bHLH Proteins Research Articles

Integrative Analysis of Metabolome and Transcriptome Identifies Potential Genes Involved in the Flavonoid Biosynthesis in Entada phaseoloides Stem.

Entada phaseoloides stem is known for its high medicinal benefits and ornamental value. Flavonoids are one of the main active constituents in E. phaseoloides stem. However, the regulatory mechanism of flavonoids accumulation in E. phaseoloides is lacking. Here, phytochemical compounds and transcripts from stems at different developmental stages in E. phaseoloides were investigated by metabolome and transcriptome analysis. The metabolite profiling of the oldest stem was obviously different from young and older stem tissues. A total of 198 flavonoids were detected, and flavones, flavonols, anthocyanins, isoflavones, and flavanones were the main subclasses. The metabolome data showed that the content of acacetin was significantly higher in the young stem and older stem than the oldest stem. Rutin and myricitrin showed significantly higher levels in the oldest stem. A total of 143 MYBs and 143 bHLHs were identified and classified in the RNA-seq data. Meanwhile, 34 flavonoid biosynthesis structural genes were identified. Based on the expression pattern of structural genes involved in flavonoid biosynthesis, it indicated that flavonol, anthocyanin, and proanthocyanin biosynthesis were first active during the development of E. phaseoloides stem, and the anthocyanin or proanthocyanin biosynthesis branch was dominant; the flavone biosynthesis branch was active at the late developmental stage of the stem. Through the correlation analysis of transcriptome and metabolome data, the potential candidate genes related to regulating flavonoid synthesis and transport were identified. Among them, the MYBs, bHLH, and TTG1 are coregulated biosynthesis of flavonols and structural genes, bHLH and transporter genes are coregulated biosynthesis of anthocyanins. In addition, the WDR gene TTG1-like (AN11) may regulate dihydrochalcones and flavonol biosynthesis in specific combinations with IIIb bHLH and R2R3-MYB proteins. Furthermore, the transport gene protein TRANSPARENT TESTA 12-like gene is positively regulated the accumulation of rutin, and the homolog of ABC transporter B family member gene is positively correlated with the content of flavone acacetin. This study offered candidate genes involved in flavonoid biosynthesis, information of flavonoid composition and characteristics of flavonoids accumulation, improved our understanding of the MYBs and bHLHs-related regulation networks of flavonoid biosynthesis in E. phaseoloides stem, and provided references for the metabolic engineering of flavonoid biosynthesis in E. phaseoloides stem.

The PGS1 basic helix-loop-helix protein regulates Fl3 to impact seed growth and grain yield in cereals.

SummaryPlant transcription factors (TFs), such as basic helix‐loop‐helix (bHLH) and AT‐rich zinc‐binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5–20 days post‐anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E‐box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand‐grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait‐associated gene for improving cereal grain yield.

Genome-wide identification and characterization of PdbHLH transcription factors related to anthocyanin biosynthesis in colored-leaf poplar (Populus deltoids)

Basic helix-loop-helix (bHLH) proteins are transcription factors (TFs) that have been shown to regulate anthocyanin biosynthesis in many plant species. However, the bHLH gene family in Populus deltoids has not yet been reported. In this study, 185 PdbHLH genes were identified in the Populus deltoids genome and were classified into 15 groups based on their sequence similarity and phylogenetic relationships. Analysis of the gene structure, chromosome location and conserved motif of the PdbHLH genes were performed by bioinformatic methods. Gene duplication analyses revealed that 114 PdbHLH were expanded and retained after WGD/segmental and proximal duplication. Investigation of cis-regulatory elements of PdbHLH genes indicated that many PdbHLH genes are involved in the regulation of anthocyanin biosynthesis. The expression patterns of PdbHLHs were obtained from previous data in two colored-leaf poplar (QHP and JHP) and green leaf poplar (L2025). Further analysis revealed that 12 candidate genes, including 3 genes (PdbHLH57, PdbHLH143, and PdbHLH173) from the subgroup III(f) and 9 gene from other groups, were positively associated with anthocyanin biosynthesis. In addition, 4 genes (PdbHLH4, PdbHLH1, PdbHLH18, and PdbHLH164) may be involved in negatively regulating the anthocyanin biosynthesis. These results provide a basis for the functional characterization of bHLH genes and investigations on the molecular mechanisms of anthocyanin biosynthesis in colored-leaf poplar.

