT8 could not recover anthocyanin deficiency but could promote PAs accumulation. Overexpression of FtTT8 in red-flowering tobacco showed that FtTT8 inhibits anthocyanin biosynthesis and accelerates proanthocyanidin biosynthesis. QRT-PCR and yeast one-hybrid assay revealed that FtTT8 might bind to the promoter of NtUFGT and suppress its expression, while binding to the promoter of NtLAR and upregulating its expression in K326 tobacco. This displayed the bidirectional regulating function of FtTT8 that negatively regulates anthocyanin biosynthesis and positively regulates proanthocyanidin biosynthesis. The results provide new insights on TT8 in Tartary buckwheat, which is inconsistent with TT8 from other plant species, and FtTT8 might be a high-quality gene resource for Tartary buckwheat breeding.

Genome-wide analysis of bHLH gene family in Coptis chinensis provides insights into the regulatory role in benzylisoquinoline alkaloid biosynthesis.

Coptis chinensis Franch is a perennial species with high medical value. The rhizome of C. chinensis is a traditional Chinese medicine widely used for more than 2000 years in China. Its principal active ingredients are benzylisoquinoline alkaloids (BIAs). The basic helix-loop-helix (bHLH) transcription factors play an important regulatory role in the biosynthesis of plant secondary metabolites. However, the bHLH genes in C. chinensis have not been described, and little is known about their roles in alkaloid biosynthesis. In this study, a total of 143 CcbHLH genes (CcbHLHs) were identified and unevenly distributed on nine chromosomes. Phylogenetic analysis divided the 143 CcbHLH proteins into 26 subfamilies by comparison with Arabidopsis thaliana bHLH proteins. The majority CcbHLHs in each subgroup had similar gene structures and conserved motifs. Furthermore, the physicochemical properties, conserved motif, intron/exon composition, and cis-acting elements of CcbHLHs were analyzed. Transcriptome analysis revealed that 30 CcbHLHs were significantly expressed in the rhizomes of C. chinensis. Co-expression analysis revealed that 11 CcbHLHs were highly positively correlated with contents of various alkaloids of C. chinensis. Moreover, yeast one-hybrid experiments verified that CcbHLH001 and CcbHLH0002 could interact with the promoters of berberine biosynthesis pathway genes CcBBE and CcCAS, suggesting their regulatory roles in BIA biosynthesis. This study provides comprehensive insights into the bHLH gene family in C. chinensis and will support in-depth functional characterization of CcbHLHs involved in the regulation of protoberberine-type alkaloid biosynthesis.

Phylogenomics of transcriptionally active AP2/ERF and bHLH transcription factors and study of their promoter regions in Nothapodytes nimmoniana (J.Graham) Mabb.

Nothapodytes nimmoniana is a medicinally important plant producing anticancer monoterpene indole alkaloid (MIA), camptothecin (CPT). The CPT is synthesised through the strictosidine intermediate following the MIA pathway; however, transcriptional regulation of CPT pathway is still elusive in N. nimmoniana. Biosynthesis of MIA is regulated by various transcription factors (TFs) belonging to AP2/ERF, bHLH, MYB, and WRKY families. The present study identified transcriptionally active full-length 105 AP2/ERF and 68 bHLH family TFs from the N. nimmoniana. AP2/ERF TFs were divided into three subfamilies along with a soloist, while bHLH TFs were divided into 10 subfamilies according to their phylogenetic similarities. Three group IXa ERFs, Nn-ERF22, Nn-ERF29, and Nn-ERF41, one subfamily IVa TF Nn-bHLH7, and three subfamilies IIIe Nn-bHLH33, Nn-bHLH51, and Nn-bHLH52 clustered with the TFs regulating alkaloid biosynthesis in Catharanthus roseus, tomato, tobacco, and Artemisia annua. Expression of these TFs in N. nimmoniana was higher in roots, which is a primary CPT accumulating tissue. Moreover, genome skimming approach was used to reconstruct the promoter regions of candidate ERF genes to identify the cis-regulatory elements. The presence of G-boxes and other jasmonic acid-responsive elements in the promoter suggests the regulation of ERFs by bHLHs. The present study effectively generated and used genomics resource for characterisation of regulatory TFs from non-model medicinal plant.

Genome-wide identification and characterisation of bHLH transcription factors in Artemisia annua.

