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SUMMARY Basic helix–loop–helix (bHLH) proteins comprise one of the largest families of transcription factors in plants, which play roles in plant development, secondary metabolism, and the response to biotic/abiotic stresses. However, the roles of bHLH proteins in thermotolerance are largely unknown. Herein, we identified a heat‐inducible member of the bHLH family in lily (Lilium longiflorum), named LlbHLH87, which plays a role in thermotolerance. LlbHLH87 was rapidly induced by transient heat stress, and its encoded protein was localized to the nucleus, exhibiting transactivation activity in both yeast and plant cells. Overexpression of LlbHLH87 in Arabidopsis enhanced basal thermotolerance, while silencing of LlbHLH87 in lily reduced basal thermotolerance. Further analysis showed that LlbHLH87 bound to the promoters of HEAT STRESS TRANSCRIPTION FACTOR A2 (LlHSFA2) and ETHYLENE‐INSENSITIVE 3 (LlEIN3) to directly activate their expression. In addition, LlbHLH87 interacted with itself and with SPATULA (LlSPT) protein. LlSPT was activated by extended heat stress and its protein competed for the homologous interaction of LlbHLH87, which reduced the transactivation ability of LlbHLH87 for target genes. Compared with that observed under LlbHLH87 overexpression alone, co‐overexpression of LlbHLH87 and LlSPT reduced the basal thermotolerance of lily to sudden heat shock, but improved its thermosensitivity to prolonged heat stress treatment. Overall, our data demonstrated that LlbHLH87 regulates thermotolerance via activation of LlEIN3 and LlHSFA2, along with an antagonistic interaction with LlSPT. [ABSTRACT FROM AUTHOR]

The basic helix–loop–helix (bHLH) gene family is a crucial regulator in plants, orchestrating various developmental processes, particularly flower formation, and mediating responses to hormonal signals. The molecular mechanism of bamboo flowering regulation remains unresolved, limiting bamboo breeding efforts. In this study, we identified 309 bHLH genes and divided them into 23 subfamilies. Structural analysis revealed that proteins in specific DlbHLH subfamilies are highly conserved. Collinearity analysis indicates that the amplification of the DlbHLH gene family primarily occurs through segmental duplications. The structural diversity of these duplicated genes may account for their functional variability. Many DlbHLHs are expressed during flower development, indicating the bHLH gene's significant role in this process. In the promoter region of DlbHLHs, different homeopathic elements involved in light response and hormone response co-exist, indicating that DlbHLHs are related to the regulation of the flower development of D. latiflorus. [ABSTRACT FROM AUTHOR]

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are key regulators of cell fate specification and differentiation during development. Their dysregulation is implicated not only in developmental abnormalities but also in various adult diseases and cancers. Recently, the abilities of bHLH factors have been exploited in reprogramming strategies for cell replacement therapy. One such factor is NEUROD1, which has been associated with the reprogramming of the epigenetic landscape and potentially possessing pioneer factor abilities, initiating neuronal developmental programs, and enforcing pancreatic endocrine differentiation. The review aims to consolidate current knowledge on NEUROD1's multifaceted roles and mechanistic pathways in human and mouse cell differentiation and reprogramming, exploring NEUROD1 roles in guiding the development and reprogramming of neuroendocrine cell lineages. The review focuses on NEUROD1's molecular mechanisms, its interactions with other transcription factors, its role as a pioneer factor in chromatin remodeling, and its potential in cell reprogramming. We also show a differential potential of NEUROD1 in differentiation of neurons and pancreatic endocrine cells, highlighting its therapeutic potential and the necessity for further research to fully understand and utilize its capabilities. [ABSTRACT FROM AUTHOR]

[Objective] Using Paeonia qiui as the experimental material, the regulatory mechanism of PqbHLH1 gene on anthocyanin synthesis during leaf development was studied. [Method] Based on the transcriptome data obtained in the early stage, primers were designed and the bHLH1 gene was cloned from the leaves of Paeonia qiui using PCR technology. Bioinformatics methods were used to analyze the physicochemical properties, hydrophobicity, theoretical isoelectric points, protein secondary structure, and evolutionary relationships of the PqbHLH1 gene. Fluorescence quantitative PCR technology was used to detect its expressions in different tissues and stages. Agrobacterium mediated method was employed to overexpress it in Arabidopsis thaliana and detect the effect of overexpressing PqbHLH1 on the anthocyanins of peony leaves. [Result] The total length of the coding region of PqbHLH1 gene was 1 920 bp, encoding 639 amino acids. The PqbHLH1 protein was hydrophilic and does not contain signal peptides or transmembrane structures, and was localized in the nucleus. Phylogenetic analysis showed that the PqbHLH1 protein in peony ovale had the closest genetic relationship with VvMYCA1 in grapes. Fluorescence quantitative PCR analysis showed that the expression of PqbHLH1 gene in peony leaves had a trend of first decreasing and then increasing, and the expression was significant in the S6 stage of leaves. The expressions of anthocyanin synthesis structural genes PqCHS, PqF3'H, PqDFR, and PqANS showed a trend of first increasing and then decreasing with leaf development. In A. thaliana strains overexpressing the PqbHLH1 gene, the expressions of structural genes related to anthocyanin synthesis was significantly higher than that of wild-type Arabidopsis, and the expression trend of anthocyanin content was consistent with that of structural genes AtCHI, AtF3'H, and AtUFGT, while the expression trend of flavonol content was consistent with that of structural gene AtFLS. [Conclusion] The PqbHLH1 gene positively regulates the synthesis of anthocyanins and flavonols in peony leaves by regulating the expressions of genes related to anthocyanin synthesis. [ABSTRACT FROM AUTHOR]

Sesame is a labor intensive crop with limited mechanized harvesting mainly due to the seed shattering (SS) trait. In this study, we performed a genetic analysis of the seed-shattering resistance (SR) trait with a SR sesame mutant 12M07. Unlike the SS type, the parenchyma cells in the abscission zone of the 12M07 mutant are arranged loosely but adhere to the seed coat. Inheritance analysis of six generations derived from 12M07 (SR)×Xiangcheng Dazibai (SS) showed that the SR trait is recessive and controlled by a single gene pair. Association mapping of the F2 population with 888,619 variants (single-nucleotide polymorphisms (SNPs) and insertion-deletion (InDels)) and 31,884 structural variations (SVs) determined that only SV12002 in the 5´ upstream region of gene Sindi0765000 (named SiHEC3) in Chr.3 was significantly associated with the SR trait. SiHEC3 encodes the bHLH transcription factor. A 1,049 bp deletion occurred in the 5´ UTR of Sihec3 in 12M07. SiHEC3 is mainly expressed in developing placental tissues, with the expression peaking in capsules at 45 days after pollination. A dual-luciferase reporter assay in tobacco confirmed that the promoter activity of Sihec3 was reduced because of the deletion of the 1,049 bp promoter sequence. Protein–protein interaction network analysis showed that HEC3 is co-expressed with nine key proteins, such as SHATTERPROOF1 (SHP1) and SEEDSTICK (STK) which participate in the secondary wall biosynthesis of the abscission layer in plants. The findings of this study show the important function of Sihec3 corresponding with the SR trait and supply the genetic information for breeding new varieties that are amenable to mechanized harvesting in sesame and other crops.

