bHLH Family Research Articles

Genome-Wide Characterization of Differentially Expressed Scent Genes in the MEP Control Network of the Flower of Lilium 'Sorbonne'.

Fragrance is an important feature of ornamental lilies. Components of volatile substances and important genes for monoterpene synthesis in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were examined in this study. Twenty volatile compounds (2 in the budding stage, 3 in the initial flowering stage, 7 in the semi-flowering stage, 17 in the full-flowering stage, and 5 in withering stage) were detected in the Oriental lily 'Sorbonne' using gas chromatography-mass spectrometry. The semi- and full-flowering stages were key periods for volatile substance production and enzyme function. Sequence assembly from samples collected during all flowering stages resulted in the detection of 274,849 genes and 129,017 transcripts. RNA sequencing and heatmapping led to the detection of genes in the MEP monoterpene metabolism pathway. Through gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we extracted key genes (LiDXS2, LiLIS, and LiMYS) and transcription factors (in the bHLH, MYB, HD-ZIP, and NAC families) associated with the MEP pathway. Tissue localization revealed that LiDXS2, LiLIS, and LiMYS were expressed in Lilium 'Sorbonne' petals in the full-flowering stage. Genes regulating the 1-deoxy-D-X-lignone-5-phosphate synthase family of rate-limiting enzymes, involved in the first step of monoterpene synthesis, showed high expression in the semi- and full-flowering stages. LiDXS2 was cloned and localized in chloroplast subcells. The relative expression of terpene-related genes in the MEP and mevalonic acid pathways of wild-type and LiLIS/LiMYS transgenic Arabidopsis thaliana, and changes in chemical composition, confirmed that LiLIS/LiMYS regulates the monoterpene synthesis pathway. The results of this study provide a theoretical basis for the synthesis of lily aromatic substances and the cultivation of new garden flower varieties.

IbMYC2 Contributes to Salt and Drought Stress Tolerance via Modulating Anthocyanin Accumulation and ROS-Scavenging System in Sweet Potato.

Basic helix-loop-helix (bHLH) transcription factors extensively affect various physiological processes in plant metabolism, growth, and abiotic stress. However, the regulation mechanism of bHLH transcription factors in balancing anthocyanin biosynthesis and abiotic stress in sweet potato (Ipomoea batata (L.) Lam.) remains unclear. Previously, transcriptome analysis revealed the genes that were differentially expressed among the purple-fleshed sweet potato cultivar 'Jingshu 6' and its anthocyanin-rich mutant 'JS6-5'. Here, we selected one of these potential genes, IbMYC2, which belongs to the bHLH transcription factor family, for subsequent analyses. The expression of IbMYC2 in the JS6-5 storage roots is almost four-fold higher than Jingshu 6 and significantly induced by hydrogen peroxide (H2O2), methyl jasmonate (MeJA), NaCl, and polyethylene glycol (PEG)6000. Overexpression of IbMYC2 significantly enhances anthocyanin production and exhibits a certain antioxidant capacity, thereby improving salt and drought tolerance. In contrast, reducing IbMYC2 expression increases its susceptibility. Our data showed that IbMYC2 could elevate the expression of anthocyanin synthesis pathway genes by binding to IbCHI and IbDFR promoters. Additionally, overexpressing IbMYC2 activates genes encoding reactive oxygen species (ROS)-scavenging and proline synthesis enzymes under salt and drought conditions. Taken together, these results demonstrate that the IbMYC2 gene exercises a significant impact on crop quality and stress resistance.

Genome-wide identification of bHLH transcription factors and functional analysis in salt gland development of the recretohalophyte sea lavender (Limonium bicolor).