BACKGROUND: A. annua (also named Artemisia annua, sweet wormwood) is the main source of the anti-malarial drug artemisinin, which is synthesised and stored in its trichomes. Members of the basic Helix-Loop-Helix (bHLH) family of transcription factors (TFs) have been implicated in artemisinin biosynthesis in A. annua and in trichome development in other plant species. RESULTS: Here, we have systematically identified and characterised 226 putative bHLH TFs in A. annua. All of the proteins contain a HLH domain, 213 of which also contain the basic motif that mediates DNA binding of HLH dimers. Of these, 22 also contained a Myc domain that permits dimerisation with other families of TFs; only two proteins lacking the basic motif contained a Myc domain. Highly conserved GO annotations reflected the transcriptional regulatory role of the identified TFs, and suggested conserved roles in biological processes such as iron homeostasis, and guard cell and endosperm development. Expression analysis revealed that three genes (AabHLH80, AabHLH96, and AaMyc-bHLH3) exhibited spatiotemporal expression patterns similar to genes encoding key enzymes in artemisinin synthesis. CONCLUSIONS: This comprehensive analysis of bHLH TFs provides a new resource to direct further analysis into key molecular mechanisms underlying and regulating artemisinin biosynthesis and trichome development, as well as other biological processes, in the key medicinal plant A. annua.

A pH-responsive metal-organic framework for the co-delivery of HIF-2α siRNA and curcumin for enhanced therapy of osteoarthritis.

The occurrence of osteoarthritis (OA) is highly correlated with the reduction of joint lubrication performance, in which persistent excessive inflammation and irreversible destruction of cartilage dominate the mechanism. The inadequate response to monotherapy methods, suboptimal efficacy caused by undesirable bioavailability, short retention, and lack of stimulus-responsiveness, are few unresolved issues. Herein, we report a pH-responsive metal-organic framework (MOF), namely, MIL-101-NH2, for the co-delivery of anti-inflammatory drug curcumin (CCM) and small interfering RNA (siRNA) for hypoxia inducible factor (HIF-2α). CCM and siRNA were loaded via encapsulation and surface coordination ability of MIL-101-NH2. Our vitro tests showed that MIL-101-NH2 protected siRNA from nuclease degradation by lysosomal escape. The pH-responsive MIL-101-NH2 gradually collapsed in an acidic OA microenvironment to release the CCM payloads to down-regulate the level of pro-inflammatory cytokines, and to release the siRNA payloads to cleave the target HIF-2α mRNA for gene-silencing therapy, ultimately exhibiting the synergetic therapeutic efficacy by silencing HIF-2α genes accompanied by inhibiting the inflammation response and cartilage degeneration of OA. The hybrid material reported herein exhibited promising potential performance for OA therapy as supported by both in vitro and in vivo studies and may offer an efficacious therapeutic strategy for OA utilizing MOFs as host materials.

bHLH010/089 Transcription Factors Control Pollen Wall Development via Specific Transcriptional and Metabolic Networks in Arabidopsis thaliana.

The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089-like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Tropaeolum majus R2R3 MYB Transcription Factor TmPAP2 Functions as a Positive Regulator of Anthocyanin Biosynthesis.

Anthocyanins are an important group of water-soluble and non-toxic natural pigments with antioxidant and anti-inflammatory properties that can be found in flowers, vegetables, and fruits. Anthocyanin biosynthesis is regulated by several different types of transcription factors, including the WD40-repeat protein Transparent Testa Glabra 1 (TTG1), the bHLH transcription factor Transparent Testa 8 (TT8), Glabra3 (GL3), Enhancer of GL3 (EGL3), and the R2R3 MYB transcription factor Production of Anthocyanin Pigment 1 (PAP1), PAP2, MYB113, and MYB114, which are able to form MYB-bHLH-WD40 (MBW) complexes to regulate the expression of late biosynthesis genes (LBGs) in the anthocyanin biosynthesis pathway. Nasturtium (Tropaeolum majus) is an edible flower plant that offers many health benefits, as it contains numerous medicinally important ingredients, including anthocyanins. By a comparative examination of the possible anthocyanin biosynthesis regulator genes in nasturtium varieties with different anthocyanin contents, we found that TmPAP2, an R2R3 MYB transcription factor gene, is highly expressed in "Empress of India", a nasturtium variety with high anthocyanin content, while the expression of TmPAP2 in Arabidopsis led to the overproduction of anthocyanins. Protoplast transfection shows that TmPAP2 functions as a transcription activator; consistent with this finding, some of the biosynthesis genes in the general phenylpropanoid pathway and anthocyanin biosynthesis pathway were highly expressed in "Empress of India" and the 35S:TmPAP2 transgenic Arabidopsis plants. However, protoplast transfection indicates that TmPAP2 may not be able to form an MBW complex with TmGL3 and TmTTG1. These results suggest that TmPAP2 may function alone as a key regulator of anthocyanin biosynthesis in nasturtiums.