A basic helix-loop-helix transcription factor, figα, is one of the earliest marker genes of oocyte differentiation in vertebrates. In the present study, we made figα knockout medaka by CRISPR/Cas9, expecting aborted progress of oogenesis, to see if differentiation of somatic ovarian tissues is affected. Figα knockout male gonads differentiated normally into testes with functional sperm. The females, on the other hand, were sterile; there are oocytes only up to pachytene. No growing oocytes in diplotene were found. The phenotype was already apparent at 10 days after hatching, when diplotene oocytes start to develop in the control. Furthermore, several putative target genes of figα were not expressed in the mutant female gonads. Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred. Figα knockout female gonads differentiated morphologically into ovaries, suggesting that in medaka, figα knockout prevents oogenesis progress into diplotene stage, and ovarian differentiation do not need developed oocytes beyond pachytene stage. This ovarian phenotype reminded us of ''pachytene checkpoint'', which blocks meiotic progress into diplotene when chromosomal abnormalities are present. Therefore, we further analyzed phenotype of oocytes in the mutant ovaries. The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage. The number of apoptotic oocytes in the knockout ovaries was small. Finally, we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining. Taken together, figα knockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than ''pachytene checkpoint'' and arrested oocytes remained in the ovary. This is in contrast to figα knockout mice where all oocytes underwent apoptosis quickly after birth.

The bHLH transcription factor family plays crucial roles in plant growth and development and their responses to adversity. In this study, a highly salt-induced bHLH gene, PagbHLH35 (Potri.018G141600), was identified from Populus alba × P. glandullosa (84K poplar). PagbHLH35 contains a highly conserved bHLH domain within the region of 52–114 amino acids. A subcellular localization result confirmed its nuclear localization. A yeast two-hybrid assay indicated PagbHLH35 lacks transcriptional activation activity, while a yeast one-hybrid assay indicated it could specifically bind to G-box and E-box elements. The expression of PagbHLH35 reached its peak at 12 h and 36 h time points under salt stress in the leaves and roots, respectively. A total of three positive transgenic poplar lines overexpressing PagbHLH35 were generated via Agrobacterium-mediated leaf disk transformation. Under NaCl stress, the transgenic poplars exhibited significantly enhanced morphological and physiological advantages such as higher POD activity, SOD activity, chlorophyll content, and proline content, and lower dehydration rate, MDA content and hydrogen peroxide (H2O2) content, compared to wild-type (WT) plants. In addition, histological staining showed that there was lower ROS accumulation in the transgenic poplars under salt stress. Moreover, the relative expression levels of several antioxidant genes in the transgenic poplars were significantly higher than those in the WT. All the results indicate that PagbHLH35 can improve salt tolerance by enhancing ROS scavenging in transgenic poplars. [ABSTRACT FROM AUTHOR]

Abstract Background bHLH transcription factors play significant roles in regulating plant growth and development, stress response, and anthocyanin biosynthesis. Sweetpotato is a pivotal food and industry crop, but little information is available on sweetpotato bHLH genes. Results Herein, 227 putative IbbHLH genes were defined on sweetpotato chromosomes, and fragment duplications were identified as the dominant driving force for IbbHLH expansion. These IbbHLHs were divided into 26 subfamilies through phylogenetic analysis, as supported by further analysis of exon-intron structure and conserved motif composition. The syntenic analysis between IbbHLHs and their orthologs from other plants depicted evolutionary relationships of IbbHLHs. Based on the transcriptome data under salt stress, the expression of 12 IbbHLHs was screened for validation by qRT-PCR, and differential and significant transcriptions under abiotic stress were detected. Moreover, IbbHLH123 and IbbHLH215, which were remarkably upregulated by stress treatments, had obvious transactivation activity in yeasts. Protein interaction detections and yeast two-hybrid assays suggested an intricate interaction correlation between IbbHLHs. Besides, transcriptome screening revealed that multiple IbbHLHs may be closely related to anthocyanin biosynthesis based on the phenotype (purple vs. white tissues), which was confirmed by subsequent qRT-PCR analysis. Conclusions These results shed light on the promising functions of sweetpotato IbbHLHs in abiotic stress response and anthocyanin biosynthesis.

In many plant species, petal abscission can be considered the final step of petal senescence. Cytokinins (CKs) are powerful suppressors of petal senescence; however, their role in petal abscission is ambiguous. Here, we observed that, in rose (Rosa hybrida), biologically active CK is accumulated during petal abscission and acts as an accelerator of the abscission process. Using a combination of reverse genetics, and molecular and biochemical techniques, we explored the roles of a LESION SIMULATING DISEASE1 (LSD1) family member RhLOL1 interacting with a bHLH transcription factor RhILR3 in CK-induced petal abscission. Silencing RhLOL1 delays rose petal abscission, while the overexpression of its ortholog SlLOL1 in tomato (Solanum lycopersicum) promotes pedicel abscission, indicating the conserved function of LOL1 in activating plant floral organ abscission. In addition, we identify a bHLH transcription factor, RhILR3, that interacts with RhLOL1. We show that RhILR3 binds to the promoters of the auxin signaling repressor auxin/indole-3-acetic acid (Aux/IAA) genes to inhibit their expression; however, the interaction of RhLOL1 with RhILR3 activates the expression of the Aux/IAA genes including RhIAA4-1. Silencing RhIAA4-1 delays rose petal abscission. Our results thus reveal a RhLOL1-RhILR3 regulatory module involved in CK-induced petal abscission via the regulation of the expression of the Aux/IAA genes.

Background: bHLH transcription factors play significant roles in regulating plant growth and development, stress response, and anthocyanin biosynthesis. Sweetpotato is a pivotal food and industry crop, but little information is available on sweetpotato bHLH genes. Results: Herein, 227 putative IbbHLH genes were defined on sweetpotato chromosomes, and fragment duplications were identified as the dominant driving force for IbbHLH expansion. These IbbHLHs were divided into 26 subfamilies through phylogenetic analysis, as supported by further analysis of exon-intron structure and conserved motif composition. The syntenic analysis between IbbHLHs and their orthologs from other plants depicted evolutionary relationships of IbbHLHs. Based on the transcriptome data under salt stress, the expression of 12 IbbHLHs was screened for validation by qRT-PCR, and differential and significant transcriptions under abiotic stress were detected. Moreover, IbbHLH123 and IbbHLH215, which were remarkably upregulated by stress treatments, had obvious transactivation activity in yeasts. Protein interaction detections and yeast two-hybrid assays suggested an intricate interaction correlation between IbbHLHs. Besides, transcriptome screening revealed that multiple IbbHLHs may be closely related to anthocyanin biosynthesis based on the phenotype (purple vs. white tissues), which was confirmed by subsequent qRT-PCR analysis. Conclusions: These results shed light on the promising functions of sweetpotato IbbHLHs in abiotic stress response and anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

The role of bHLH transcription factors in plant response to abiotic stress and regulation of flavonoid metabolism is well documented. However, to date, the bHLH transcription factor family in Iris domestica remains unreported, impeding further research on flavonoid metabolism in this plant. To address this knowledge gap, we employed bioinformatics to identify 39 IdbHLH genes and characterised their phylogenetic relationships and gene expression patterns under both drought and copper stress conditions. Our evolutionary tree analysis classified the 39 IdbHLHs into 17 subfamilies. Expression pattern analysis revealed that different IdbHLH transcription factors had distinct expression trends in various organs, suggesting that they might be involved in diverse biological processes. We found that IdbHLH36 was highly expressed in all organs (Transcripts Per Million (TPM) > 10), while only 12 IdbHLH genes in the rhizome and four in the root were significantly upregulated under drought stress. Of these, four genes (IdbHLH05, -37, -38, -39) were co-upregulated in both the rhizome and root, indicating their potential role in drought resistance. With regards to copper stress, we found that only 12 genes were upregulated. Further co-expression analysis revealed that most bHLH genes were significantly correlated with key enzyme genes involved in isoflavone biosynthesis. Thereinto, IdbHLH06 showed a significant positive correlation with IdC4H1 and Id4CL1 (p < 0.05). Furthermore, a transient expression assay confirmed that the IdbHLH06 protein was localised in the nucleus. Our findings provide new insights into the molecular basis and regulatory mechanisms of bHLH transcription factors in isoflavone biosynthesis in I. domestica. [ABSTRACT FROM AUTHOR]

