Integrated Analysis of Basic Helix Loop Helix Transcription Factor Family and Targeted Terpenoids Reveals Candidate AarbHLH Genes Involved in Terpenoid Biosynthesis in Artemisia argyi

Artemisia argyi is a valuable traditional medicinal plant in Asia. The essential oil from its leaves is rich in terpenoids and has been used to enhance health and well-being. In China, the market scale of industries related to A. argyi has attained tens of billions of Chinese Yuan. The basic helix-loop-helix (bHLH) family is one of the largest transcription factors families in plants that plays crucial roles in diverse biological processes and is an essential regulatory component of terpenoid biosynthesis. However, the bHLH TFs and their regulatory roles in A. argyi remain unknown. Here, 53 AarbHLH genes were identified from the transcriptome of A. argyi and were classified into 15 subfamilies based on the classification of bHLH proteins in Arabidopsis thaliana. The MEME analysis showed that the conserved motif 1 and motif 2 constituted the most conserved bHLH domain and distributed in most AarbHLH proteins. Additionally, integrated analysis of the expression profiles of AarbHLH genes and the contents of targeted terpenoids in different tissues group and JA-treated group were performed. Eleven up-regulated AarbHLHs and one down-regulated AarbHLH were screened as candidate genes that may participate in the regulation of terpenoid biosynthesis (TPS-AarbHLHs). Correlation analysis between gene expression and terpenoid contents indicated that the gene expression of these 12 TPS-AarbHLHs was significantly correlated with the content changes of 1,8-cineole or β-caryophyllene. Protein–protein interaction networks further illustrated that these TPS-AarbHLHs might be involved in terpenoid biosynthesis in A. argyi. This finding provides a basis to further investigate the regulation mechanism of AarbHLH genes in terpenoid biosynthesis, and will be helpful to improve the quality of A. argyi.

Phenotypical Characterization of a Mutant dea1 With Developmental Defect in Female Inflorescence and Mutantion Impact on Yield in Maize

The establishment of female inflorescence morphology is of great significance to the formation of final maize yield. defective ear1 (dea1) is a novel maize mutant with developmental defect of female inflorescence caused by natural variation. Morphological analysis revealed that the mutant dea1 was characterized as a “scar-like” crack on the adaxial side of the top of the ear, accounting for 28.6-100.0% of the ear length, with an average of 32.4%. The results of scanning electron microscope showed that there was collapse in the formation of paired spikelet primordium at the base of the axillary meristem. Most of investigated botanical and agronomical traits of dea1 were lower than those of wild type, except for ear length and hundred grain weight. The grain yield per ear of mutant dea1 was 35.93% lower than that of wild type, and the width of mutation crack contributed the most to the yield loss per ear. The identification of the mutant dea1 and the characteristically phenotypic analysis provide a theoretical basis for the study of the molecular regulation mechanism of ear development and the application of high-yield breeding in maize.The establishment of female inflorescence morphology is of great significance to the formation of final maize yield. defective ear1 (dea1) is a novel maize mutant with developmental defect of female inflorescence caused by natural variation. Morphological analysis revealed that the mutant dea1 was characterized as a “scar-like” crack on the adaxial side of the top of the ear, accounting for 28.6-100.0% of the ear length, with an average of 32.4%. The results of scanning electron microscope showed that there was collapse in the formation of paired spikelet primordium at the base of the axillary meristem. Most of investigated botanical and agronomical traits of dea1 were lower than those of wild type, except for ear length and hundred grain weight. The grain yield per ear of mutant dea1 was 35.93% lower than that of wild type, and the width of mutation crack contributed the most to the yield loss per ear. The identification of the mutant dea1 and the characteristically phenotypic analysis provide a theoretical basis for the study of the molecular regulation mechanism of ear development and the application of high-yield breeding in maize.