Transcription factors with basic helix-loop-helix (bHLH) structures regulate plant growth, epidermal structure development, metabolic processes, and responses to stress extensively. Sea lavender (Limonium bicolor) is a recretohalophyte with unique salt glands in the epidermis that make it highly resistant to salt stress, contributing to the improvement of saline lands. However, the features of the bHLH transcription factor family in L. bicolor are largely unknown. Here, we systematically analyzed the characteristics, localization, and phylogenetic relationships of 187 identified bHLH family genes throughout the L. bicolor genome, as well as their cis-regulatory promoter elements, expression patterns, and key roles in salt gland development or salt tolerance by genetic analysis. Nine verified L. bicolor bHLH genes are expressed and the encoded proteins function in the nucleus, among which the proteins encoded by Lb2G14060 and Lb1G07934 also localize to salt glands. Analysis of CRISPR-Cas9-generated knockout mutants and overexpression lines indicated that the protein encoded by Lb1G07934 is involved in the formation of salt glands, salt secretion, and salt resistance, indicating that bHLH genes strongly influence epidermal structure development and stress responses. The current study lays the foundation for further investigation of the effects and functional mechanisms of bHLH genes in L. bicolor and paves the way for selecting salt-tolerance genes that will enhance salt resistance in crops and for the improvement of saline soils.

Transcriptome-Wide Identification and Expression Analysis of bHLH Family Genes in Iris domestica under Drought and Cu Stress.

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.

Basic helix-loop-helix (bHLH) gene family in rye (Secale cereale L.): genome-wide identification, phylogeny, evolutionary expansion and expression analyses

BackgroundRye (Secale cereale), one of the drought and cold-tolerant crops, is an important component of the Triticae Dumortier family of Gramineae plants. Basic helix-loop-helix (bHLH), an important family of transcription factors, has played pivotal roles in regulating numerous intriguing biological processes in plant development and abiotic stress responses. However, no systemic analysis of the bHLH transcription factor family has yet been reported in rye.ResultsIn this study, 220 bHLH genes in S. cereale (ScbHLHs) were identified and named based on the chromosomal location. The evolutionary relationships, classifications, gene structures, motif compositions, chromosome localization, and gene replication events in these ScbHLH genes are systematically analyzed. These 220 ScbHLH members are divided into 21 subfamilies and one unclassified gene. Throughout evolution, the subfamilies 5, 9, and 18 may have experienced stronger expansion. The segmental duplications may have contributed significantly to the expansion of the bHLH family. To systematically analyze the evolutionary relationships of the bHLH family in different plants, we constructed six comparative genomic maps of homologous genes between rye and different representative monocotyledonous and dicotyledonous plants. Finally, the gene expression response characteristics of 22 ScbHLH genes in various biological processes and stress responses were analyzed. Some candidate genes, such as ScbHLH11, ScbHLH48, and ScbHLH172, related to tissue developments and environmental stresses were screened.ConclusionsThe results indicate that these ScbHLH genes exhibit characteristic expression in different tissues, grain development stages, and stress treatments. These findings provided a basis for a comprehensive understanding of the bHLH family in rye.

A Comprehensive Analysis of Auxin Response Factor Gene Family in Melastoma dodecandrum Genome.

Auxin Response Factors (ARFs) mediate auxin signaling and govern diverse biological processes. However, a comprehensive analysis of the ARF gene family and identification of their key regulatory functions have not been conducted in Melastoma dodecandrum, leading to a weak understanding of further use and development for this functional shrub. In this study, we successfully identified a total of 27 members of the ARF gene family in M. dodecandrum and classified them into Class I-III. Class II-III showed more significant gene duplication than Class I, especially for MedARF16s. According to the prediction of cis-regulatory elements, the AP2/ERF, BHLH, and bZIP transcription factor families may serve as regulatory factors controlling the transcriptional pre-initiation expression of MedARF. Analysis of miRNA editing sites reveals that miR160 may play a regulatory role in the post-transcriptional expression of MeARF. Expression profiles revealed that more than half of the MedARFs exhibited high expression levels in the stem compared to other organs. While there are some specific genes expressed only in flowers, it is noteworthy that MedARF16s, MedARF7A, and MedARF9B, which are highly expressed in stems, also demonstrate high expressions in other organs of M. dodecandrum. Further hormone treatment experiments revealed that these MedARFs were sensitive to auxin changes, with MedARF6C and MedARF7A showing significant and rapid changes in expression upon increasing exogenous auxin. In brief, our findings suggest a crucial role in regulating plant growth and development in M. dodecandrum by responding to changes in auxin. These results can provide a theoretical basis for future molecular breeding in Myrtaceae.