Anthocyanins are secondary metabolites induced by environmental stimuli and developmental signals. The positive regulators of anthocyanin biosynthesis have been reported, whereas the anthocyanin repressors have been neglected. Although the signal transduction pathways of gibberellin (GA) and jasmonic acid (JA) and their regulation of anthocyanin biosynthesis have been investigated, the cross‐talk between GA and JA and the antagonistic mechanism of regulating anthocyanin biosynthesis remain to be investigated. In this study, we identified the anthocyanin repressor MdbHLH162 in apple and revealed its molecular mechanism of regulating anthocyanin biosynthesis by integrating the GA and JA signals. MdbHLH162 exerted passive repression by interacting with MdbHLH3 and MdbHLH33, which are two recognized positive regulators of anthocyanin biosynthesis. MdbHLH162 negatively regulated anthocyanin biosynthesis by disrupting the formation of the anthocyanin‐activated MdMYB1‐MdbHLH3/33 complexes and weakening transcriptional activation of the anthocyanin biosynthetic genes MdDFR and MdUF3GT by MdbHLH3 and MdbHLH33. The GA repressor MdRGL2a antagonized MdbHLH162‐mediated inhibition of anthocyanins by sequestering MdbHLH162 from the MdbHLH162‐MdbHLH3/33 complex. The JA repressors MdJAZ1 and MdJAZ2 interfered with the antagonistic regulation of MdbHLH162 by MdRGL2a by titrating the formation of the MdRGL2a‐MdbHLH162 complex. Our findings reveal that MdbHLH162 integrates the GA and JA signals to negatively regulate anthocyanin biosynthesis. This study provides new information for discovering more anthocyanin biosynthesis repressors and explores the cross‐talk between hormone signals. [ABSTRACT FROM AUTHOR]

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are key regulators of cell fate specification and differentiation during development. Their dysregulation is implicated not only in developmental abnormalities but also in various adult diseases and cancers. Recently, the abilities of bHLH factors have been exploited in reprogramming strategies for cell replacement therapy. One such factor is NEUROD1, which has been associated with the reprogramming of the epigenetic landscape and potentially possessing pioneer factor abilities, initiating neuronal developmental programs, and enforcing pancreatic endocrine differentiation. The review aims to consolidate current knowledge on NEUROD1’s multifaceted roles and mechanistic pathways in human and mouse cell differentiation and reprogramming, exploring NEUROD1 roles in guiding the development and reprogramming of neuroendocrine cell lineages. The review focuses on NEUROD1’s molecular mechanisms, its interactions with other transcription factors, its role as a pioneer factor in chromatin remodeling, and its potential in cell reprogramming. We also show a differential potential of NEUROD1 in differentiation of neurons and pancreatic endocrine cells, highlighting its therapeutic potential and the necessity for further research to fully understand and utilize its capabilities.

Fruit development has been central in the evolution and domestication of flowering plants. In common bean (Phaseolus vulgaris), the principal global grain legume staple, two main production categories are distinguished by fibre deposition in pods: dry beans, with fibrous, stringy pods; and stringless snap/green beans, with reduced fibre deposition, which frequently revert to the ancestral stringy state. Here, we identify genetic and developmental patterns associated with pod fibre deposition. Transcriptional, anatomical, epigenetic and genetic regulation of pod strings were explored through RNA-seq, RT-qPCR, fluorescence microscopy, bisulfite sequencing and whole-genome sequencing. Overexpression of the INDEHISCENT ('PvIND') orthologue was observed in stringless types compared with isogenic stringy lines, associated with overspecification of weak dehiscence-zone cells throughout the pod vascular sheath. No differences in DNA methylation were correlated with this phenotype. Nonstringy varieties showed a tandemly direct duplicated PvIND and a Ty1-copia retrotransposon inserted between the two repeats. These sequence features are lost during pod reversion and are predictive of pod phenotype in diverse materials, supporting their role in PvIND overexpression and reversible string phenotype. Our results give insight into reversible gain-of-function mutations and possible genetic solutions to the reversion problem, of considerable economic value for green bean production.

Abstract Background The basic helix-loop-helix (bHLH) gene family is one of plants’ largest transcription factor families. It plays an important role in regulating plant growth and abiotic stress response. Results In this study, we determined that the PavbHLH28 gene participated in cold resistance. The PavbHLH28 gene was located in the nucleus and could be induced by low temperature. Under the treatment of ABA, PEG, and GA3, the transcript level of PavbHLH28 was affected. At low temperature, overexpression of the PavbHLH28 gene enhanced the cold resistance of plants with higher proline content, lower electrolyte leakage (EL) and malondialdehyde (MDA) content. Compared with the WT plants, the transgenic plants accumulated fewer reactive oxygen species (ROS), and the activity and expression levels of antioxidant enzymes were significantly increased. The expression of proline synthesis enzyme genes was up-regulated, and the transcripts levels of degradation genes were significantly down-regulated. The transcripts abundance of the cold stressed-related genes in the C-repeat binding factor (CBF) pathway was not significantly different between WT plants and transgenic plants after cold stress. Moreover, the PavbHLH28 could directly bind to the POD2 gene promoter and promote its gene expression. Conclusions Overall, PavbHLH28 enhanced the cold resistance of transgenic plants through a CBF-independent pathway, which may be partly related to ROS scavenging.

【Objective】The bHLH transcription factor family is an important family of transcription factors in eukaryotes, which plays an important role in plant growth and development, secondary metabolism and stress response. Analysis of the response pattern of bHLH transcription factor gene OsbHLH109 (LOC_Os01g67480) to stress and hormones in rice (Oryza sativa L.) could provide a theoretical basis for further researches on the functions of OsbHLH109 in stress and hormone responses.【Method】The bioinformatics of OsbHLH109 was analyzed, and the expression patterns of OsbHLH109 in different tissues of Rice Zhonghua 11 under different hormone and abiotic stresses were analyzed by quantitative PCR (qRT-PCR).【Result】The encoding region of OsbHLH109 was 1 164 bp in length, containing 6 exons and encoding 388 amino acids, which was a bHLH transcription factor containing a conserved domain of HLH. Phylogenetic analysis showed that OsbHLH109 was closely related to the homologous proteins in monocots and Dicotyledones. The analysis of cis-acting elements showed that the promoter region of OsbHLH109 contained 24 plant hormone response cis-elements and 25 environmental stress response cis-elements. The qRT-PCR analysis showed that OsbHLH109 was expressed in roots, stems, leaves and young panicles of rice, but the expression level was the highest in leaves. The results of hormone treatment response expression profile analysis showed that the response expression differences of OsbHLH109 in leaves to 6-Benzylaminopurine (6-BA), indole acetic acid (IAA), gibberellic acid (GA3) and abscisic acid (ABA) treatments all reached significant levels. The results of stress treatment response expression profile analysis showed that the response of OsbHLH109 to low temperature stress, drought stress, high temperature stress and salt stress were significantly different in leaves, indicating that the expression of OsbHLH109 was induced by 6-BA, GA3, IAA, ABA, high temperature, low temperature, PEG6000 and NaCl, which suggested that OsbHLH109 was essential for the abiotic stress responses in rice.【Conclusion】Rice OsbHLH109 is a member of the bHLH transcription factor family and is highly expressed in leaves. The expression of OsbHLH109 gene is regulated by high temperature, low temperature and salt stress, and responds to hormone signals such as 6-BA, IAA, GA3 and ABA. It is predicted that it may be involved in abiotic stress response of rice through ABA and other hormone signal transduction pathways.

