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

ABSTRACT: 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.

SUBMITTER: Duan Z

PROVIDER: S-EPMC8633956 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Radish Basic Helix-Loop-Helix Transcription Factor, RsTT8 Acts a Positive Regulator for Anthocyanin Biosynthesis.

Project description:The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.

| S-EPMC5682339 | biostudies-literature

Genome-wide analysis of basic helix-loop-helix superfamily members related to anthocyanin biosynthesis in eggplant (Solanum melongena L.).

Project description:The basic helix-loop-helix (bHLH) superfamily is considered the second largest transcription factor (TF) family. It plays regulatory roles in the developmental processes of plants and in their defense responses. In recent years, many bHLH superfamily genes have been identified and characterized in herbaceous and woody plants. However, the comprehensive genomic and functional analyses of these genes in eggplant (Solanum melongena L.) have not been reported. In this study, 121 bHLH TFs were identified in the recently released eggplant genome. The phylogeny, gene structure and conserved motifs of the SmbHLH gene were comprehensively studied. Subsequently, the phylogenetic relationship between the bHLH of eggplant and the bHLH of other species was analyzed, and the proteins were classified into 17 subfamilies. Among these protein sequences, 16 subgroups were clustered into the functional clades of Arabidopsis. Two candidate genes (SmbHLH1, SmbHLH117) that may be involved in anthocyanin biosynthesis were screened. The tissue specificity or differential expression of the bHLH genes in different tissues and under various light and temperature conditions suggested the differential regulation of tissue development and metabolism. This study not only provides a solid foundation for the functional dissection of the eggplant bHLH gene family but may also be useful for the future synthesis of anthocyanins in eggplant.

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Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop.

Project description:Cell elongation is promoted by different environmental and hormonal signals, involving light, temperature, brassinosteroid (BR), and gibberellin, that inhibit the atypical basic helix-loop-helix (bHLH) transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1). Ectopic accumulation of IBH1 causes a severe dwarf phenotype, but the cell elongation suppression mechanism is still not well understood. Here, we identified a close homolog of IBH1, IBH1-LIKE1 (IBL1), that also antagonized BR responses and cell elongation. Genome-wide expression analyses showed that IBH1 and IBL1 act interdependently downstream of the BRASSINAZOLE-RESISTANT1 (BZR1)-PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-DELLA module. Although characterized as non-DNA binding, IBH1 repressed direct IBL1 transcription, and they both acted in tandem to suppress the expression of a common downstream helix-loop-helix (HLH)/bHLH network, thus forming an incoherent feed-forward loop. IBH1 and IBL1 together repressed the expression of PIF4, known to stimulate skotomorphogenesis synergistically with BZR1. Strikingly, PIF4 bound all direct and down-regulated HLH/bHLH targets of IBH1 and IBL1. Additional genome-wide comparisons suggested a model in which IBH1 antagonized PIF4 but not the PIF4-BZR1 dimer.

| S-EPMC3932932 | biostudies-literature

Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant.

Project description:As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics for the diploid woodland strawberry Fragaria vesca. In addition, transcription profiles of 113 orthologous bHLH genes from various tissues were analyzed for the cultivar 'Benihoppe', its white-flesh mutant 'Xiaobai', and the 'Snow Princess' from their fruit development to the ripening, as well as those under either the ABA or Eth treatment. Both the RT-PCR and qRT-PCR results show that seven selected FabHLH genes (FabHLH17, FabHLH25, FabHLH27, FabHLH29, FabHLH40, FabHLH80, FabHLH98) are responsive to the fruit anthocyanin biosynthesis and hormone signaling according to transcript profiles where three color modes are observed for strawberry's fruit skin and flesh. Further, prediction for the protein interaction network reveals that four bHLHs (FabHLH25, FabHLH29, FabHLH80, FabHLH98) are involved in the fruit anthocyanin biosynthesis and hormone signaling transduction. These bioinformatics and expression profiles provide a good basis for a further investigation of strawberry bHLH genes.

| S-EPMC5807450 | biostudies-literature

Alternative Splicing of the Basic Helix-Loop-Helix Transcription Factor Gene CmbHLH2 Affects Anthocyanin Biosynthesis in Ray Florets of Chrysanthemum (Chrysanthemum morifolium).

Project description:Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of CmbHLH2 were detected between the florets of different colors. CmbHLH2 generated two alternatively spliced transcripts, designated CmbHLH2Full and CmbHLH2Short . Compared with CmbHLH2Full , CmbHLH2Short encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2Short localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2Short failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2Full promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2Full and CmMYB6 also enhanced the promoter activities of CmCHS and CmDFR. Notably, the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2Full, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2Short did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.

| S-EPMC8222801 | biostudies-literature

Basic helix-loop-helix transcription factor gene family phylogenetics and nomenclature.