Berberine inhibited the formation of metastasis by intervening the secondary homing of colorectal cancer cells in the blood circulation to the lung and liver through HEY2.

BACKGROUND: Metastasis is the leading cause of death in patients with colorectal cancer (CRC). The 5-year survival rate of CRC patients in whom the cancer has spread to distant sites is 13.5%. The most common sites of CRC metastasis are liver and lung. The principal therapies for CRC metastatic disease are surgery, but its benefits are limited. PURPOSE: This study aimed to reveal the regulatory mechanism of berberine on secondary homing of CRC cells to form metastatic focus. This was more valuable than the previous direct study of the migration and metastasis characteristics of CRC cells. METHODS: In this study, we used the functional enrichment analysis of differentially expressed genes after berberine treatment and investigated co-expression modules related with CRC metastasis by WGCNA. PPI and survival analyses of significant modules were also conducted. The biological functions of berberine in CRC lung and liver metastasis were investigated by a series of in vitro and in vivo experiments: MTT, colony formation and mouse tail vein injection. And we scanned through the entire extracellular domain of HEY2 protein for autodocking analysis with berberine. RESULTS: We found the differentially expressed genes (DEGs) after berberine treatment were related with cancer progression and metastasis related pathways. Through WGCNA analysis, four cancer progression and metastasis related modules were detected. After PPI and survival analysis, we identified and validated HEY2 as a hub gene, high expression and poor survival at the metastatic stage. Functionally, berberine inhibited the survival, invasion and migration of CRC cells in vitro and in vivo. Mechanistically, berberine treatment down-regulated the expression of HEY2, metastasis related protein E-cadherin, ß-catenin and Cyclin D1 during Mesenchymal epithelial transformation (MET). Berberine and HEY2 showed a significant interaction, and berberine binded to HEY2 protein at the residue HIS-99 interface with a hydrogen-bond distance of 1.9A. CONCLUSIONS: We revealed that berberine could significantly inhibit the expression of hub gene HEY2 and metastasis related proteins E-cadherin and ß-catenin and Cyclin D1 during MET in CRC lung and liver metastases. In total, HEY2 was a promising candidate biomarker for prognosis and molecular characteristics in CRC metastasis.

[Cloning and expression analysis of 8 bHLH transcription factors in Panax quinquefolius].

A total of 8 bHLH transcription factors were cloned from Panax quinquefolius and the response of them to methyl jasmonate(MeJA) was studied.To be specific, based on the preliminary transcriptome screening, 8 bHLH transcription factors were cloned with seedlings which had been cultured for 3 weeks.The content of ginsenosides Rg_1, Re, and Rb_1, and total saponins in the adventitious roots of P.quinquefolius was determined at different time of MeJA treatment by high performance liquid chromatography(HPLC) and spectrophotometry.Real-time quantitative polymerase chain reaction(PCR) was used to detect the relative expression of 8 transcription factors after MeJA treatment.The correlation between the relative expression of the 8 transcription factors and the saponin content after MeJA treatment was checked by Pearson's correlation analysis.The results showed that the PCR products(Pq-bHLH21-Pq-bHLH28) of the 8 bHLH transcription factors were 762-2 013 bp in length.They were submitted to NCBI to obtain the Genbank access numbers.The proteins yielded from Pq-bHLH21-Pq-bHLH28 showed amino acid sequence identity of 24.90%, and each amino acid sequence had the bHLH(Basic Helix-loop-helix) conserved domain and belonged to the bHLH family.The 5 amino acid sequences of Pq-bHLH22 and Pq-bHLH24-Pq-bHLH27 contained the bHLH-MYC N domain, which belonged to the MYC transcription factors.Pq-bHLH21-Pq-bHLH28 responded to MeJA within 48 h of treatment.At 72 h, the expression of Pq-bHLH24 reached 106.53 folds the highest in the treatment group.Pq-bHLH25, Pq-bHLH27, and Pq-bHLH28 showed synergic expression.Pq-bHLH21 may re-gulate the biosynthetic pathway of ginsenoside Rb_1, while Pq-bHLH22, Pq-bHLH25, and Pq-bHLH28 were in significantly positive correlation with the biosynthetic pathway of ginsenoside Re.The result lays a foundation for further verifying the regulation of ginsenoside biosynthesis by bHLH transcription factors.