The basic helix–loop–helix (bHLH) gene family is a crucial regulator in plants, orchestrating various developmental processes, particularly flower formation, and mediating responses to hormonal signals. The molecular mechanism of bamboo flowering regulation remains unresolved, limiting bamboo breeding efforts. In this study, we identified 309 bHLH genes and divided them into 23 subfamilies. Structural analysis revealed that proteins in specific DlbHLH subfamilies are highly conserved. Collinearity analysis indicates that the amplification of the DlbHLH gene family primarily occurs through segmental duplications. The structural diversity of these duplicated genes may account for their functional variability. Many DlbHLHs are expressed during flower development, indicating the bHLH gene’s significant role in this process. In the promoter region of DlbHLHs, different homeopathic elements involved in light response and hormone response co-exist, indicating that DlbHLHs are related to the regulation of the flower development of D. latiflorus.

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【Objective】Peppermint essential oil is an important raw material in the cosmetics, pharmaceutical, and food industries. bHLH transcription factors（TFs）play a crucial role in regulating the biosynthesis of volatile secondary metabolites（VSMs）in plants. Exploring the role of bHLH TFs in the regulation of VSM biosynthesis in peppermint may provide important gene resources for improving peppermint germplasm through metabolic or genetic engineering, and expand our understanding of the pathways and regulatory mechanisms of plant VSM biosynthesis.【Method】The expression pattern of bHLH96 in peppermint roots, stems, and leaves was analyzed using transcriptome sequencing and qRT-PCR techniques. The full-length of bHLH96 coding sequence（CDS）was cloned using the RT-PCR method. Subsequently, a plant expression vector, pBI121-bHLH96, was constructed using DNA recombination technology, and the overexpression of bHLH96 in the peppermint leaves was achieved through Agrobacterium-mediated transformation. The effects of bHLH96 overexpression on the content of terpenoid compounds and the expression of related genes involved in terpenoid biosynthesis in the peppermint leaves were examined.【Result】The results showed that the full length of bHLH96 CDS was 861 bp encoding 286 amino acids. Peppermint bHLH96 protein localized at the nucleus, and the sequence similarity among plant bHLH96 proteins ranged from 45.45% to 73.68%. Peppermint bHLH96 had a close relationship with plant bHLH proteins from the Labiatae family such as Lavandula angustifolia LaMYC4 and Salvia miltiorrhiza SmbHLH94. Transient overexpression of the bHLH96 gene in peppermint leaves significantly influenced relative contents of 15 terpenoid compounds. bHLH96 overexpression significantly upregulated the expressions of 5 terpene synthesis-related genes, such as OC2, SM1, KS1, ND, and EAO1, while downregulated the expressions of NLS1, LS1 and iPR. 【Conclusion】Peppermint bHLH96 might regulate terpene synthesis by modulating the expressions of terpene synthesis-related genes and influencing the activities of terpene synthases. [ABSTRACT FROM AUTHOR]

The basic/helix-loop-helix (bHLH) transcription factors play important regulatory roles in plant growth and development, secondary metabolism, signal transduction, and stress responses. The cDNA of cotton GhbHLH71 gene is 996 bp in length, encoding 331 amino acid residues. Protein sequence of GhbHLH71 contains a conserved bHLH structural domain, which is a member of the bHLH transcription factor family. The qRT-PCR showed that GhbHLH71 gene was relatively highly expressed at 3--12 DPA (days post anthesis, DPA) at rapid elongation stage in cotton fiber, implying that it mainly played a role in cotton fiber development. A CRISPR/Cas9 gene editing vector of GhbHLH71 gene was constructed, and then was transferred into upland cotton (Gossypium hirsutum L.). Six T0 mutant plants were obtained after PCR detection of Cas9 gene and mutation detection of target loci. The phenotypic traits of different T1 GhbHLH71 gene mutant plants were not significantly different from the control during cotton growth and development, but the fiber length of T1 mutants was significantly shorter compared with the control, and T2 generation mutant lines stably inherited the shortened fiber phenotype of T1 generation lines. The shortened fiber lengths of the 4# and 8# mutant lines in two consecutive generations were more than 20% shorter compared with the control, indicating that the mutation of GhbHLH71 gene mainly affected the elongation of cotton fiber cells. This study provides some insight into the biological functions of bHLH transcription factors in cotton and the molecular mechanism of cotton fiber development. [ABSTRACT FROM AUTHOR]

Background: The basic helix-loop-helix (bHLH) gene family is one of plants' largest transcription factor families. It plays an important role in regulating plant growth and abiotic stress response. Results: In this study, we determined that the PavbHLH28 gene participated in cold resistance. The PavbHLH28 gene was located in the nucleus and could be induced by low temperature. Under the treatment of ABA, PEG, and GA3, the transcript level of PavbHLH28 was affected. At low temperature, overexpression of the PavbHLH28 gene enhanced the cold resistance of plants with higher proline content, lower electrolyte leakage (EL) and malondialdehyde (MDA) content. Compared with the WT plants, the transgenic plants accumulated fewer reactive oxygen species (ROS), and the activity and expression levels of antioxidant enzymes were significantly increased. The expression of proline synthesis enzyme genes was up-regulated, and the transcripts levels of degradation genes were significantly down-regulated. The transcripts abundance of the cold stressed-related genes in the C-repeat binding factor (CBF) pathway was not significantly different between WT plants and transgenic plants after cold stress. Moreover, the PavbHLH28 could directly bind to the POD2 gene promoter and promote its gene expression. Conclusions: Overall, PavbHLH28 enhanced the cold resistance of transgenic plants through a CBF-independent pathway, which may be partly related to ROS scavenging. [ABSTRACT FROM AUTHOR]

While bHLH transcription factors have been linked to the regulation of various abiotic stressors, research on drought-related bHLH proteins and their molecular processes in Agropyron mongolicum has remained limited. In this study, a bHLH gene from A. mongolicum, designated as AmbHLH148, was successfully cloned and isolated. AmbHLH148 was exclusively localized within the nucleus. Additionally, qRT-PCR analysis demonstrated a significant upregulation of AmbHLH148 in response to drought stress. When transferred into tobacco (Nicotiana benthamiana), the heterologous expression of AmbHLH148 led to enhanced drought tolerance. Under drought stress conditions, AmbHLH148-OE transgenic tobacco plants exhibited increased activities of antioxidant defense enzymes, such as SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase). These enzymes efficiently mitigated the accumulation of reactive oxygen species (ROS) compared to wild-type plants. Furthermore, AmbHLH148-OE transgenic tobacco showed elevated levels of PRO (proline) and reduced MDA (malondialdehyde) content, contributing to enhanced stability in the plant's cell membrane system during drought stress. In summary, this study underscores that the overexpression of AmbHLH148 in transgenic tobacco acts as a positive regulator under drought stress by enhancing the plant's antioxidant capacity. These findings shed light on the molecular mechanisms involved in bHLH transcription factors' role in drought resistance, contributing to the discovery and utilization of drought-resistant genes in A. mongolicum for enhancing crop drought resistance. [ABSTRACT FROM AUTHOR]