Overexpression of HLH4 Inhibits Cell Elongation and Anthocyanin Biosynthesis in Arabidopsis thaliana.

In plants, many basic helix-loop-helix (bHLH) transcription factors are involved in controlling cell elongation. Three bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1), Cryptochrome Interacting Basic Helix-loop-helix 5 (CIB5), and Arabidopsis ILI1 binding bHLH1 (IBH1) form a triantagonistic system that antagonistically regulates cell elongation in a competitive manner. In this study, we identified a new player, HLH4, related to IBH1, that negatively regulates cell elongation in Arabidopsis thaliana. Overexpression of HLH4 causes dwarf and dark green phenotypes and results in the downregulation of many key regulatory and enzymatic genes that participate in the anthocyanin biosynthetic pathway. HLH4 interacts with CIB5 and PRE1. By interacting with CIB5, HLH4 interferes with the activity of CIB5, and thus inhibiting the transcription of cell elongation-related genes regulated by CIB5, including EXPANSINS8 and 11 (EXP8 and EXP11) and indole-3-acetic acid 7 and 17 (IAA7 and IAA17). The interference of HLH4 on CIB5 is counteracted by PRE1, in which these bHLH proteins form a new tri-antagonistic system.

Genome-Wide Identification of Apple Atypical bHLH Subfamily PRE Members and Functional Characterization of MdPRE4.3 in Response to Abiotic Stress.

Paclobutrazol Resistance (PRE) genes encode atypical basic helix–loop–helix (bHLH) transcription factor family. Typical bHLH proteins contain a bifunctional structure with a basic region involved in DNA binding and an adjacent helix–loop–helix domain involved in protein–protein interaction. PRE members lack the basic region but retain the HLH domain, which interacts with other typical bHLH proteins to suppress or enhance their DNA-binding activity. PRE proteins are involved in phytohormone responses, light signal transduction, and fruit pigment accumulation. However, apple (Malus domestica) PRE protein functions have not been studied. In this study, nine MdPRE genes were identified from the apple GDDH13 v1.1 reference genome and were mapped to seven chromosomes. The cis-acting element analysis revealed that MdPRE promoters possessed various elements related to hormones, light, and stress responses. Expression pattern analysis showed that MdPRE genes have different tissue expression profiles. Hormonal and abiotic stress treatments can induce the expression of several MdPRE genes. Moreover, we provide molecular and genetic evidence showing that MdPRE4.3 increases the apple’s sensitivity to NaCl, abscisic acid (ABA), and indoleacetic acid (IAA) and improves tolerance to brassinosteroids (BR); however, it does not affect the apple’s response to gibberellin (GA). Finally, the protein interaction network among the MdPRES proteins was predicted, which could help us elucidate the molecular and biological functions of atypical bHLH transcription factors in the apple.

Chili Pepper AN2 (CaAN2): A Visible Selection Marker for Nondestructive Monitoring of Transgenic Plants.

Selecting transformed plants is generally time consuming and laborious. To develop a method for transgenic plant selection without the need for antibiotics or herbicides, we evaluated the suitability of the R2R3 MYB transcription factor gene CaAN2 from purple chili pepper (Capsicum annuum) for use as a visible selection marker. CaAN2 positively regulates anthocyanin biosynthesis. Transient expression assays in tobacco (Nicotiana tabacum) leaves revealed that CaAN2 actively induced sufficient pigment accumulation for easy detection without the need for a basic helix-loop-helix (bHLH) protein as a cofactor; similar results were obtained for tobacco leaves transiently co-expressing the anthocyanin biosynthesis regulators bHLH B-Peru from maize and R2R3 MYB mPAP1D from Arabidopsis. Tobacco plants harboring CaAN2 were readily selected based on their red color at the shoot regeneration stage due to anthocyanin accumulation without the need to impose selective pressure from herbicides. Transgenic tobacco plants harboring CaAN2 showed strong pigment accumulation throughout the plant body. The ectopic expression of CaAN2 dramatically promoted the transcription of anthocyanin biosynthetic genes as well as regulators of this process. The red coloration of tobacco plants harboring CaAN2 was stably transferred to the next generation. Therefore, anthocyanin accumulation due to CaAN2 expression is a useful visible trait for stable transformation, representing an excellent alternative selection system for transgenic plants.

