Genome-Wide Identifification and Characterization of RsbHLH Transcription Factors Involved in Flower development of R. simsii

Genome-Wide Identifification and Characterization of RsbHLH Transcription Factors Involved in Flower development of R. simsii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Identifification and Characterization of RsbHLH Transcription Factors Involved in Flower development of R. simsii Shenghui Tu, Xuechun zhao, Xiaojing wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4524787/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Basic helix-loop-helix (bHLH) gene family, as one of the largest transcription factor families in plants, played essential roles in regulating plant growth and development as well as in response to various environmental stress. However, the bHLH gene family in R. simsii has not yet been reported. In this study, 116 RsbHLH genes were identified in the R. simsii genome, which were divided into 13 groups based on the sequence similarity and phylogenetic relationships. Analysis of gene/protein structure, chromosome location, cis -acting elements and synteny of the RsbHLH family genes were conducted using the bioinformatic methods. Gene duplication analysis showed that 99 RsbHLH genes were expanded and retained after dispersed and WGD/segmental duplication events. Investigation of cis -acting elements within promoters of RsbHLHs indicated that many RsbHLH genes might be involved in regulating the diverse physiological processes in R. simsii. The expression patterns of RsbHLHs in different tissues and in different stages of flower development were investigated based on the RNA-seq data. Further analysis revealed that 13 candidate RsbHLH genes might be involved in regulating the flower development of R. simsii. In addition, the results of qRT-PCR showed that five RsbHLH genes ( RsbHLH033 , RsbHLH016 , RsbHLH048 , RsbHLH114 , and RsbHLH115 ) may be involved in positively and negatively regulating the high- and low-temperature stress. These results provide a basis for the functional characterization of RsbHLH genes and investigations on the molecular mechanisms of flower development of R. simsii . R. simsii Flower development RsbHLH genes Phylogenetic analysis Structure analysis Expression patterns Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Transcription factors (TFs), as the key transcriptional regulation factors in Eukaryote, control the expression of specific genes by binding specific DNA sequences[ 1 ]. The Basic helix-loop-helix (bHLH) gene family, as one of the largest TF families, plays important roles in regulating plant growth and development as well as in response to various environmental stress[ 2 – 4 ]. The bHLH protein contains the conserved bHLH domain of approximately 60 amino acids, which has two functionally regions: a basic region and a HLH region[ 5 ]. The basic region of approximately 10–15 amino acids was located at the N-terminus, which is used for recognizing and binding to the specific E-box motif (CANNTG)[ 6 ]. The HLH domain of ~ 40 amino acids located at the C-terminus acts as a dimerization domain, which is used for promoting the dimerization between proteins[ 7 ]. With the sequencing and release of plant genomes, the bHLH gene family has been widely identified and characterized in many plants, such as Arabidopsis , Pyrus ussuriensis , grape, potato, apple, mango, cabbage, and cotton[ 8 – 15 ]. Numerous studies have revealed that the bHLH family genes not only play important roles in regulating plants growth and metabolism such as light signal transduction, photomorphogenesis, and secondary metabolism, but also participate in responding to various stresses[ 4 , 10 , 16 – 19 ]. For example, GhPAS1, encoding a bHLH transcription factor, was involved in regulates plant development and architecture in cotton through mediating BR signaling[ 20 ]. Makkena and Lamb (2013) reported that the Arabidopsis SPT gene encoding a bHLH transcription factor participated in regulating root growth by controlling the size of the root meristem[ 21 ]. The tomato bHLH transcription factor (SlPRE2) was involved in regulating the fruit development by responding to gibberellin signals, and a rice bHLH transcription factor (DTD) functioned coordinately with TDR to control tapetum function and pollen development[ 22 ]. Moreover, the bHLH family genes play important roles in