【Objective】The bHLH transcription factor family is an important family of transcription factors in eukaryotes, which plays an important role in plant growth and development, secondary metabolism and stress response. Analysis of the response pattern of bHLH transcription factor gene OsbHLH109 (LOC_Os01g67480) to stress and hormones in rice (Oryza sativa L.) could provide a theoretical basis for further researches on the functions of OsbHLH109 in stress and hormone responses.【Method】The bioinformatics of OsbHLH109 was analyzed, and the expression patterns of OsbHLH109 in different tissues of Rice Zhonghua 11 under different hormone and abiotic stresses were analyzed by quantitative PCR (qRT-PCR).【Result】The encoding region of OsbHLH109 was 1 164 bp in length, containing 6 exons and encoding 388 amino acids, which was a bHLH transcription factor containing a conserved domain of HLH. Phylogenetic analysis showed that OsbHLH109 was closely related to the homologous proteins in monocots and Dicotyledones. The analysis of cisacting elements showed that the promoter region of OsbHLH109 contained 24 plant hormone response cis-elements and 25 environmental stress response cis-elements. The qRT-PCR analysis showed that OsbHLH109 was expressed in roots, stems, leaves and young panicles of rice, but the expression level was the highest in leaves. The results of hormone treatment response expression profile analysis showed that the response expression differences of OsbHLH109 in leaves to 6-Benzylaminopurine (6-BA), indole acetic acid (IAA), gibberellic acid (GA3) and abscisic acid (ABA) treatments all reached significant levels. The results of stress treatment response expression profile analysis showed that the response of OsbHLH109 to low temperature stress, drought stress, high temperature stress and salt stress were significantly different in leaves, indicating that the expression of OsbHLH109 was induced by 6-BA, GA3, IAA, ABA, high temperature, low temperature, PEG6000 and NaCl, which suggested that OsbHLH109 was essential for the abiotic stress responses in rice.【Conclusion】Rice OsbHLH109 is a member of the bHLH transcription factor family and is highly expressed in leaves. The expression of OsbHLH109 gene is regulated by high temperature, low temperature and salt stress, and responds to hormone signals such as 6-BA, IAA, GA3 and ABA. It is predicted that it may be involved in abiotic stress response of rice through ABA and other hormone signal transduction pathways. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Chilling-induced accumulation of reactive oxygen species (ROS) is harmful to plants, which usually produce anthocyanins to scavenge ROS as protection from chilling stress. As a tropical crop, cassava is hypersensitive to chilling, but the biochemical basis of this hypersensitivity remains unclear. We previously generated MeMYB2-RNAi transgenic cassava with increased chilling tolerance. Here we report that MeMYB2-RNAi transgenic cassava accumulated less ROS but more cyanidin-3-O-glucoside than the wild type under early chilling stress. Under this stress, the anthocyanin biosynthesis pathway was more active in MeMYB2-RNAi lines than in the wild type, and several genes involved in the pathway, including MeTT8, were up-regulated by MeMYB2-RNAi in the transgenic cassava. MeMYB2 bound to the MeTT8 promoter and blocked its expression under both normal and chilling conditions, thereby inhibiting anthocyanin accumulation. MeTT8 was shown to bind to the promoter of Dihydroflavonol 4-reductase (MeDFR-2) and increased MeDFR-2 expression. MeMYB2 appears to act as an inhibitor of chilling-induced anthocyanin accumulation during the rapid response of cassava to chilling stress.

The bHLH transcription factor family plays crucial roles in plant growth and development and their responses to adversity. In this study, a highly salt-induced bHLH gene, PagbHLH35 (Potri.018G141600), was identified from Populus alba × P. glandullosa (84K poplar). PagbHLH35 contains a highly conserved bHLH domain within the region of 52–114 amino acids. A subcellular localization result confirmed its nuclear localization. A yeast two-hybrid assay indicated PagbHLH35 lacks transcriptional activation activity, while a yeast one-hybrid assay indicated it could specifically bind to G-box and E-box elements. The expression of PagbHLH35 reached its peak at 12 h and 36 h time points under salt stress in the leaves and roots, respectively. A total of three positive transgenic poplar lines overexpressing PagbHLH35 were generated via Agrobacterium-mediated leaf disk transformation. Under NaCl stress, the transgenic poplars exhibited significantly enhanced morphological and physiological advantages such as higher POD activity, SOD activity, chlorophyll content, and proline content, and lower dehydration rate, MDA content and hydrogen peroxide (H2O2) content, compared to wild-type (WT) plants. In addition, histological staining showed that there was lower ROS accumulation in the transgenic poplars under salt stress. Moreover, the relative expression levels of several antioxidant genes in the transgenic poplars were significantly higher than those in the WT. All the results indicate that PagbHLH35 can improve salt tolerance by enhancing ROS scavenging in transgenic poplars.

Abstract Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana.

Abstract Chinese wild rice (Zizania latifolia) is rich in phenolic compounds, particularly flavonoids. This study identified 203 basic helix‐loop‐helix (bHLHs) in Z. latifolia and showed that ZlbHLH196 (Zla16G011250) corresponds to the ZlRc gene in Z. latifolia, with its protein product localizing to the nucleus. Notably, the pericarps of ZlRc‐overexpressing (OE) rice are brown, whereas those of wild‐type (WT) rice are nonpigmented. The total phenolic, flavonoid, and proanthocyanidin contents, antioxidant activity, as well as enzyme inhibitory effects of ZlRc‐OE rice were significantly higher than those of WT rice. Overall, 221 differential phenolic metabolites were identified between ZlRc‐OE and WT rice, among which 198 were upregulated in the former. Additionally, a total of 227 differentially expressed genes were identified between ZlRc‐OE and WT rice, with 173 upregulated. Kyoto Encyclopedia of Genes and Genomes annotation and enrichment analysis of phenolic metabolites revealed enhanced isoflavonoid, flavone, flavonol, and flavonoid biosynthesis pathways in ZlRc‐OE rice, which, furthermore, showed a markedly upregulated expression and significantly higher activities of four key flavonoid biosynthesis–related enzymes (phenylalanine ammonia‐lyase, chalcone synthase, chalcone‐flavanone isomerase, and dihydroflavonol 4‐reductase). These findings show that ZlRc‐overexpression promotes phenolic compound accumulation in rice seeds and can be used to bioaugment rice phenolic content.

The basic helix–loop–helix (bHLH) transcription factor family is the second-largest transcription factor family in plants. Members of this family are involved in the processes of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which has been used as ornamental architecture and traditional Chinese herbal medicine plants. However, the bHLH transcription factors in Loropetalum chinense var. rubrum (L. chinense var. rubrum) have not yet been systematically demonstrated, and their role in the biosynthesis of anthocyanin is still unclear. Here, we identified 165 potential LcbHLHs genes by using two methods, and they were unequally distributed on chromosomes 1 to 12 of the genome of L. chinense var. rubrum. Based on an evolutionary comparison with proteins from Arabidopsis and Oryza sativa, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a particular subfamily had similar gene structures and conserved motifs. The Gene Ontology annotation and Cis-elements predicted that LcbHLHs had many molecular functions and were involved in processes of plant growth, including the biosynthesis of flavonoids and anthocyanins. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars of L. chinense var. rubrum. Many LcbHLHs were expressed in the leaves, and only a few genes were highly expressed in the flowers. Six LcbHLHs candidate genes were identified by bioinformatics analysis and expression analysis. Further Real-time quantitative PCR analysis and protein interaction network analysis showed that LcbHLH156, which is one of the candidate proteins belonging to the IIIf subfamily, could interact with proteins related to anthocyanin synthesis. Therefore, LcbHLH156 was transiently expressed in L. chinense var. rubrum to verify its function in regulating anthocyanin synthesis. Compared with the control group, red pigment accumulation appeared at the wound after injection, and the total anthocyanin content increased at the wound of leaves. These results lay a foundation for the research of the regulation mechanism of leaf colors in L. chinense var. rubrum and also provide a basis for the function of the LcbHLH family. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

bHLH（basic helix-loop-helix）transcription factors play crucial roles in the biosynthesis of flavonoids in plants. In order to explore the function and molecular regulation mechanism of bHLH transcription factor in flavonoid biosynthesis in tartary buckwheat, RTPCR was used to clone the FtbHLH3, one bHLH transcription factor. Then the bioinformatics subcellular localization, transcriptional activation activity, gene expression, gene co-expression, and the correlation between gene expression level and total flavonoid content of it were conducted. As results, the CDS length of FtbHLH3 was total 783 bp and it encoded total 260 amino acids. Conserved domain and phylogenetic analysis showed that FtbHLH3 was a member of non-IIIf subfamily of bHLH transcription factor family. Subcellular localization and transcriptional activation assay suggested that FtbHLH3 was located in the nucleus and had no transcriptional activation activity. Gene co-expression analysis revealed that FtbHLH3 was co-expressed with the Arabidopsis TT8 homologous gene FtTT8 and 12 structural genes of flavonoid biosynthesis in the tartary buckwheat. The correlation analysis between gene expression level and total flavonoids content in different tissues indicated that there was a highly positive correlation between the expressions of FtbHLH3 and the contents of total flavonoids. All these results suggest that FtbHLH3 is a non-IIIf subfamily bHLH transcription factor that was located in the nucleus and has no transcriptional activation activity. It may regulate the flavonoids biosynthesis in tartary buckwheat through interacting with other transcription factors. [ABSTRACT FROM AUTHOR]