TRIPTYCHON-LIKE regulates aspects of both fruit flavor and color in citrus

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix-loop-helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase, responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator-repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing.

Ethylene Response Factor109 Attunes Immunity, Photosynthesis, and Iron Homeostasis in Arabidopsis Leaves.

Iron (Fe) is an essential micronutrient element for all organisms including plants. Chlorosis of young leaves is a common symptom of Fe deficiency, reducing the efficiency of photosynthesis, and, ultimately, crop yield. Previous research revealed strong responsiveness of the putative key transcription factor ERF109 to the Fe regime. To elucidate the possible role of ERF109 in leaf Fe homeostasis and photosynthesis, we subjected Arabidopsis thaliana erf109 knockout lines and Col-0 wild-type plants to transcriptome profiling via RNA-seq. The transcriptome profile of Fe-sufficient erf109 leaves showed a 71% overlap with Fe-deficient Col-0 plants. On the other hand, genes that were differentially expressed between Fe-deficient and Fe-sufficient Col-0 plants remained unchanged in erf109 plants under conditions of Fe deficiency. Mutations in ERF109 increased the expression of the clade Ib bHLH proteins bHLH38, bHLH39, bHLH101, the nicotianamine synthase NAS4, and the Fe storage gene FER1. Moreover, mutations in ERF109 led to significant down-regulation of defense genes, including CML37, WRKY40, ERF13, and EXO70B2. Leaves of erf109 exhibited increased Fe levels under both Fe-sufficient and Fe-deficient conditions. Reduced Fv/Fm and Soil Plant Analysis Development (SPAD) values in erf109 lines under Fe deficiency indicate curtailed ability of photosynthesis relative to the wild-type. Our findings suggest that ERF109 is a negative regulator of the leaf response to Fe deficiency. It further appears that the function of ERF109 in the Fe response is critical for regulating pathogen defense and photosynthetic efficiency. Taken together, our study reveals a novel function of ERF109 and provides a systematic perspective on the intertwining of the immunity regulatory network and cellular Fe homeostasis.

The bHLH family NITROGEN‐REPLETION INSENSITIVE1 represses nitrogen starvation‐induced responses in Chlamydomonas reinhardtii

The use of nitrogen (N) fertilizers has contributed to a quadrupling of agricultural production. However, > 50% of N fertilizers remain unabsorbed by crops and leak into the environment, demanding strategies to improve the N use efficiency. To identify the regulatory mechanisms of N assimilation, we performed a genetic screen using a Chlamydomonas reinhardtii strain whose motility depends upon the transcription of nitrate reductase-encoding NIT1, which is upregulated in response to N starvation. A persistently swimming mutant, nitrogen repletion insensitive 1 (nri1), was isolated and found to express N starvation-induced genes in N-replete culture. NRI1, previously reported as NSG17 (nitrogen starvation-induced gene 17), encodes a basic helix-loop-helix (bHLH) family transcription factor homologous to BES-interacting myc-like (BIM) in plants. Transcriptome analysis of N-replete nri1 culture revealed a concerted upregulation of the genes involved in the uptake and assimilation of external N sources. Many transcription factor-encoding genes in the bHLH and SBP families were also upregulated in nri1 culture. The DNA-binding sites of bHLH and SBP proteins were enriched in the promoters of the upregulated genes in nri1, suggesting that NRI1 suppresses transcriptional activators to regulate N starvation-induced genes. Consistent with the ammonium-insensitive gene expression pattern, N-starved nri1 gametes could not dedifferentiate back to vegetative cells when incubated in ammonium-containing media. Taken together, we conclude that NRI1 represses N starvation-specific responses when sufficient N is supplied in C. reinhardtii. Further investigation of how cellular N conditions regulate NRI1 activity will unravel the elusive mechanisms by which plant cells sense and respond to N starvation.