Rice PIFs: Critical regulators in rice development and stress response.

Phytochrome-interacting factors (PIFs) belong to a subfamily of the basic helix-loop-helix (bHLH) family of transcription factors, which serve as a "hub" for development and growth of plants. They have the capability to regulate the expression of many downstream genes, integrate multiple signaling pathways, and act as a signaling center within the cell. In rice (Oryza sativa), the PIF family genes, known as OsPILs, play a crucial part in many different aspects. OsPILs play a crucial role in regulating various aspects of photomorphogenesis, skotomorphogenesis, plant growth, and development in rice. These vital processes include chlorophyll synthesis, plant gravitropism, plant height, flowering, and response to abiotic stress factors such as low temperature, drought, and high salt. Additionally, OsPILs are involved in controlling several important agronomic traits in rice. Some OsPILs members coordinate with each other to function. This review summarizes and prospects the latest research progress on the biological functions of OsPILs transcription factors and provides a reference for further exploring the functions and mechanism of OsPILs.

Genome-wide identification of the bHLH transcription factor family in Rosa persica and response to low-temperature stress.

Basic helix-loop-helix (bHLH) transcription factors are involved in plant growth and development, secondary metabolism, and abiotic stress responses have been studied in a variety of plants. Despite their importance in plant biology, the roles and expression patterns of bHLH family genes in Rosa persica have not been determined. In this study, the RbebHLH family genes were systematically analyzed using bioinformatics methods, and their expression patterns under low-temperature stress were analyzed by transcriptome and related physiological index measurements. In total, 142 RbebHLHs were identified in the genome of R. persica, distributed on seven chromosomes. Phylogenetic analysis including orthologous genes in Arabidopsis divided RbebHLHs into 21 subfamilies, with similar structures and motifs within a subfamily. A collinearity analysis revealed seven tandem duplications and 118 segmental duplications in R. persica and 127, 150, 151, 172, and 164 segmental duplications between R. persica and Arabidopsis thaliana, Prunus mume, Fragaria vesca, Rosa chinensis, and Prunus persica, respectively. A number of cis-regulatory elements associated with abiotic stress response and hormone response were identified in RbebHLHs, and 21 RbebHLHs have potential interactions with the CBF family. In addition, the expression results showed that part of bHLH may regulate the tolerance of R. persica to low-temperature stress through the jasmonic acid and pathway. Transcriptomic data showed that the expression levels of different RbebHLHs varied during overwintering, and the expression of some RbebHLHs was significantly correlated with relative conductivity and MDA content, implying that RbebHLHs play important regulatory roles in R. persica response to low-temperature stress. Overall, this study provides valuable insights into the study of RbebHLHs associated with low-temperature stress.

Genome-wide analysis of the bHLH family and identification of bHLH genes involved in fruit development and ripening of cultivated octoploid strawberry

Abstract Objectives The basic helix–loop–helix (bHLH) transcription factors (TFs) regulate fruit growth in many plants. However, there is no available study on the bHLH gene family in the haplotype-resolved genome of cultivated strawberry (Fragaria × ananassa). Materials and Methods The 131 FabHLH genes identified in the strawberry cultivar ‘Yanli’ haplotype-resolved genome were classified into 24 subfamilies according to their phylogenetic relationships. Gene structure, conserved motifs, and chromosomal locations were investigated using bioinformatics. Results In total, 15 FabHLH genes potentially involved in fruit development were screened based on transcriptome analysis of different stages of fruit development. We also identified the cis-regulatory elements of these 15 FabHLH genes, predicted upstream transcription factors, and identified protein–protein interactions. Conclusions The findings of this study improve our understanding of the regulation mediated by bHLH TFs during strawberry fruit growth and maturation.

A comprehensive analysis of transcriptomic data for comparison of cold tolerance in two Brassica napus genotypes.