BackgroundThe basic helix-loop-helix (bHLH) proteins, a large transcription factors family, are involved in plant growth and development, and defensive response to various environmental stresses. The resurrection plant Myrothamnus flabellifolia is known for its extremely strong drought tolerance, but few bHLHs taking part in abiotic stress response have been unveiled in M. flabellifolia.ResultsIn the present research, we cloned and characterized a dehydration-inducible gene, MfbHLH38, from M. flabellifolia. The MfbHLH38 protein is localized in the nucleus, where it may act as a transcription factor. Heterologous expression of MfbHLH38 in Arabidopsis improved the tolerance to drought and salinity stresses, as determined by the studies on physiological indexes, such as contents of chlorophyll, malondialdehyde (MDA), proline (Pro), soluble protein, and soluble sugar, water loss rate of detached leaves, reactive oxygen species (ROS) accumulation, as well as antioxidant enzyme activities. Besides, MfbHLH38 overexpression increased the sensitivity of stomatal closure to mannitol and abscisic acid (ABA), improved ABA level under drought stress, and elevated the expression of genes associated with ABA biosynthesis and ABA responding, sucha as NCED3, P5CS, and RD29A.ConclusionsOur results presented evidence that MfbHLH38 enhanced tolerance to drought and salinity stresses in Arabidopsis through increasing water retention ability, regulating osmotic balance, decreasing stress-induced oxidation damage, and possibly participated in ABA-dependent stress-responding pathway.

The role of bHLH transcription factors in plant response to abiotic stress and regulation of flavonoid metabolism is well documented. However, to date, the bHLH transcription factor family in Iris domestica remains unreported, impeding further research on flavonoid metabolism in this plant. To address this knowledge gap, we employed bioinformatics to identify 39 IdbHLH genes and characterised their phylogenetic relationships and gene expression patterns under both drought and copper stress conditions. Our evolutionary tree analysis classified the 39 IdbHLHs into 17 subfamilies. Expression pattern analysis revealed that different IdbHLH transcription factors had distinct expression trends in various organs, suggesting that they might be involved in diverse biological processes. We found that IdbHLH36 was highly expressed in all organs (Transcripts Per Million (TPM) > 10), while only 12 IdbHLH genes in the rhizome and four in the root were significantly upregulated under drought stress. Of these, four genes (IdbHLH05, -37, -38, -39) were co-upregulated in both the rhizome and root, indicating their potential role in drought resistance. With regards to copper stress, we found that only 12 genes were upregulated. Further co-expression analysis revealed that most bHLH genes were significantly correlated with key enzyme genes involved in isoflavone biosynthesis. Thereinto, IdbHLH06 showed a significant positive correlation with IdC4H1 and Id4CL1 (p < 0.05). Furthermore, a transient expression assay confirmed that the IdbHLH06 protein was localised in the nucleus. Our findings provide new insights into the molecular basis and regulatory mechanisms of bHLH transcription factors in isoflavone biosynthesis in I. domestica.

Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana. [ABSTRACT FROM AUTHOR]

Neuronal networks in the nervous system play a central role in various behaviors essential for living organisms, such as movement, breathing, posture, and balance. Synaptic connections between different types of neurons during development form the basic architecture of neural networks that facilitate vital functions. The spinal cord contains a balanced number of excitatory (Glutamatergic) and inhibitory (GABAergic) neurons that connect to each other to create neural networks. Studies to date have investigated transcription factor (TF) networks that are expressed and function throughout central nervous system (CNS) development to identify and guide the development of diverse populations of neurons in the spinal cord. The balance between inhibitory and excitatory neurons in the dorsal spinal cord is established early in development in a process dominated by the interplay between basic helix-loop-helix (bHLH) transcriptional activators and a PRDM repressor PRDM13. The bHLH TFs, ASCL1 and PTF1A, initiate excitatory and inhibitory neuron gene expression programs, respectively, while PRDM13 is necessary to silence alternative cell fates. The central premise is that bHLH and PRDM factors sit poised at critical fate choice points in multiple lineages throughout the embryo, including those generating neuronal diversity. Although substantial progress has been made in identifying these factors and their functions in neuronal sub-type specification, the mechanisms how regulate specific gene programs are not yet clear. Uncovering these mechanisms will shed light on future research on the solution of disorder in the development of the nervous system and the treatment of clinical problems resulting from developmental anomalies. [ABSTRACT FROM AUTHOR]

Improvement of low nitrogen (LN) tolerance or nitrogen use efficiency (NUE) in crops is imperative for environment-friendly agriculture development. The basic helix-loop-helix (bHLH) transcription factors are involved in multiple abiotic stresses and are suitable as candidate genes for improving LN tolerance. Few studies were performed on the characterization of the HvbHLH gene family and their function in response to LN stress in barley. In this study, 103 HvbHLH genes were identified through genome-wide analysis. HvbHLH proteins were classified into 20 subfamilies based on phylogenetic analysis in barley, which was supported by conserved motifs and gene structure analysis. The stress-related cis-element analysis in the promoters showed that HvbHLHs are probably involved in multiple stress responses. By phylogenetic analysis of HvbHLHs and bHLHs in other plants, some HvbHLHs were predicted to play roles in response to nutrition deficiency stress. Furthermore, at least 16 HvbHLHs were differentially expressed in two barley genotypes differing in LN tolerance under LN stress. Finally, overexpression of HvbHLH56 enhanced LN stress tolerance in transgenic Arabidopsis, suggesting it is an important regulator in LN stress response. The differentially expressed HvbHLHs identified herein may be valuable for the breeding of barley cultivars with LN tolerance. [ABSTRACT FROM AUTHOR]

Anthocyanin biosynthesis determines the leaf color of Acer palmatum as a widely-planted landscape tree. Previously, ApMYB1 has been characterized as a positive regulator of anthocyanin biosynthesis. To further elucidate the mechanism of leaf coloration, the present study identified a basic helix-loop-helix (bHLH) transcription factor (ApbHLH1) through the phylogenetic analysis of 156 putative bHLH proteins in Acer palmatum and eight reference bHLHs which were known to be involved in the anthocyanin biosynthesis of selected plants. Protein structure comparison showed that ApbHLH1 has a conserved bHLH domain, and its N-terminal contains an MYB-interacting region. The expression of ApbHLH1 in leaves was found to not be correlated with anthocyanin contents either in green, semi-red leaves or during leaf autumnal senescence when anthocyanin content increased. ApbHLH1 expression in detached leaves was induced by exogenous senescence-promoting chemicals, including H2O2, SA, MeJA, ACC and ABA, with certain durations. In particular, either high light or low temperature induced ApbHLH1 expression significantly, and combination of high light and low temperatures seemed more effective in inducing ApbHLH1 expression. Luciferase complementation imaging assays confirmed the physical interaction between ApbHLH1 and ApMYB1, which could be abolished by either the truncating MYB-interacting region of ApbHLH1 or the deleting bHLH interacting domain of ApMYB1. The transient expression of ApbHLH1 could not induce anthocyanin production, while the co-expression of ApbHLH1 and ApMYB1 resulted in a higher accumulation of anthocyanins compared to the expression of ApMYB1 alone in tobacco leaves. Collectively, our results revealed that ApbHLH1 participated in leaf coloration through binding with ApMYB1 and enhancing the ApMYB1 function of promoting anthocyanin biosynthesis during leaf autumnal reddening in Acer palmatum. ApbHLH1 could have the potential for breeding color-leafed plants through co-transformation with ApMYB1. [ABSTRACT FROM AUTHOR]

