bHLH Gene Family Research Articles

Analyses of the bHLH gene family in Populus trichocarpa reveal roles of four PtbHLHs in regulating the drought stress response

As one of the largest families of transcription factors in plants, the basic helix-loop-helix (bHLH) transcription factor family regulates a wide range of functions in plants. However, little is known about the functions of bHLH family members in Populus trichocarpa during plant growth and in the response to drought stress. In our study, 190 PtbHLH genes were identified in the P. trichocarpa genome and classified into 21 groups. Analyses of microarray datasets showed that most PtbHLH members not only have multiple functions in poplar growth, but also respond rapidly to drought stress in the leaves or roots. We selected four genes, PtbHLH35, PtbHLH121, PtbHLH137, and PtbHLH152, which were highly expressed in leaves or roots under drought stress, for functional validation analyses. These genes encoded nucleus-localized bHLH transcription factors. Transient expression of PtbHLH35, PtbHLH121, and PtbHLH152 in P. trichocarpa improved drought tolerance by activating the antioxidant system to eliminate reactive oxygen species and reduce the degree of cell damage in the leaves under drought stress. Overexpression of PtbHLH137 improved drought tolerance by activating antioxidant enzymes in the roots to eliminate reactive oxygen species, and by increasing the abscisic acid content in the roots in response to drought stress. Together, our findings provide insights into the functions of PtbHLH family members in growth and in the response to drought.

Genome-Wide Identification and Characterization of bHLH Gene Family in Hevea brasiliensis

The basic helix-loop-helix (bHLH) transcription factors play crucial roles in plant growth, development, and stress responses. However, their identification and insights into the understanding of their role in rubber trees remain largely uncovered. In this study, the bHLH gene family was explored and characterized in rubber trees using systematic bioinformatics approaches. In total, 180 bHLH genes were identified in the rubber tree genome, distributed unevenly across 18 chromosomes, and phylogenetic analysis classified these genes into 23 distinct subfamilies. Promoter regions revealed a high density of cis-elements responsive to light and hormones. Enrichment analysis indicated involvement in numerous biological processes, including growth, development, hormone responses, abiotic stress resistance, and secondary metabolite biosynthesis. Protein interaction network analysis identified extensive interactions between HbbHLH genes and other functional genes, forming key clusters related to iron homeostasis, plant growth, and stomatal development. Expression profiling of HbbHLH genes have demonstrated varied responses to endogenous and environmental changes. RT-qPCR of eleven HbbHLH genes in different tissues and under ethylene, jasmonic acid, and cold treatments revealed tissue-specific expression patterns and significant responses to these stimuli, highlighting the roles of these genes in hormone and cold stress responses. These findings establish a framework for exploring the molecular functions of bHLH transcription factors in rubber trees.

Genome-Wide Identification of the bHLH Gene Family in Callerya speciosa Reveals Its Potential Role in the Regulation of Isoflavonoid Biosynthesis.

Callerya speciosa (Champ. ex Benth.) Schot is a significant leguminous plant valued for its edible tuberous roots, which are a plentiful source of isoflavonoids. Basic helix-loop-helix (bHLH) transcription factors (TFs) have been reported to regulate secondary metabolism in plants, especially flavonoid biosynthesis. However, the bHLH genes in C. speciosa have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 CsbHLH genes were identified in the C. speciosa genome, classifying them into 23 subfamilies based on the gene structures and phylogenetic relationships. All the CsbHLH proteins contained both motifs 1 and 2, whereas motif 8 was only distributed in subgroup III (d + e). Collinearity analysis demonstrated that fragmental replications are the primary driver of CsbHLH evolution, with the majority of duplicated CsbHLH gene pairs experiencing selective pressure. Nine candidate CsbHLH genes were found to play a potential role in regulating isoflavonoid biosynthesis through a combination of gene-to-metabolite correlation analysis and weighted gene co-expression network analysis (WGCNA). Additionally, the cis-regulatory elements and response to MeJA of these nine genes were characterized and confirmed through quantitative real-time PCR (qRT-PCR) analysis. Among them, three CsbHLHs (CsbHLH9, CsbHLH89, and CsbHLH95) were selected for further investigation. Yeast two-hybrid (Y2H), dual-luciferase (LUC) assays, bimolecular fluorescence complementation (BiFC) assays, and transient transformation demonstrated that CsbHLH9 acted as a transcriptional activator through its interaction with CsMYB36 and binding to the promoters of isoflavonoid biosynthesis genes in a MeJA-induced manner, such as CsIFR2, CsI3'H2, and CsCHS4, to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of bHLH genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in C. speciosa.

Genome-Wide Identification and Role of the bHLH Gene Family in Dendrocalamus latiflorus Flowering Regulation.

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.

Genome-Wide Identification of the bHLH Gene Family in Rhododendron delavayi and Its Expression Analysis in Different Floral Tissues.