Brassica napus is an important oil crop and cold stress severely limits its productivity. To date, several studies have reported the regulatory genes and pathways involved in cold-stress responses in B. napus. However, transcriptome-scale identification of the regulatory genes is still lacking. In this study, we performed comparative transcriptome analysis of cold-tolerant C18 (CT - C18) and cold-sensitive C6 (CS - C6) Brassica napus genotypes under cold stress for 7 days, with the primary purpose of identifying cold-responsive transcription in B. napus. A total of 6061 TFs belonging to 58 families were annotated in the B. napus genome, of which 3870 were expressed under cold stress in both genotypes. Among these, 451 TFs were differentially expressed (DE), with 21 TF genes expressed in both genotypes. Most TF members of the MYB (26), bHLH (23), and NAC (17) families were significantly expressed in the CT - C18 genotype compared with the CS - C6 B. napus genotype. GO classification showed a significant role in transcription regulation, DNA-binding transcription factor activity, response to chitin, and the ethylene-activated signaling pathway. KEGG pathway annotation revealed these TFs are involved in regulating more pathways, resulting in more tolerance. In conclusion, the results provide insights into the molecular regulation mechanisms of B. napus in response to freezing treatment, expanding our understanding of the complex molecular mechanisms in plants' response to freezing stress.

RNA-seq analysis reveals transcriptome reprogramming and alternative splicing during early response to salt stress in tomato root.

Salt stress is one of the dominant abiotic stress conditions that cause severe damage to plant growth and, in turn, limiting crop productivity. It is therefore crucial to understand the molecular mechanism underlying plant root responses to high salinity as such knowledge will aid in efforts to develop salt-tolerant crops. Alternative splicing (AS) of precursor RNA is one of the important RNA processing steps that regulate gene expression and proteome diversity, and, consequently, many physiological and biochemical processes in plants, including responses to abiotic stresses like salt stress. In the current study, we utilized high-throughput RNA-sequencing to analyze the changes in the transcriptome and characterize AS landscape during the early response of tomato root to salt stress. Under salt stress conditions, 10,588 genes were found to be differentially expressed, including those involved in hormone signaling transduction, amino acid metabolism, and cell cycle regulation. More than 700 transcription factors (TFs), including members of the MYB, bHLH, and WRKY families, potentially regulated tomato root response to salt stress. AS events were found to be greatly enhanced under salt stress, where exon skipping was the most prevalent event. There were 3709 genes identified as differentially alternatively spliced (DAS), the most prominent of which were serine/threonine protein kinase, pentatricopeptide repeat (PPR)-containing protein, E3 ubiquitin-protein ligase. More than 100 DEGs were implicated in splicing and spliceosome assembly, which may regulate salt-responsive AS events in tomato roots. This study uncovers the stimulation of AS during tomato root response to salt stress and provides a valuable resource of salt-responsive genes for future studies to improve tomato salt tolerance.

Automatic annotation of the bHLH gene family in plants

BackgroundThe bHLH transcription factor family is named after the basic helix-loop-helix (bHLH) domain that is a characteristic element of their members. Understanding the function and characteristics of this family is important for the examination of a wide range of functions. As the availability of genome sequences and transcriptome assemblies has increased significantly, the need for automated solutions that provide reliable functional annotations is emphasised.ResultsA phylogenetic approach was adapted for the automatic identification and functional annotation of the bHLH transcription factor family. The bHLH_annotator, designed for the automated functional annotation of bHLHs, was implemented in Python3. Sequences of bHLHs described in literature were collected to represent the full diversity of bHLH sequences. Previously described orthologs form the basis for the functional annotation assignment to candidates which are also screened for bHLH-specific motifs. The pipeline was successfully deployed on the two Arabidopsis thaliana accessions Col-0 and Nd-1, the monocot species Dioscorea dumetorum, and a transcriptome assembly of Croton tiglium. Depending on the applied search parameters for the initial candidates in the pipeline, species-specific candidates or members of the bHLH family which experienced domain loss can be identified.ConclusionsThe bHLH_annotator allows a detailed and systematic investigation of the bHLH family in land plant species and classifies candidates based on bHLH-specific characteristics, which distinguishes the pipeline from other established functional annotation tools. This provides the basis for the functional annotation of the bHLH family in land plants and the systematic examination of a wide range of functions regulated by this transcription factor family.