Gene silencing, knockout and over-expression of a transcription factor ABORTED MICROSPORES (SlAMS) strongly affects pollen viability in tomato (Solanum lycopersicum).

BACKGROUND: The tomato (Solanum lycopersicum L.) is an economically valuable crop grown worldwide. Because the use of sterile males reduces the cost of F1 seed production, the innovation of male sterility is of great significance for tomato breeding. The ABORTED MICROSPORES gene (AMS), which encodes for a basic helix-loop-helix (bHLH) transcription factor, has been previously indicated as an essential gene for tapetum development in Arabidopsis and rice. To determine the function of the SlAMS gene (AMS gene from S. lycopersicum) and verify whether it is a potential candidate gene for generating the male sterility in tomato, we used virus-induced gene silencing (VIGS), CRISPR/Cas9-mediated genome editing and over-expression technology to transform tomato via Agrobacterium infection. RESULTS: Here, the full-length SlAMS gene with 1806 bp from S. lycopersicum (Accession No. MK591950.1) was cloned from pollen cDNA. The results of pollen grains staining showed that, the non-viable pollen proportions of SlAMS-silenced (75%), -knockouted (89%) and -overexpressed plants (60%) were significantly higher than the wild type plants (less than 10%; P < 0.01). In three cases, the morphology of non-viable pollen grains appeared tetragonal, circular, atrophic, shriveled, or otherwise abnormally shaped, while those of wild type appeared oval and plump. Furthermore, the qRT-PCR analysis indicated that SlAMS in anthers of SlAMS-silenced and -knockouted plants had remarkably lower expression than in that of wild type (P < 0.01), and yet it had higher expression in SlAMS-overexpressed plants (P < 0.01). CONCLUSION: In this paper, Our research suggested alternative approaches to generating male sterility in tomato, among which CRISPR/Cas9-mediated editing of SlAMS implied the best performance. We also demonstrated that the downregulation and upregulation of SlAMS both affected the pollen formation and notably led to reduction of pollen viability, suggesting SlAMS might be essential for regulating pollen development in tomato. These findings may facilitate studies on clarifying the SlAMS-associated molecular regulatory mechanism of pollen development in tomato.

Identification, Molecular Characteristic, and Expression Analysis of PIFs Related to Chlorophyll Metabolism in Tea Plant (Camellia sinensis).

The phytochrome-interacting factors (PIFs) proteins belong to the subfamily of basic helix-loop-helix (bHLH) transcription factors and play important roles in chloroplast development and chlorophyll biosynthesis. Currently, knowledge about the PIF gene family in Camellia sinensis remains very limited. In this study, seven PIF members were identified in the C. sinensis genome and named based on homology with AtPIF genes in Arabidopsis thaliana. All C. sinensis PIF (CsPIF) proteins have both the conserved active PHYB binding (APB) and bHLH domains. Phylogenetic analysis revealed that CsPIFs were clustered into four groups-PIF1, PIF3, PIF7, and PIF8-and most CsPIFs were clustered in pairs with their corresponding orthologs in Populus tremula. CsPIF members in the same group tended to display uniform or similar exon-intron distribution patterns and motif compositions. CsPIF genes were differentially expressed in C. sinensis with various leaf colors and strongly correlated with the expression of genes involved in the chlorophyll metabolism pathway. Promoter analysis of structural genes related to chlorophyll metabolism found DNA-binding sites of PIFs were abundant in the promoter regions. Protein-protein interaction networks of CsPIFs demonstrated a close association with phytochrome, PIF4, HY5, TOC1, COP1, and PTAC12 proteins. Additionally, subcellular localization and transcriptional activity analysis suggested that CsPIF3b was nuclear localized protein and possessed transcriptional activity. We also found that CsPIF3b could activate the transcription of CsHEMA and CsPOR in Nicotiana benthamiana leaves. This work provides comprehensive research of CsPIFs and would be helpful to further promote the regulation mechanism of PIF on chlorophyll metabolism in C. sinensis.