The bHLH genes play a crucial role in plant growth, development, and stress responses. However, there is currently limited research on bHLH genes in the important horticultural plant Rhododendron delavayi Franch. In this study, we conducted a comprehensive genome-wide identification and in-depth analysis of the bHLH gene family in R. delavayi using bioinformatics approaches. A total of 145 bHLH family members were identified, encoding proteins ranging from 98 to 3300 amino acids in length, with molecular weights ranging from 11.44 to 370.51 kDa and isoelectric points ranging from 4.22 to 10.80. These 145 bHLH genes were unevenly distributed across 13 chromosomes, with three bHLH genes located on contig 52. Chromosome 8 contained the highest number of bHLH family members with 19 genes, while chromosomes 9 and 13 had the lowest, with 7 genes each. Phylogenetic analysis revealed a close evolutionary relationship between bHLH genes in R. delavayi and Arabidopsis thaliana. Subcellular localization analysis indicated that most bHLH genes were located in the nucleus. Promoter analysis of R. delavayi bHLH genes revealed the presence of various cis-regulatory elements associated with light responses, methyl jasmonate responses, low-temperature responses, and coenzyme responses, suggesting that bHLH genes are involved in multiple biological processes in R. delavayi. Through transcriptome analysis, we identified three key functional genes-Rhdel02G0041700, Rhdel03G0013600, and Rhdel03G0341200-that may regulate flower color in R. delavayi. In conclusion, our study comprehensively identified and analyzed the bHLH gene family in R. delavayi and identified three bHLH genes related to flower color, providing a foundation for molecular biology research and breeding in R. delavayi.

Comparative genomics and transcriptomics analysis of the bHLH gene family indicate their roles in regulating flavonoid biosynthesis in Sophora flavescens.

The basic helix-loop-helix (bHLH) transcription factors play crucial roles in various processes, such as plant development, secondary metabolism, and response to biotic/abiotic stresses. Sophora flavescens is a widely used traditional herbal medicine in clinical practice, known for its abundant flavonoids as the main active compounds. However, there has been no comprehensive analysis of S. flavescens bHLH (SfbHLH) gene family reported currently. In this study, we identified 167 SfbHLH genes and classified them into 23 subfamilies based on comparative genomics and phylogenetic analysis. Furthermore, widespread duplications significantly contributed to the expansion of SfbHLH family. Notably, SfbHLH042 was found to occupy a central position in the bHLH protein-protein interaction network. Transcriptome analysis of four tissues (leaf, stem, root and flower) revealed that most SfbHLH genes exhibited high expression levels exclusively in specific tissues of S. flavescens. The integrated analysis of transcriptomics and metabolomics during pod development stages revealed that SfbHLH042 may play a central role in connecting SfbHLH genes, flavonoids, and key enzymes involved in the biosynthesis pathway. Moreover, we also checked the expression of 8 SfbHLH genes using RT-qPCR analysis to realize the expression profiles of these genes among various tissues at different cultivated periods and root development. Our study would aid to understand the phylogeny and expression profile of SfbHLH family genes, and provide a promising candidate gene, SfbHLH042, for regulating the biosynthesis of flavonoids in S. flavescens.

Identification of the sweet orange (Citrus sinensis) bHLH gene family and the role of CsbHLH55 and CsbHLH87 in regulating salt stress.

Salt stress is one of the primary environmental stresses limiting plant growth and production and adversely affecting the growth, development, yield, and fruit quality of Citrus sinensis. bHLH (basic helix-loop-helix) genes are involved in many bioregulatory processes in plants, including growth and development, phytohormone signaling, defense responses, and biosynthesis of specific metabolites. In this study, by bioinformatics methods, 120 CsbHLHgenes were identified, and phylogenetic analysis classified them into 18 subfamilies that were unevenly distributed on nine chromosomes. The cis-acting elements of the CsbHLH genes were mainly hormone-related cis-acting elements. Seventeen CsbHLH genes exhibited significant differences in expression under salt stress. Six CsbHLH genes with significant differences in expression were randomly selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The qRT-PCR results showed a strong correlation with the transcriptome data. Phytohormones such as jasmonic acid (JA) are essential for biotic and abiotic stress responses in plants, and CsbHLH55 and CsbHLH87 are considered candidate target genes for sweet orange MYC2 transcription factors involved in the JA signaling pathway. These genes are the main downstream effectors in the JA signaling pathway and can be activated to participate in the JA signaling pathway. Activation of the JA signaling pathway inhibits the production of reactive oxygen species and improves the salt tolerance of sweet orange plants. The CsbHLH55 and CsbHLH87 genes could be candidate genes for breeding new transgenic salt-resistant varieties of sweet orange.

Genome-wide identification of bHLH gene family and its response to cadmium stress in Populus × canescens.