Multi-omics responses of barley seedlings to low and high linear energy transfer irradiation

Barley is a resilient crop with high nutritional value and adaptability, making it a promising candidate for phytoremediation and space agriculture. The study presents a comprehensive multi-omics analysis of the impact of ionising radiation (IR) on barley seedlings, intending to identify candidate pathways for creating radiation-resilient barley plants. We found that different IR treatments (gamma, electron, proton, neutron) increased the intensity of protein catabolism and led to the attenuation of translation. The impact of IRs on protein synthesis and degradation was accompanied by rearrangements in energy metabolism and reallocation of nitrogen, probably due to enhanced protein catabolism. At least partially, those changes seem to fuel secondary metabolites production, including riboflavin, various phytoalexins, phytosiderophores, ferulic and sinapic acids, kaempferol, quercetin, nictoflorin, gallate, and podophyllotoxin. Many of these compounds have antioxidant or radioprotective properties. To focus on possible targets for gene editing, we identified genes differentially regulated after all types of IR exposure and potential transcription factors regulating secondary metabolism, including AP2/ERF, WRKY, bHLH, bZIP, MYB, and NAC families.

Genome-Wide Analysis of the Universal Stress Protein Gene Family in Blueberry and Their Transcriptional Responses to UV-B Irradiation and Abscisic Acid

Universal stress proteins (USPs) play essential roles in plant development, hormonal regulation, and abiotic stress responses. However, the characteristics and functional divergence of USP family members have not been studied in blueberry (Vaccinium corymbosum). In this study, we identified 72 VcUSP genes from the Genome Database for Vaccinium. These VcUSPs could be divided into five groups based on their phylogenetic relationships. VcUSPs from groups Ⅰ, Ⅳ, and Ⅴ each possess one UspA domain; group Ⅰ proteins also contain an ATP-binding site that is not present in group Ⅳ and Ⅴ proteins. Groups Ⅱ and Ⅲ include more complex proteins possessing one to three UspA domains and UspE or UspF domains. Prediction of cis-regulatory elements in the upstream sequences of VcUSP genes indicated that their protein products are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of RNA deep sequencing data showed that 21 and 7 VcUSP genes were differentially expressed in response to UV-B radiation and exogenous abscisic acid (ABA) treatments, respectively. VcUSP41 and VcUSP68 expressions responded to both treatments, and their encoded proteins may integrate the UV-B and ABA signaling pathways. Weighted gene co-expression network analysis revealed that VcUSP22, VcUSP26, VcUSP67, VcUSP68, and VcUSP41 were co-expressed with many transcription factor genes, most of which encode members of the MYB, WRKY, zinc finger, bHLH, and AP2 families, and may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B and exogenous ABA treatments. Our study provides a useful reference for the further functional analysis of VcUSP genes and blueberry molecular breeding.

The apple transcription factor MdbHLH4 regulates plant morphology and fruit development by promoting cell enlargement

The bHLH family, the second largest transcription factor (TF) family in plants, plays a crucial role in regulating plant growth and development processes. However, the biological functions and mechanisms of most bHLH proteins remain unknown, particularly in apples. In this study, we found that MdbHLH4 positively modulates plant growth and development by enhancing cell expansion. Overexpression (OE) of MdbHLH4 resulted in increased biomass, stem and root length, leaf area, and larger areas of pith, xylem, and cortex with greater cell size compared with wild-type apple plants. Conversely, RNA interference (RNAi)-mediated silencing of MdbHLH4 led to reduced xylem and phloem as well as smaller cell size compared to wild-type apple plants. Ectopic expression of MdbHLH4 in tomatoes resulted in enlarged fruits with impaired color appearance, decreased accumulation of soluble solids, and decreased flesh firmness along with larger seeds. Subsequent investigations have shown that MdbHLH4 directly binds to the promoters of MdARF6b and MdPIF4b, enhancing their expression levels. These findings suggest that MdbHLH4 potentially regulates plant cell expansion through auxin and light signaling pathways. These study results not only provide new insights into the roles of bHLH transcription factors in regulating plant growth and development but also contribute to a deeper understanding of their underlying mechanisms.