The bHLH/HLH transcription factors GhFP2 and GhACE1 antagonistically regulate fiber elongation in cotton.

Basic helix-loop-helix/helix-loop-helix (bHLH/HLH)transcription factors play important roles in cell elongation in plants. However, little is known about how bHLH/HLH transcription factors antagonistically regulate fiber elongation in cotton (Gossypium hirsutum). In this study, we report that two bHLH/HLH transcription factors, fiber-related protein 2 (GhFP2) and ACTIVATOR FOR CELL ELONGATION 1 (GhACE1), function in fiber development of cotton. GhFP2 is an atypical bHLH protein without the basic region, and its expression is regulated by brassinosteroid (BR)-related BRASSINAZOLE RESISTANT 1 (GhBZR1). Overexpression of GhFP2 in cotton hindered fiber elongation, resulting in shortened fiber length. In contrast, suppression of GhFP2 expression in cotton promoted fiber development, leading to longer fibers compared with the wild-type. GhFP2 neither contains a DNA-binding domain nor has transcriptional activation activity. Furthermore, we identified GhACE1, a bHLH protein that interacts with GhFP2 and positively regulates fiber elongation. GhACE1 could bind to promoters of plasma membrane intrinsic protein 2;7 (GhPIP2;7) and expansions 8 (GhEXP8) for directly activating their expression, but the interaction between GhFP2 and GhACE1 suppressed transcriptional activation of these target genes by GhACE1. Taken together, our results indicate that GhACE1 promotes fiber elongation by activating expressions of GhPIP2;7 and GhEXP8, but its transcription activation on downstream genes may be obstructed by BR-modulated GhFP2. Thus, our data reveal a key mechanism for fiber cell elongation through a pair of antagonizing HLH/bHLH transcription factors in cotton.

Comparative transcriptome and weighted correlation network analyses reveal candidate genes involved in chlorogenic acid biosynthesis in sweet potato

Chlorogenic acids (CGAs) are important secondary metabolites produced in sweet potato. However, the mechanisms of their biosynthesis and regulation remain unclear. To identify potential genes involved in CGA biosynthesis, analysis of the dynamic changes in CGA components and RNA sequencing were performed on young leaves (YL), mature leaves (ML), young stems (YS), mature stems (MS) and storage roots (SR). Accordingly, we found that the accumulation of six CGA components varied among the different tissues and developmental stages, with YS and YL recording the highest levels, while SR exhibited low levels. Moreover, the transcriptome analysis yielded 59,287 unigenes, 3,767 of which were related to secondary-metabolite pathways. The differentially expressed genes (DEGs) were identified based on CGA content levels by comparing the different samples, including ML vs. YL, MS vs. YS, SR vs. YL and SR vs. YS. A total of 501 common DEGs were identified, and these were mainly implicated in the secondary metabolites biosynthesis. Additionally, eight co-expressed gene modules were identified following weighted gene co-expression network analysis, while genes in darkgrey module were highly associated with CGA accumulation. Darkgrey module analysis revealed that 12 unigenes encoding crucial enzymes (PAL, 4CL, C4H, C3H and HCT/HQT) and 42 unigenes encoding transcription factors (MYB, bHLH, WD40, WRKY, ERF, MADS, GARS, bZIP and zinc finger protein) had similar expression patterns with change trends of CGAs, suggesting their potential roles in CGA metabolism. Our findings provide new insights into the biosynthesis and regulatory mechanisms of CGA pathway, and will inform future efforts to build a genetically improve sweet potato through the breeding of high CGA content varieties.