Introduction: Alkaloids are one of the main medicinal components of Dendrobium species. Dendrobium alkaloids are mainly composed of terpene alkaloids. Jasmonic acid (JA) induce the biosynthesis of such alkaloids, mainly by enhancing the expression of JA-responsive genes to increase plant resistance and increase the content of alkaloids. Many JA-responsive genes are the target genes of bHLH transcription factors (TFs), especially the MYC2 transcription factor. Methods: In this study, the differentially expressed genes involved in the JA signaling pathway were screened out from Dendrobium huoshanense using comparative transcriptomics approaches, revealing the critical roles of basic helix-loop-helix (bHLH) family, particularly the MYC2 subfamily. Results and discussion: Microsynteny-based comparative genomics demonstrated that whole genome duplication (WGD) and segmental duplication events drove bHLH genes expansion and functional divergence. Tandem duplication accelerated the generation of bHLH paralogs. Multiple sequence alignments showed that all bHLH proteins included bHLH-zip and ACT-like conserved domains. The MYC2 subfamily had a typical bHLH-MYC_N domain. The phylogenetic tree revealed the classification and putative roles of bHLHs. The analysis of cis-acting elements revealed that promoter of the majority of bHLH genes contain multiple regulatory elements relevant to light response, hormone responses, and abiotic stresses, and the bHLH genes could be activated by binding these elements. The expression profiling and qRT-PCR results indicated that bHLH subgroups IIIe and IIId may have an antagonistic role in JA-mediated expression of stress-related genes. DhbHLH20 and DhbHLH21 were considered to be the positive regulators in the early response of JA signaling, while DhbHLH24 and DhbHLH25 might be the negative regulators. Our findings may provide a practical reference for the functional study of DhbHLH genes and the regulation of secondary metabolites. [ABSTRACT FROM AUTHOR]

Panicle development is crucial to increase the grain yield of rice (Oryza sativa). The molecular mechanisms of the control of panicle development in rice remain unclear. In this study, we identified a mutant with abnormal panicles, termed branch one seed 1-1 (bos1-1). The bos1-1 mutant showed pleiotropic defects in panicle development, such as the abortion of lateral spikelets and the decreased number of primary panicle branches and secondary panicle branches. A combined map-based cloning and MutMap approach was used to clone BOS1 gene. The bos1-1 mutation was located in chromosome 1. A T-to-A mutation in BOS1 was identified, which changed the codon from TAC to AAC, resulting in the amino acid change from tyrosine to asparagine. BOS1 gene encoded a grassspecific basic helix–loop–helix transcription factor, which is a novel allele of the previously cloned LAX PANICLE 1 (LAX1) gene. Spatial and temporal expression profile analyses showed that BOS1 was expressed in young panicles and was induced by phytohormones. BOS1 protein was mainly localized in the nucleus. The expression of panicle development-related genes, such as OsPIN2, OsPIN3, APO1, and FZP, was changed by bos1-1 mutation, suggesting that the genes may be the direct or indirect targets of BOS1 to regulate panicle development. The analysis of BOS1 genomic variation, haplotype, and haplotype network showed that BOS1 gene had several genomic variations and haplotypes. These results laid the foundation for us to further dissect the functions of BOS1. [ABSTRACT FROM AUTHOR]

Key message: Transcriptomic, physiological, and qRT-PCR analysis revealed the potential mechanism by which SlPRE2 regulates plant growth and stomatal size via multiple phytohormone pathways in tomato. Paclobutrazol resistance proteins (PREs) are atypical members of the basic/helix-loop-helix (bHLH) transcription factor family that regulate plant morphology, cell size, pigment metabolism and abiotic stress in response to different phytohormones. However, little is known about the network regulatory mechanisms of PREs in plant growth and development in tomato. In this study, the function and mechanism of SlPRE2 in tomato plant growth and development were investigated. The quantitative RT-PCR results showed that the expression of SlPRE2 was regulated by multiple phytohormones and abiotic stresses. It showed light-repressed expression during the photoperiod. The RNA-seq results revealed that SlPRE2 regulated many genes involved in photosynthesis, chlorophyll metabolism, phytohormone metabolism and signaling, and carbohydrate metabolism, suggesting the role of SlPRE2 in gibberellin, brassinosteroid, auxin, cytokinin, abscisic acid and salicylic acid regulated plant development processes. Moreover, SlPRE2 overexpression plants showed widely opened stomata in young leaves, and four genes involved in stomatal development showed altered expression. Overall, the results demonstrated the mechanism by which SlPRE2 regulates phytohormone and stress responses and revealed the function of SlPRE2 in stomatal development in tomato. These findings provide useful clues for understanding the molecular mechanisms of SlPRE2-regulated plant growth and development in tomato. [ABSTRACT FROM AUTHOR]

Stress response in plant is regulated by a large number of genes co-operating in diverse networks that serve multiple adaptive process. To understand how gene regulatory networks (GRNs) modulating abiotic stress responses, we compare the GRNs underlying drought and cold stresses using samples collected at 4 or 6 h intervals within 48 h in Chinese bayberry (Myrica rubra). We detected 7,583 and 8,840 differentially expressed genes (DEGs) under drought and cold stress respectively, which might be responsive to environmental stresses. Drought- and cold-responsive GRNs, which have been built according to the timing of transcription under both abiotic stresses, have a conserved trans-regulator and a common regulatory network. In both GRNs, basic helix-loop-helix family transcription factor (bHLH) serve as central nodes. MrbHLHp10 transcripts exhibited continuous increase in the two abiotic stresses and acts upstream regulator of ASCORBATE PEROXIDASE (APX) gene. To examine the potential biological functions of MrbHLH10, we generated a transgenic Arabidopsis plant that constitutively overexpresses the MrbHLH10 gene. Compared to wild-type (WT) plants, overexpressing transgenic Arabidopsis plants maintained higher APX activity and biomass accumulation under drought and cold stress. Consistently, RNAi plants had elevated susceptibility to both stresses. Taken together, these results suggested that MrbHLH10 mitigates abiotic stresses through the modulation of ROS scavenging. [ABSTRACT FROM AUTHOR]