The characterization and candidate gene isolation for a novel male-sterile mutant ms40 in maize.

KEY MESSAGE: A novel genic male-sterile mutant ms40 was obtained from EMS treated RP125. The key candidate gene ZmbHLH51 located on chromosome 4 was identified by map-based cloning. This study further enriched the male sterile gene resources for both production applications and theoretical studies of abortion mechanisms. Maize male-sterile mutant 40 (ms40) was obtained from the progeny of the ethyl methanesulfonate (EMS) treated inbred line RP125. Genetic analysis indicated that the sterility was controlled by a single recessive nuclear gene. Cytological observation of anthers revealed that the cuticles of ms40 anthers were abnormal, and no Ubisch bodies were observed on the inner surface of ms40 anthers through scanning electron microscopy(SEM). Moreover, its tapetum exhibited delayed degradation and then blocked the formation of normal microspores. Using map-based cloning strategy, the ms40 locus was found to locate in a 282-kb interval on chromosome 4, and five annotated genes were predicted within this region. PCR-based sequencing detected a single non-synonymous SNP (G > A) that changed glycine (G) to arginine (A) in the seventh exon of Zm00001d053895, while no sequence difference between ms40 and RP125 was found for the other four genes. Zm00001d053895 encodes the bHLH transcription factor ZmbHLH51 which is localized in the nucleus. Phylogenetic analysis showed that ZmbHLH51 had the highest homology with Sb04g001650, a tapetum degeneration retardation (TDR) bHLH transcription factor in Sorghum bicolor. Co-expression analysis revealed a total of 1192 genes co-expressed with ZmbHLH51 in maize, 647 of which were anther-specific genes. qRT-PCR results suggested the expression levels of some known genes related to anther development were affected in ms40. In summary, these findings revealed the abortion characteristics of ms40 anthers and lay a foundation for further studies on the mechanisms of male fertility.

Single Exposure to the Cathinones MDPV and α-PVP Alters Molecular Markers of Neuroplasticity in the Adult Mouse Brain.

Synthetic cathinones have gained popularity among young drug users and are widely used in the clandestine market. While the cathinone-induced behavioral profile has been extensively investigated, information on their neuroplastic effects is still rather fragmentary. Accordingly, we have exposed male mice to a single injection of MDPV and α-PVP and sacrificed the animals at different time points (i.e., 30 min, 2 h, and 24 h) to have a rapid readout of the effect of these psychostimulants on neuroplasticity in the frontal lobe and hippocampus, two reward-related brain regions. We found that a single, low dose of MDPV or α-PVP is sufficient to alter the expression of neuroplastic markers in the adult mouse brain. In particular, we found increased expression of the transcription factor Npas4, increased ratio between the vesicular GABA transporter and the vesicular glutamate transporter together with changes in the expression of the neurotrophin Bdnf, confirming the widespread impact of these cathinones on brain plasticity. To sum up, exposure to low dose of cathinones can impair cortical and hippocampal homeostasis, suggesting that abuse of these cathinones at much higher doses, as it occurs in humans, could have an even more profound impact on neuroplasticity.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development.

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.

Genome-wide identification of the tea plant bHLH transcription factor family and discovery of candidate regulators of trichome formation.

Leaf trichomes play vital roles in plant resistance and the quality of tea. Basic helix-loop-helix (bHLH) transcription factors (TFs) play an important role in regulating plant development and growth. In this study, a total of 134 CsbHLH proteins were identified in the Camellia sinensis var. sinensis (CSS) genome. They were divided into 17 subgroups according to the Arabidopsis thaliana classification. Phylogenetic tree analysis indicated that members of subgroups IIIc-I and IIIc-II might be associated with trichome formation. The expression patterns of CsbHLH116, CsbHLH133, CsbHLH060, CsbHLH028, CsbHLH024, CsbHLH112 and CsbHLH053 from clusters 1, 3 and 5 were similar to the trichome distribution in tea plants. CsbHLH024 and CsbHLH133 were located in the cell nucleus and possessed transcriptional activation ability. They could interact with CsTTG1, which is a regulator of tea trichome formation. This study provides useful information for further research on the function of CsbHLHs in trichome formation.

CircMEMO1 modulates the promoter methylation and expression of TCF21 to regulate hepatocellular carcinoma progression and sorafenib treatment sensitivity.

BACKGROUND: Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples. METHODS: The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity. RESULTS: We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment. CONCLUSION: Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.