The Basic/Helix-Loop-Helix Transcription Factor Family Gene RcbHLH112 Is a Susceptibility Gene in Gray Mould Resistance of Rose (Rosa Chinensis).

The basic/helix-loop-helix (bHLH) family is a major family of transcription factors in plants. Although it has been reported that bHLH plays a defensive role against pathogen infection in plants, there is no comprehensive study on the bHLH-related defence response in rose (Rosa sp.). In this study, a genome-wide analysis of bHLH family genes (RcbHLHs) in rose was carried out, including their phylogenetic relationships, gene structure, chromosome localization and collinearity analysis. Via phylogenetic analysis, a total of 121 RcbHLH genes in the rose genome were divided into 21 sub-groups. These RcbHLHs are unevenly distributed in all 7 chromosomes of rose. The occurrence of gene duplication events indicates that whole-genome duplication and segmental duplication may play a key role in gene duplication. Ratios of non-synonymous to synonymous mutation frequency (Ka/Ks) analysis showed that the replicated RcbHLH genes mainly underwent purification selection, and their functional differentiation was limited. Gene expression analysis showed that 46 RcbHLHs were differentially expressed in rose petals upon B. cinerea infection. It is speculated that these RcbHLHs are candidate genes that regulate the response of rose plants to B. cinerea infection. Virus-induced gene silencing (VIGS) confirmed that RcbHLH112 in rose is a susceptibility factor for infection with B. cinerea. This study provides useful information for further study of the functions of the rose bHLH gene family.

Transplantation of Wnt3a-modified neural stem cells promotes neural regeneration and functional recovery after spinal cord injury via Wnt-Gli2 pathway.

Neural stem cell (NSCs) transplantation has great potential in the treatment of spinal cord injury (SCI). Previous studies have indicated that the Wnt pathway could regulate the expression of basic helix-loop-helix (bHLH) family factor Hes5 and Mash1 in NSCs, but not through the notch intracellular domain. This suggests that there are other signals involved in this process. The aim of this study was to investigate the role of Wnt-Gli2 pathway in the treatment of SCI by transplanting neural stem cells. NSCs were isolated from the striata of embryonic day 14 mice. Activation of the Wnt pathway was achieved using Wnt3a protein, while Gli2 was inhibited using Gli2-siRNA. Expression levels of Gli2 and bHLH factors were assessed using western blotting. NSCs proliferation was evaluated using CCK-8 assay, and neural differentiation was determined by immunofluorescence staining. Finally, the modified NSCs were transplanted into mice with SCI, and their effects were assessed using behavioral and histological tests. Our results demonstrated that Wnt3a promoted the expression of Mash1 through Gli2. Moreover, the expression of Ngn1 and Hes1 was up-regulated, while Hes5 was down-regulated. Wnt3a also promoted NSCs proliferation and neural differentiation through this signaling pathway. In vivo experiments showed that NSCs transplantation mediated by Wnt3a-Gli2 signaling increased the number of neurons and resulted in improved Basso Mouse Scale scores. In conclusion, our findings suggest that Gli2 plays a role in mediating the regulation of Wnt3a signaling on promoting NSCs proliferation and neural differentiation. This pathway is therefore important in NSCs-mediated SCI recovery.

Genome-Wide Identification and Characterization of the bHLH Gene Family and Its Response to Abiotic Stresses in Carthamus tinctorius.