Coloration of plant organs is governed by a combination of MYB- and bHLH-type transcription factors (TFs) and WD repeats or WD40 proteins in a MYB-bHLH-WDR (MBW) ternary protein complex for anthocyanin synthesis. Here, the MYB-type gene Rosea1 (ROS1) and the bHLH-type gene Delila (DEL) from Antirrhinum majus were expressed separately or simultaneously in Arabidopsis and Brassica napus to determine the influence on anthocyanin biosynthesis in different plant species. Overexpression of ROS1 alone substantially improved anthocyanin production in the leaves and seeds in Arabidopsis and triggered the transcription of late biosynthetic genes, such as AtDFR and AtANS. However, overexpression of DEL alone in Arabidopsis failed to produce any anthocyanin in the whole plants. Interestingly, DEL and ROS1 co-expression strengthened the anthocyanins biosynthesis and modified petal color from white into purple in Arabidopsis. The anthocyanidin content was consistent with the organ coloration in Arabidopsis. The main anthocyanin compounds in transgenic ROS1 leaves were cyanidin 3,5-O-diglucoside (28.0 µg/g DW) and cyanidin 3-O-glucoside (17.5 µg/g DW). In flowers of DEL and ROS1 co-expressed transgenic Arabidopsis, the dominating anthocyanin compounds were delphinidin 3-O-glucoside (33.8 µg/g DW), cyanidin 3,5-O-diglucoside (20.1 µg/g DW), and cyanidin 3-O-glucoside (10.5 µg/g DW). However in B. napus, neither separate nor co-expression of DEL and ROS1 produced any organ coloration. Most of the early and the late biosynthetic genes had no significant induction in the transgenic B. napus. [ABSTRACT FROM AUTHOR]

NEUROD2 , encoding the neurogenic differentiation factor 2, is essential for neurodevelopment. To date, heterozygous missense variants in this gene have been identified in eight patients (from six unrelated families) with epileptic encephalopathy and developmental delay. We describe a child with initial clinical suspicion of Rett/Rett-like syndrome, in whom exome sequencing detected a novel de novo variant (c.388G > A, p.Glu130Lys) in NEUROD2. Interestingly, a missense change affecting the same codon, c.388G > C (p.Glu130Gln), was previously identified in other two patients. Our results suggest that Glu130 might represent a potential mutational hotspot of NEUROD2. Furthermore, the clinical findings (especially the absence of clinically overt seizures) strengthen the NEUROD2- phenotypic spectrum, implying that developmental delay may also manifest isolatedly. We suggest inclusion of NEUROD2 -associated developmental and epileptic encephalopathies (DEEs) in the differential diagnosis of atypical Rett syndrome as well as gene panels related to autism spectrum disorder. [ABSTRACT FROM AUTHOR]

The storage roots of purple-fleshed sweetpotato rich in anthocyanins are considered nutrient-rich foods with health effects. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation remains to be revealed. In this study, IbMYB1-2 was isolated from purple-fleshed sweetpotato "Xuzishu8". The phylogenetic and sequence analysis indicated that IbMYB1-2 belongs to the SG6 subfamily with a conserved bHLH motif. Subcellular localization analysis and transcriptional activity assay revealed that IbMYB1-2 is a key transcriptional activator and is specific to the nucleus. Agrobacterium rhizogenes -mediated overexpression of IbMYB1-2 in sweetpotato through in vivo root transgenic system led to an increase in anthocyanins in the root of sweetpotato. qRT-PCR and transcriptome analysis depicted that the transcript levels of IbMYB1-2, IbbHLH42, and eight structural genes that are associated with the synthesis of anthocyanin were upregulated in overexpressed IbMYB1-2 transgenic roots. Dual-luciferase reporter (DLR) assay and yeast one-hybrid (Y1H) assay demonstrated IbMYB1-2 binding to the promoter regions of IbbHLH42 and other anthocyanin biosynthetic genes, including IbCHS , IbCHI, IbF3H , IbDFR , IbANS , IbGSTF12 , IbUGT78D2 , and IbUF3GT. Moreover, IbbHLH42 was shown to be an active enhancer for the formation of MYB-bHLH-WD40 (MBW) complex, which strongly supports the promoter activities of the IbCHS , IbANS , IbUGT78D2 , and IbGSTF12 genes to induce anthocyanin accumulation. Taken together, our findings not only revealed the underlying regulatory molecular mechanism of IbMYB1-2 for anthocyanin accumulation in the storage roots of sweetpotato but also uncovered a potential mechanism by which IbbHLH42 modulated anthocyanin biosynthesis through a positive feedback regulatory loop. • IbMYB1-2 belongs to R2R3-MYB subfamily SG6 and acts as a transcriptional activator. • Overexpression of IbMYB1-2 induces anthocyanin accumulation in sweetpotato roots. • Overexpression of IbMYB1-2 in roots activates multiple anthocyanin biosynthetic genes. • IbMYB1-2 binds to the promoters of IbbHLH42 and eight anthocyanin biosynthetic genes. • IbMYB1-2 and IbbHLH42 coregulate promoter activity to promote anthocyanin accumulation. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation. [ABSTRACT FROM AUTHOR]

Chrysanthemum morifolium Ramat. 'Huaihuang' is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. 'Huaiju 2#' being bred from 'Huaihuang', shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in 'Huaiju 2#'. On the basis of the transcriptome database of 'Huaiju 2#' after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus. [ABSTRACT FROM AUTHOR]

Xinjiang wild apple (Malus sieversii) is an ancient relic; a plant with abundant genetic diversity and disease resistance. Several transcription factors were studied in response to different biotic and abiotic stresses on the wild apple. Basic/helix–loop–helix (bHLH) is a large plant transcription factor family that plays important roles in plant responses to various biotic and abiotic stresses and has been extensively studied in several plants. However, no study has yet been conducted on the bHLH gene in M. sieversii. Based on the genome of M. sieversii, 184 putative MsbHLH genes were identified, and their physicochemical properties were studied. MsbHLH covered 23 subfamilies and lacked two subfamily genes of Arabidopsis thaliana based on the widely used classification method. Moreover, MsbHLH exon–intron structures matched subfamily classification, as evidenced by the analysis of their protein motifs. The analysis of cis-acting elements revealed that many MsbHLH genes share stress- and hormone-related cis-regulatory elements. These MsbHLH transcription factors were found to be involved in plant defense responses based on the protein–protein interactions among the differentially expressed MsbHLHs. Furthermore, 94 MsbHLH genes were differentially expressed in response to pathogenic bacteria. The qRT-PCR results also showed differential expression of MsbHLH genes. To further verify the gene function of bHLH, our study used the transient transformation method to obtain the overexpressed MsbHLH155.1 transgenic plants and inoculated them. Under Valsa canker infection, the lesion phenotype and physiological and biochemical indexes indicated that the antioxidant capacity of plants could increase and reduce the damage caused by membrane peroxidation. This study provides detailed insights into the classification, gene structure, motifs, chromosome distribution, and gene expression of bHLH genes in M. sieversii and lays a foundation for a better understanding disease resistance in plants, as well as providing candidate genes for the development of M. sieversii resistance breeding. [ABSTRACT FROM AUTHOR]

While bHLH transcription factors have been linked to the regulation of various abiotic stressors, research on drought-related bHLH proteins and their molecular processes in Agropyron mongolicum has remained limited. In this study, a bHLH gene from A. mongolicum, designated as AmbHLH148, was successfully cloned and isolated. AmbHLH148 was exclusively localized within the nucleus. Additionally, qRT-PCR analysis demonstrated a significant upregulation of AmbHLH148 in response to drought stress. When transferred into tobacco (Nicotiana benthamiana), the heterologous expression of AmbHLH148 led to enhanced drought tolerance. Under drought stress conditions, AmbHLH148-OE transgenic tobacco plants exhibited increased activities of antioxidant defense enzymes, such as SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase). These enzymes efficiently mitigated the accumulation of reactive oxygen species (ROS) compared to wild-type plants. Furthermore, AmbHLH148-OE transgenic tobacco showed elevated levels of PRO (proline) and reduced MDA (malondialdehyde) content, contributing to enhanced stability in the plant’s cell membrane system during drought stress. In summary, this study underscores that the overexpression of AmbHLH148 in transgenic tobacco acts as a positive regulator under drought stress by enhancing the plant’s antioxidant capacity. These findings shed light on the molecular mechanisms involved in bHLH transcription factors’ role in drought resistance, contributing to the discovery and utilization of drought-resistant genes in A. mongolicum for enhancing crop drought resistance.