regulating the biosynthesis of various secondary metabolites. In Actinidia chinensis , AcbHLH42 interacting with AcMYB123 can specifically regulate the biosynthesis of anthocyanins in the endocarp of kiwifruit by binding and activating the promoters of AcF3GT1 and AcANS genes[ 23 ]. Li et al. (2017) reported that the DhbHLH1 promoted the accumulation of anthocyanins in lip tissue of Dendrobium hybrid orchids [ 24 ]. In addition, the bHLH transcription factors are involved in response to various environmental stresses. In apple, MdbHLH104 regulates activity of the plasma membrane H + -ATPase by the MdAHA8 gene, thereby enhancing the tolerance of apple to Fe deficiency[ 25 ]. Overexpression of SlICE1 (a MYC-type bHLH transcription factor) in transgenic tomato increased the expression of cold-responsive genes and enhanced cold tolerance in tomatoes[ 26 ]. The Arabidopsis AtbHLH92 transcription factor was involved in plant response to osmotic stresses[ 27 ], and AtbHLH17 (AtAIB) had been reported to confer the drought tolerance of transgenic plants via regulating of ABA signaling[ 28 ]. Rhododendron simsii , also called the Indian Azalea, belongs to the Rhododendron genus of the Ericaceae family, wihich is a valuable horticultural and medicinal plant species due to the flowers with brilliant red corollas. However, several questions are still needed to be explored. Are there bHLH transcription factors involved in regulating flower development of R. simsii ? Which bHLH transcription factors are involved in regulating anthocyanin biosynthesis in R. simsii flower. What’s the difference among the different RsbHLH transcription factors involving the anthocyanin biosynthesis in R. simsii ? The genome-wide identification and characterization of RsbHLH family genes can help us answers these questions. In the present study, we comprehensively evaluated the RsbHLH gene family and performed phylogenetic analysis, gene/protein structure analysis, gene duplication, and chromosome location. Moreover, the expression pattern of RsbHLH genes in different tissue and the different stages of flower development was evaluated with the released RNA-seq data. The expression patterns of candidate RsbHLH genes associated with flower development in R. simsii under extreme temperature treatment was further investigated. Our study provides a reference for further functional studies of the RsbHLH family and offers the targeted gene resources for cultivating superior R. simsii ornamental varieties via genetic engineering techniques. Methods Plant Materials In the present study, two-year-old R. simsii seedlings were grown in a single growth chamber, which was programmed a photoperiod of 12 h of light at a temperature of 22°C, followed by 12 h of darkness at 18°C, relative humidity 70%, and light intensity 4000 Lux. 12 R. simsii seedlings were moved into the incubator at 4.0 ± 0.5°C for 0, 3, and 6 d for cold stress treatment, while another 12 seedlings were moved into the incubator at 40 ± 0.5°C for 0, 3, and 6 d for heat stress treatment. The leaves were quickly cut into pieces, frozen in liquid nitrogen, and then stored at − 80°C. Identification of RsbHLH family genes in R. simsii The genome sequences and corresponding annotations of three Rhododendron species ( R. simsii , R. irroratum , and R. williamsianum ) were obtained from the Ericaceae Genome Resource database (TEGR, http://www.tegr.com.cn ). The HMM file of bHLH domain (PF00010) downloaded from the Pfam database was applied for identifying the RsbHLH family genes from the genomes of three Rhododendron species through the HMMER software. Furthermore, the redundant sequences located at the same chromosome and short proteins (less than 100 aa) were removed based on the physical localizations. We further verify the presence of core domain for all candidate proteins using the SMART and Pfam databases[ 29 , 30 ]. Finally, the identified proteins with the conserved domain (bHLH) were regarded as the RsbHLH family genes and used in this study. Sequence features, subcellular localization prodiction and structural analysis of RsbHLH genes in R. simsii The ProtParam online tool ( https://web.expasy.org/protparam/ ) was used to analyze the sequence features of RsbHLH family genes in R. simsii , including the number of amino acids, molecular weight, isoelectric point, instability index, aliphatic index, and hydrophilicity. Moreover, the