Potential HNF1A-AS1/miR-214/INHBA signal regulation mechanism in colorectal cancer based on bioinformatics analysis and validation

Preceding studies have identified that noncoding RNA plays a significant role in the occurrence and development of tumors. Colorectal cancer (CRC) has attracted increasing attention due to its high incidence and mortality rate. Based on Cancer Genome Atlas (TCGA) database analysis, it was found that compared with normal tissues, HNF1A-AS1 and INHBA were highly expressed in CRC tissues; miR-214 was relatively low expressed, and it is predicted to specifically target the3' untranslated region (3'UTR region) of INHBA. Besides, the result was consistent with the quantitative reverse transcription PCR (RT-qPCR) verification results of 17 CRC cases and adjacent tissues collected clinically. Western Blot (WB) manifested that INHBA protein was highly expressed in CRC tissues, which was consistent with the results of CRC cell lines (HT29, SW480). Immunohistochemical (IHC) staining demonstrated that INHBA protein was brownish yellow, overwhelming majority of INHBA protein were located in the cytoplasm, and expression level was significantly higher than that in adjacent tissues. Based on previous studies, the biological process of INHBA-mediated TGF-β/Smad signaling pathway inducing the proliferation and invasion of CRC has been partially confirmed, but the upstream signaling molecules and mechanisms which regulating INHBA remain unclear. Herein, benefiting from bioinformatics, preliminary experimental results and previous research, they provide basis for the follow-up study on the regulation of HNF1A-AS1/miR-214/INHBA signal axis in CRC.

The Paeonia qiui R2R3-MYB Transcription Factor PqMYB113 Positively Regulates Anthocyanin Accumulation in Arabidopsis thaliana and Tobacco

Paeonia qiui is a wild species of tree peony native to China. Its leaves are purplish red from the bud germination to the flowering stage, and anthocyanin is the main pigment in purplish red leaves. However, the anthocyanin synthesis regulation mechanism in tree peony leaves remains unclear. In this study, an R2R3-MYB, PqMYB113 was identified from the leaves of P. qiui. Phylogenetic analysis revealed that PqMYB113 clustered with Liquidambar LfMYB113 and grape VvMYBA6. Subcellular location analysis showed that PqMYB113 was located in the cell nucleus. The transient reporter assay suggested that PqMYB113 was a transcriptional activator. The overexpression of PqMYB113 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) resulted in increased anthocyanin accumulation and the upregulation of CHS, F3H, F3’H, DFR, and ANS. The dual luciferase reporter assay showed that PqMYB113 could activate the promoters of PqDFR and PqANS. Bimolecular fluorescence complementation assays and yeast two-hybrid assays suggested that PqMYB113 could form a ternary MBW complex with PqbHLH1 and PqWD40 cofactors. These results provide insight into the regulation of anthocyanin biosynthesis in tree peony leaves.

Disposable Stainless-Steel Wire-Based Electrochemical Microsensor for In Vivo Continuous Monitoring of Hydrogen Peroxide in Vein of Tomato Leaf

As one of the pivotal signal molecules, hydrogen peroxide (H2O2) has been demonstrated to play important roles in many physiological processes of plants. Continuous monitoring of H2O2 in vivo could help understand its regulation mechanism more clearly. In this study, a disposable electrochemical microsensor for H2O2 was developed. This microsensor consists of three parts: low-cost stainless-steel wire with a diameter of 0.1 mm modified by gold nanoparticles (disposable working electrode), an untreated platinum wire with a diameter of 0.1 mm (counter electrode), and an Ag/AgCl wire with a diameter of 0.1 mm (reference electrode), respectively. The microsensor could detect H2O2 in levels from 10 to 1000 µM and exhibited excellent selectivity. On this basis, the dynamic change in H2O2 in the vein of tomato leaf under high salinity was continuously monitored in vivo. The results showed that the production of H2O2 could be induced by high salinity within two hours. This study suggests that the disposable electrochemical microsensor not only suits continuously detecting H2O2 in microscopic plant tissue in vivo but also reduces the damage to plants. Overall, our strategy will help to pave the foundation for further investigation of the generation, transportation, and elimination mechanism of H2O2 in plants.

Screening and Identification of Transcription Factors Potentially Regulating Foxl2 Expression in Chlamys farreri Ovary

Foxl2 is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamysfarreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 107 CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of C. farreri Foxl2 (Cf-Foxl2) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary.

Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores’ production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

Homologous BHLH transcription factors induce distinct deformations of torsionally-stressed DNA: a potential transcription regulation mechanism.

Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein we address the impact of torsional stress on DNA complexation with homologous human basic-helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax, and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequences, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of ±5°/b.p. step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

A Glutathione Peroxidase Gene from Litopenaeus vannamei Is Involved in Oxidative Stress Responses and Pathogen Infection Resistance

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.