Intragenic suppressors unravel the role of the SCREAM ACT-like domain for bHLH partner selectivity in stomatal development

Multicellular organisms develop specialized cell types to achieve complex functions of tissues and organs. The basic helix-loop-helix (bHLH) proteins act as master regulatory transcription factors of such specialized cell types. Plant stomata are cellular valves in the aerial epidermis for efficient gas exchange and water control. Stomatal differentiation is governed by sequential actions of three lineage-specific bHLH proteins, SPEECHLESS (SPCH), MUTE, and FAMA, specifying initiation and proliferation, commitment, and terminal differentiation, respectively. A broadly expressed bHLH, SCREAM (SCRM), heterodimerizes with SPCH/MUTE/FAMA and drives stomatal differentiation via switching its partners. Yet nothing is known about its heterodimerization properties or partner preference. Here, we report the role of the SCRM C-terminal ACT-like (ACTL) domain for heterodimerization selectivity. Our intragenic suppressor screen of a dominant scrm-D mutant identified the ACTL domain as a mutation hotspot. Removal of this domain or loss of its structural integrity abolishes heterodimerization with MUTE, but not with SPCH or FAMA, and selectively abrogates the MUTE direct target gene expression. Consequently, the scrm-D ACTL mutants confer massive clusters of arrested stomatal precursor cells that cannot commit to differentiation when redundancy is removed. Structural and biophysical studies further show that SPCH, MUTE, and FAMA also possess the C-terminal ACTL domain, and that ACTL•ACTL heterodimerization is sufficient for partner selectivity. Our work elucidates a role for the SCRM ACTL domain in the MUTE-governed proliferation-differentiation switch and suggests mechanistic insight into the biological function of the ACTL domain, a module uniquely associated with plant bHLH proteins, as a heterodimeric partner selectivity interface.

R3-MYB transcription factor LcMYBx from Litchi chinensis negatively regulates anthocyanin biosynthesis by ectopic expression in tobacco.

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.

Initial assessment of Id protein antagonist (aHLH) shows anti-proliferative activity on HEK293T, LS174T and HCT116 cells

Id proteins (1–4) which are dominant-negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors function in a variety of cellular processes. They bind to the ubiquitously expressed ‘E box’ protein of the class A bHLH proteins (E12, E47, E2–2, and HEB). Each Id protein has distinct preferences and specificities for their E protein targets. A mutant version of E47 protein, an Id protein antagonist known as aHLH which is defective in DNA binding functions is known to suppress growth in human carcinoma cell lines. This work investigates the effects of the Id protein antagonist aHLH on growth and on pro-apoptotic functions in vitro using immortalized (highly tumorigenic) transformed human embryonic kidney cells (HEK293T), human epithelial colon adenocarcinoma cell line (LS174T) and human epithelial colon carcinoma cell line (HCT116). Plasmid encoding the aHLH proteins was transiently over-expressed in cultured HEK293T, LS174T and HCT116. Cell viability assay with MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was performed. Apoptotic and cell cycle assays were also performed on the transfected cells thereafter. Here, the results demonstrate that Id inhibition by aHLH impairs cell viability, inhibits cell proliferation and induces apoptosis in HEK293T, LS174T and HCT116 cell lines. These findings on the role of aHLH in cell growth and survival could help manipulate their functions as targets for developing anti-cancer agents and could also serve as molecular markers of human cancers.

Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum.

Stem mechanical strength is one of the most important agronomic traits that affects the resistance of plants against insects and lodging, and plays an essential role in the quality and yield of plants. Several transcription factors regulate mechanical strength in crops. However, mechanisms of stem strength formation and regulation remain largely unexplored, especially in ornamental plants. In this study, we identified an atypical bHLH transcription factor CmHLB (HLH PROTEIN INVOLVED IN LIGNIN BIOSYNTHESIS) in chrysanthemum, belonging to a small bHLH sub-family - the PACLOBUTRAZOL RESISTANCE (PRE) family. Overexpression of CmHLB in chrysanthemum significantly increased mechanical strength of the stem, cell wall thickness, and lignin content, compared with the wild type. In contrast, CmHLB RNA interference lines exhibited the opposite phenotypes. RNA-seq analysis indicated that CmHLB promoted the expression of genes involved in lignin biosynthesis. Furthermore, we demonstrated that CmHLB interacted with Chrysanthemum KNOTTED ARABIDOPSIS THALIANA7 (CmKNAT7) through the KNOX2 domain, which has a conserved function, i.e. it negatively regulates secondary cell wall formation of fibres and lignin biosynthesis. Collectively, our results reveal a novel role for CmHLB in regulating lignin biosynthesis by interacting with CmKNAT7 and affecting stem mechanical strength in Chrysanthemum.