Project description:A phylogenetic analysis of the basic helix-loop-helix (bHLH) gene superfamily was performed using seven different species (human, mouse, rat, worm, fly, yeast, and plant Arabidopsis) and involving over 600 bHLH genes (Stevens et al., 2008). All bHLH genes were identified in the genomes of the various species, including expressed sequence tags, and the entire coding sequence was used in the analysis. Nearly 15% of the gene family has been updated or added since the original publication. A super-tree involving six clades and all structural relationships was established and is now presented for four of the species. The wealth of functional data available for members of the bHLH gene superfamily provides us with the opportunity to use this exhaustive phylogenetic tree to predict potential functions of uncharacterized members of the family. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique elements of the evolution and functional relationships of the different genes in the bHLH gene family.

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

Project description: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.

| S-EPMC8408231 | biostudies-literature

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2

Project description:Math2 (NEX-1/NeuroD6) is a member of the bHLH transcription factor family and is involved in neuronal differentiation and maturation. In the present study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. Examining the promoter region of rat Prg1, we found four E-boxes designated -E1 to -E4 (CANNTG) which were recognized by the bHLH transcription factor. Using chromatin immunoprecipitation (ChIP) assays, we found that Math2 directly bound to the E-box(es) in the Prg1 promoter. The reporter assay of Prg1 showed that -E1 was critical for the regulation of the Prg1 expression by Math2. Then, the functional role of Math2 and Prg1 was investigated in PC12 cells. Seventy-two hours after transfection of Math2 or Prg1, neurite length and number was significantly induced in PC12 cells. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

2008-07-27 | GSE11829 | GEO

TcMYC2a, a Basic Helix-Loop-Helix Transcription Factor, Transduces JA-Signals and Regulates Taxol Biosynthesis in Taxus chinensis.

Project description:The multitherapeutic taxol, which can be obtained from Taxus spp., is the most widely used anticancer drug. Taxol biosynthesis is significantly regulated by jasmonate acid (JA), one of the most important endogenous hormones in land plants. Nevertheless, the JA-inducing mechanism remains poorly understood. MYC2 is one of the key regulators of JA signal transfer and the biosynthesis of various secondary metabolites. Here, TcMYC2a was identified to contain a basic helix-loop-helix (bHLH)-leucine zipper domain, a bHLH-MYC_N domain, and a BIF/ACT-like domain. TcMYC2a was also found to bind with TcJAZ3 in yeast, which was a homolog of Arabidopsis JASMONATE ZIM-domain JAZ proteins, indicating that TcMYC2a had a similar function to AtMYC2 of JA signal transduction. TcMYC2a was able to affect the expression of GUS reporter gene by binding with the T/G-box, G-box, and E-box, which were the key cis-elements of TASY and TcERF12/15 promoter. TcMYC2a overexpression also led to significantly increased expression of TASY, tat, dbtnbt, t13h, and t5h genes. Additionally, TcERF15, which played the positive role to regulate tasy gene, was up-regulated by TcMYC2a. All these results revealed that TcMYC2a can regulate taxol biosynthesis either directly or via ERF regulators depending on JA signaling transduction.

| S-EPMC6021540 | biostudies-literature

Overexpression of bHLH95, a basic helix-loop-helix transcription factor family member, impacts trichome formation via regulating gibberellin biosynthesis in tomato.

Project description:Trichomes are epidermal protuberances on aerial parts of plants known to play an important role in biotic and abiotic stresses. To date, our knowledge of the regulation of trichome formation in crop species is very limited. Through phenotyping of the Solanum pennellii×S. lycopersicum (cv. M82) introgression population, we identified the SlbHLH95 transcription factor as a negative regulator of trichome formation in tomato. In line with this negative role, SlbHLH95 displayed a very low expression in stems where trichomes are present at high density. Overexpression of SlbHLH95 resulted in a dramatically reduced trichome density in stems and a significant down-regulation of a set of trichome-related genes. In addition to the lower trichome density, overexpressing lines also showed pleiotropic alterations affecting both vegetative and reproductive development. While most of these phenotypes were reminiscent of gibberellin (GA)-deficient phenotypes, expression studies showed that two GA biosynthesis genes, SlGA20ox2 and SlKS5, are significantly down-regulated in SlbHLH95-OE plants. Moreover, in line with a decrease in active GA content, the glabrous and dwarf phenotypes were rescued by exogenous GA treatment. In addition, yeast one-hybrid and transactivation assays revealed that SlbHLH95 represses the expression of SlGA20ox2 and SlKS5 via direct binding to their promoters. Taken together, our study established a link between SlbHLH95, GA, and trichome formation, and uncovered the role of this gene in modulating GA biosynthesis in tomato.

| S-EPMC7475245 | biostudies-literature

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