The basic helix-loop-helix (bHLH) gene family is integral to various aspects of plant development and the orchestration of stress response. This study focuses on the bHLH genes within Populus × canescens, a poplar species noted for its significant tolerance to cadmium (Cd) stress. Through our comprehensive genomic analysis, we have identified and characterized 170 bHLH genes within the P. canescens genome. These genes have been systematically classified into 22 distant subfamilies based on their evolutionary relationships. A notable conservation in gene structure and motif compositions were conserved across these subfamilies. Further analysis of the promoter regions of these genes revealed an abundance of essential cis-acting element, which are associated with plant hormonal regulation, development processes, and stress response pathway. Utilizing quantitative PCR (qPCR), we have documented the differential regulation of PcbHLHs in response to elevated Cd concentrations, with distinct expression patterns observed across various tissues. This study is poised to unravel the molecular mechanism underpinning Cd tolerance in P. canescens, offering valuable insights for the development of new cultivars with enhanced Cd accumulation capacity and tolerance. Such advancements are crucial for implementing effective phytoremediation strategies to mitigate soil pollution caused by Cd.

Genome-wide identification of bHLH gene family and screening of candidate gene in response to salt stress in kiwifruit

As the second largest family of transcription factors in plants, bHLH is widely involved in the growth and development of organisms and their response to stress. In the past, there was no systematic division of this gene family in kiwifruit. In this study, we identified 206 AcbHLH genes, divided into 22 subfamilies, and analyzed chromosomal localization, physicochemical properties, conserved domains, and intron/exon composition. In addition, the repeat events of the AcbHLH gene in kiwifruit and the evolutionary relationship with Arabidopsis thaliana and Oryza sativa were further analyzed. We analyzed the expression levels of the AcbHLH genes under biotic and abiotic stresses (heat, low temperature, drought, and Bacillus anthracis infection). In addition, we also investigated the biological function of AcbHLH146 under salt stress using a VIGS-mediated gene silencing method. In the AcbHLH146-VIGS line, the content of H2O2 and O2- was lower and the damage was less. SOD, POD, and CAT enzyme activities are higher. These results suggest that AcbHLH146 may play a negative moderating role in the response of kiwifruit to salt stress. Our findings provide new insights into the potential role of AcbHLH146 under abiotic stress, and provide a theoretical basis for the molecular mechanism of Kiwifruit adaptation to salt stress.

Exploring the guardian of abiotic stress: Genome-wide identification of the basic helix-loop-helix transcription factor family in Juglans mandshurica

The Basic helix-loop-helix (bHLH) transcription factors are wildly involved in many physiological processes in plants, such as tissue/organ development, metabolite synthesis, signal transduction and abiotic stress response. However, the structure, characteristics, and functions of bHLH genes remain unknown in Juglans mandshurica. This study identified 118 bHLH gene family members using a genome-wide database and classified into 16 subfamilies. Gene structure and conserved motifs analysis revealed that the protein structure of each JmbHLH subfamily was relatively conserved. Four pairs of tandem replication genes (JmbHLH015-JmbHLH016, JmbHLH022-JmbHLH023-JmbHLH024, JmbHLH092-JmbHLH093, and JmbHLH108-JmbHLH109) were identified, which could explain the functional diversity of JmbHLH genes. The Ka/Ks values of the intraspecific collinear gene pairs were less than 1, indicating that the genes were relatively conserved during evolution. Protein interaction network analysis predicted the key genes in the JmbHLH functional network. JmbHLH genes that regulate tissues/organs development and light stress were screened using transcriptomic data, the expression pattern of JmbHLH genes differed significantly. Additionally, twelve JmbHLH genes were expressed differently under drought, salt, and cadmium stress, demonstrating that these genes may have multiple biological functions. The results provide new insights into the characteristics and regulatory functions of JmbHLH genes in J. mandshurica.

Genome-wide characterization of the bHLH gene family in Gynostemma pentaphyllum reveals its potential role in the regulation of gypenoside biosynthesis

BackgroundGynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated.ResultsThis study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees.ConclusionsIn conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

Systematic identification and expression analysis of bHLH gene family reveal their relevance to abiotic stress response and anthocyanin biosynthesis in sweetpotato

BackgroundbHLH 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.ResultsHerein, 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.ConclusionsThese results shed light on the promising functions of sweetpotato IbbHLHs in abiotic stress response and anthocyanin biosynthesis.

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.

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 ofbHLHgenes involved in fruit development and ripening of cultivated octoploid strawberry

AbstractObjectivesThe 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 MethodsThe 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.ResultsIn 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.ConclusionsThe findings of this study improve our understanding of the regulation mediated by bHLH TFs during strawberry fruit growth and maturation.

Overexpression of PavbHLH28 from Prunus avium enhances tolerance to cold stress in transgenic Arabidopsis

BackgroundThe 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.ResultsIn 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.ConclusionsOverall, PavbHLH28 enhanced the cold resistance of transgenic plants through a CBF-independent pathway, which may be partly related to ROS scavenging.

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.

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.