R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.).

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

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.

Atoh8 in Development and Disease.

Simple SummarybHLH transcription factors control a variety of functions during development and disease. Atoh8 is one such bHLH factor with unique characteristics in the ‘atonal’ family. Consistent with the other homologs of its family, Atoh8 was identified to regulate cell fate and differentiation during development. Furthermore, it was also found to regulate other mechanisms in adult and mature organisms such as cancer, iron homeostasis and stress response. This review systematically outlines the role of Atoh8 in development and pathologies. At the same time, this review also anticipates its potential involvement in basic cellular mechanisms such as proliferation, metabolism, apoptosis and autophagy.Atoh8 belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) proteins. bHLH proteins have been identified in a wide range of organisms from yeast to humans. The members of this special group of transcription factors were found to be involved not only in embryonic development but also in disease initiation and its progression. Given their importance in several fundamental processes, the translation, subcellular location and turnover of bHLH proteins is tightly regulated. Alterations in the expression of bHLH proteins have been associated with multiple diseases also in context with Atoh8 which seems to unfold its functions as both transcriptional activator and repressor. Like many other bHLH transcription factors, so far, Atoh8 has also been observed to be involved in both embryonic development and carcinogenesis where it mainly acts as tumor suppressor. This review summarizes our current understanding of Atoh8 structure, function and regulation and its complex and partially controversial involvement in development and disease.

Cellular experiments to study the inhibition of c-Myc/MAX heterodimerization.

The c-Myc oncogene is a master regulator of cancer cell metabolism, which controls a variety of pathways, including cell proliferation, cell cycle, apoptosis, and epigenetics. Belonging to the bHLH family of transcription factors, c-Myc forms a heterodimeric complex with another bHLH family protein MAX. c-Myc deregulation is reported in most cancers. This heterodimeric complex is a potent transcription factor that controls the expression of the target gene by binding to the E-box sequence and thereby controlling cancer cell proliferation. c-Myc in isolation has a partially folded structure and cannot carry the transcription. However, its heterodimerization provides the ability to bind DNA and carry out the regulatory function. Therefore, heterodimerization of c-Myc and Max is of great interest for cancers, and it has always been considered a target for cancer therapy. This book chapter will present a detailed protocol of cellular experiments employed to validate the in vitro potency of c-Myc inhibitor candidates to search for a novel c-Myc-targeted neoplastic drug.

Genome-wide identification and characterization of basic helix-loop-helix transcription factors in Spodoptera litura upon pathogen infection.

Basic helix-loop-helix (bHLH) transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes, and bHLH proteins also participate in immune responses, especially in plants. However, their roles in insects upon entomopathogen infection are unknown. In this study, 54 bHLH genes in 41 families were identified in a polyphagous pest, Spodoptera litura, including a new bHLH gene in group B, which is specifically present in Lepidoptera and was thus named Lep. The conserved amino acids in the bHLH domain, structural architecture, and chromosomal distribution of bHLH genes in S. litura were analyzed. The bHLH genes in Plutella xylostella and Apis mellifera were also updated, and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed. The expression profiles of S. litura bHLH (SlbHLH) genes in 3 tissues at different developmental stages and their responses to S. litura nucleopolyhedrovirus (SpltNPV), Nomuraea rileyi (Nr), and Bacillus thuringiensis (Bt) infection were investigated. More SlbHLHs in group B were expressed and differentially expressed during pathogen infections, and SlbHLHs tended to be downregulated in the midgut of S. litura larvae after B. thuringiensis treatment. Our study provides an overview of bHLH family members in S. litura and their responses to different pathogens used for pest biocontrol. These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.