The basic helix-loop-helix (bHLH) transcription factors possess DNA-binding and dimerization domains and are involved in various biological and physiological processes, such as growth and development, the regulation of secondary metabolites, and stress response. However, the bHLH gene family in C. tinctorius has not been investigated. In this study, we performed a genome-wide identification and analysis of bHLH transcription factors in C. tinctorius. A total of 120 CtbHLH genes were identified, distributed across all 12 chromosomes, and classified into 24 subfamilies based on their phylogenetic relationships. Moreover, the 120 CtbHLH genes were subjected to comprehensive analyses, including protein sequence alignment, evolutionary assessment, motif prediction, and the analysis of promoter cis-acting elements. The promoter region analysis revealed that CtbHLH genes encompass cis-acting elements and were associated with various aspects of plant growth and development, responses to phytohormones, as well as responses to both abiotic and biotic stresses. Expression profiles, sourced from transcriptome databases, indicated distinct expression patterns among these CtbHLH genes, which appeared to be either tissue-specific or specific to certain cultivars. To further explore their functionality, we determined the expression levels of fifteen CtbHLH genes known to harbor motifs related to abiotic and hormone responses. This investigation encompassed treatments with ABA, salt, drought, and MeJA. The results demonstrated substantial variations in the expression patterns of CtbHLH genes in response to these abiotic and hormonal treatments. In summary, our study establishes a solid foundation for future inquiries into the roles and regulatory mechanisms of the CtbHLH gene family.

Genome-Wide Analysis of bHLH Family Genes and Identification of Members Associated with Cold/Drought-Induced Photoinhibition in Kandelia obovata.

Plant basic helix-loop-helix (bHLH) transcription factors play pivotal roles in responding to stress, including cold and drought. However, it remains unclear how bHLH family genes respond to these stresses in Kandelia obovata. In this study, we identified 75 bHLH members in K. obovata, classified into 11 subfamilies and unevenly distributed across its 18 chromosomes. Collineation analysis revealed that segmental duplication primarily drove the expansion of KobHLH genes. The KobHLH promoters were enriched with elements associated with light response. Through RNA-seq, we identified several cold/drought-associated KobHLH genes. This correlated with decreased net photosynthetic rates (Pn) in the leaves of cold/drought-treated plants. Weighted gene co-expression network analysis (WGCNA) confirmed that 11 KobHLH genes were closely linked to photoinhibition in photosystem II (PS II). Among them, four Phytochrome Interacting Factors (PIFs) involved in chlorophyll metabolism were significantly down-regulated. Subcellular localization showed that KobHLH52 and KobHLH30 were located in the nucleus. Overall, we have comprehensively analyzed the KobHLH family and identified several members associated with photoinhibition under cold or drought stress, which may be helpfulfor further cold/drought-tolerance enhancement and molecular breeding through genetic engineering in K. obovata.

Screening and expression analysis of transcription factors involved in genuineness of Codonopsis pilosula in Shanxi

This study aims to mine the transcription factors that affect the genuineness of Codonopsis pilosula in Shanxi based on the transcriptome data of C. pilosula samples collected from Shanxi and Gansu, and then analyze the gene expression patterns, which will provide a theoretical basis for the molecular assisted breeding of C. pilosula. Gene ontology(GO) functional annotation, conserved motif prediction, and gene expression pattern analysis were performed for the differential transcription factors predicted based on the transcriptome data of C. pilosula from different habitats. A total of 61 differentially expressed genes(DEGs) were screened out from the transcriptome data. Most of the DEGs belonged to AP2/ERF-ERF family, with the conserved motif of [2X]-[LG]-[3X]-T-[3X]-[AARAYDRAA]-[3X]-[RG]-[2X]-A-[2X]-[NFP]. Forty-three of the DEGs showed significantly higher gene expression in C. pilosula samples from Shanxi than in the samples from Gansu, including 11 genes in the AP2/ERF-ERF family, 5 genes in the NAC fa-mily, 1 gene in the bHLH family, and 2 genes in the RWP-RK family, while 18 transcription factors showed higher expression levels in the samples from Gansu. GO annotation predicted that most of the DEGs were enriched in GO terms related to transcriptional binding activity(103), metabolic process(26), and stress response(23). The expression of transcription factor genes, CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 was higher in the samples from Shanxi and in the roots of C. pilosula. CpNAC92, CpbHLH128, and CpRAP2-7 responded to the low temperature, temperature difference, and iron stresses, while CpNAC100 only responded to low temperature and iron stresses. The screening and expression analysis of the specific transcription factors CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 in C. pilosula in Shanxi laid a theoretical foundation for further research on the mechanism of genuineness formation of C. pilosula.