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.

Identification of Abscisic Acid-Dependent Phosphorylated Basic Helix-Loop-Helix Transcription Factors in Guard Cells of Vicia faba by Mass Spectrometry

An unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard-cell protoplasts (GCPs) from Vicia faba. Subsequently, ABA-dependent phosphorylated proteins were identified as basic helix–loop–helix transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, whether the 61 kDa protein in Vicia GCPs is an AKS is unclear. We performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in V. faba (VfAKSs) by mass spectrometry. The 61 kDa protein was identified as VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated at Ser residues, which are important for 14-3-3 protein binding and monomerisation, in response to ABA in GCPs. Orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain and a regulator of stomatal movement, also co-immunoprecipitated with 14-3-3 protein from guard cells.

Lyl-1 regulates primitive macrophages and microglia development

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/”early EMP” and transient-definitive/”late EMP” waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

The basic helix–loop–helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell’s fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

Genome-Wide Identification and Functional Analysis of the Basic Helix-Loop-Helix (bHLH) Transcription Family Reveals Candidate PtFBH Genes Involved in the Flowering Process of Populus trichocarpa

As one of the largest TF families+ in plants, the basic helix-loop-helix (bHLH) family plays an important part in the growth and development of many plants. FLOWERING BHLH (FBH) encodes a bHLH-type transcriptional factor related to the flowering process. Poplar is a model woody plant as well as an important economic tree species with a small genome. However, the characteristics of the bHLHs and FBHs gene family in the newest version of Populustrichocarpa genome have not been analyzed yet. We identified 233 PtbHLHs and 10 PtFBHs in the newest version genome, and PtbHLHs were classified into 21 groups with FBH subfamily occupying one, supported by phylogenetic analysis, exon–intron patterns, and conserved protein motifs. These PtHLHs were distributed on 19 chromosomes unevenly and expressed in nucleus mainly. Gene duplication and synteny analysis have indicated that the PtbHLHs gene family has undergone strong purification selection during the evolution process. The cis-elements analysis has suggested that PtbHLHs may be related to the growth and development. Conserved residues of FBHs among Arabidopsis and poplar were also identified. Expression of 227 PtHLH genes (6 unmatched, 13 no expressed) showed diverse patterns in different tissues, implying their multiple functions. Protein–protein interaction network prediction and expression patterns in three states of the flowering process (Flowers-Dormant, Flowers-Expanding and Flowers-Expanded) suggested that some members of PtbHLH and PtFBH family may be involved in the flowering process. Our comprehensive and systematic analysis can provide some valuable clues and basic reference toward further investigations on physiological and molecular functions of PtbHLHs.

The effector FEP3/IRON MAN1 modulates interaction between BRUTUS-LIKE1 and bHLH subgroup IVb and IVc proteins

Plants use the micronutrient iron (Fe) efficiently to balance the requirements for Fe during growth with its potential cytotoxic effects. A cascade of basic helix-loop-helix (bHLH) transcription factors is initiated by bHLH proteins of the subgroups IVb and IVc. This induces more than 50 genes in higher plants that can be grouped in co-expression clusters. Gene co-expression networks contain information on functional protein interactomes. We conducted a targeted yeast two-hybrid screen with pairwise combinations of 23 proteins stemming from previously characterized Fe-deficiency-induced gene co-expression clusters and regulators. We identified novel and described interactions, as well as interaction hubs with multiple interactions within the network. We found that BRUTUS-LIKE E3 ligases (BTSL1, BTSL2) interacted with basic helix-loop-helix (bHLH) transcription factors of the subgroups IVb and IVc including PYE, bHLH104 and ILR3, and with small FE UPTAKE-INDUCING PEPTIDE3/IRON MAN1 (FEP3/IMA1). Through deletion studies and with support of molecular docking, we mapped the interaction sites to three-amino-acid regions in BTSL1 and FEP3/IMA1. The FEP3/IMA1 active residues are present in interacting sites of the bHLH IVc factors. FEP3/IMA1 attenuated interaction of BTSL1 with bHLH proteins in a quantitative yeast three-hybrid assay suggesting that it is an inhibitor. Co-expression of BTSL1 and bHLH IVb and IVc factors uncovered unexpected patterns of subcellular localization. Combining deletion mapping, protein interaction and physiological analysis, we discuss the model that FEP3/IMA1 is a small effector protein inhibiting BTSL1/BTSL2-mediated degradation of bHLH subgroup IVb and IVc proteins.

The basic helix-loop-helix transcription factor TabHLH1 increases chlorogenic acid and luteolin biosynthesis in Taraxacum antungense Kitag

Polyphenols are the main active components of the anti-inflammatory compounds in dandelion, and chlorogenic acid (CGA) is one of the primary polyphenols. However, the molecular mechanism underlying the transcriptional regulation of CGA biosynthesis remains unclear. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT2) is the last rate-limiting enzyme in chlorogenic acid biosynthesis in Taraxacum antungense. Therefore, using the TaHQT2 gene promoter as a probe, a yeast one-hybrid library was performed, and a basic helix-loop-helix (bHLH) transcription factor, TabHLH1, was identified that shared substantial homology with Gynura bicolor DC bHLH1. The TabHLH1 transcript was highly induced by salt stress, and the TabHLH1 protein was localized in the nucleus. CGA and luteolin concentrations in TabHLH1-overexpression transgenic lines were significantly higher than those in the wild type, while CGA and luteolin concentrations in TabHLH1-RNA interference (RNAi) transgenic lines were significantly lower. Quantitative real-time polymerase chain reaction demonstrated that overexpression and RNAi of TabHLH1 in T. antungense significantly affected CGA and luteolin concentrations by upregulating or downregulating CGA and luteolin biosynthesis pathway genes, especially TaHQT2, 4-coumarate-CoA ligase (Ta4CL), chalcone isomerase (TaCHI), and flavonoid-3′-hydroxylase (TaF3′H). Dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays indicated that TabHLH1 directly bound to the bHLH-binding motifs of proTaHQT2 and proTa4CL. This study suggests that TabHLH1 participates in the regulatory network of CGA and luteolin biosynthesis in T. antungense and might be useful for metabolic engineering to promote plant polyphenol biosynthesis.