subcellular localization of RsbHLHs was predicted using the Protein Subcellular Localization Prediction Tool[ 31 ] (PSORT, https://www.genscript.com/psort.html ). The gene structure of RsbHLH genes was graphically displayed by the GSDS 2.0 based on the R. simsii genome annotation. The MEME software ( http://meme-suite.org/tools/meme ) was applied for identification the conserved motifs of RsbHLH proteins[ 32 ]. The parameters were set as follows: maximum numbers of motifs, 20; minimum motif width, 6 bp; maximum motif width, 50 bp. The RsbHLH family genes were renamed from RsbHLH001 to RsbHLH116 based on based on their chromosomal distribution, Phylogenetic analysis of RsbHLH family genes in R. simsii The bHLH protein sequences of four plant species, including RsbHLH, RibHLH, RwbHLH, and AtbHLH proteins from R. simsii , R. irroratum , R. williamsianum , and Arabidopsis , were aligned using the clustal W algorithm with default parameters in MEGA 11 software[ 33 ]. The alignment results were used to construct a phylogenetic tree using the neighbor-joining (NJ) method, and the reliability of the tree nodes was evaluated using 1000 bootstrap replicates. The ChiPlot online tool ( https://www.chiplot.online/ ) was used to draw the phylogenetic tree, and the RdbHLH gene family were furtehr classified into different groups based on the sequence suimilarity and identified Arabidopsis bHLH genes. Identification of the cis -regulatory elements in promoter of RsbHLH genes The 1500 bp upstream of the transcription start site (TSS) of each RsbHLH gene was extracted and used to identify the conserved cis -regulatory elements (CRE) in the promoter region of RsbHLH genes using the PlantCARE tool[ 34 ]. Chromosomal localization, gene duplication, and synteny analysis of RsbHLH genes in R. simsii The genome annotation information was used to extract the location information of RsbHLH genes on chromosomes, and the chromosomal distribution of RsbHLH genes was graphically visualized through MapChart software. Gene duplication analysis for R. simsii was performed using the MCScanX software[ 35 ]. Firstly, the potential homologous gene pairs across the R. simsii were detected using BLASTp software with e-value less than 1e − 5 . The MCScanX software was applied for identifying syntenic blocks based on the identified homologous gene pairs. Five genome duplication models, including whole-genome duplication (WGD)/segmental duplication, tandem duplication, dispersed, and proximal, were detected using the MCScanX program. The homologous gene pairs in syntenic blocks were used to estimate the Ka and Ks values using PAML package. In addition, the MCScanX program was used to perform collinearity analysis across four plant species ( R. simsii , Arabidopsis , R. irroratum , and R. williamsianum ) to identified the orthologous gene pairs. The Dual Systeny Plot program was used to visualize the evolutionary relationships of bHLH genes between R. simsii and the other three plant species. GO (Gene Ontology) enrichment analysis GO annotations of the RsbHLH family genes were performed by the GOATOOLS software ( http://github.com/tanghaibao/GOatools )[ 36 ]. Fisher’s exact test was applied for performing the GO term enrichment analysis for RsbHLHs . The Bonferroni multiple testing correction was used to minimize false positives, and functions were considered to be significantly enriched when their Bonferroni-corrected P-values (Padjust) were < 0.05. Expression analyses of RsbHLH genes in different organs and/or different stages of flower development of R. simsii RNA-Seq data of different organs (stem, leaf, and flower) and the different stages of flower develoment (T1-T5) of R. simsii was downloaded from the NCBI SAR database (SRP229032). After analysis and normalization, the expression patterns of RsbHLH family genes were displayed and visualized in heat maps. The transcript abundance of RsbHLH genes was calculated as fragments per kilobase of exon model per million mapped reads (FPKM). The log 2 (FPKM) from the RNA-seq data were subjected to hierarchical clustering with Cluster 3.0, and the results were graphically displayed using Java TreeView. qRT-PCR analysis of RsbHLHs The PrimerScript® RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used to reverse transcribe the RNA into cDNA. RT-qPCR was conducted with three biological replicates and three technical replicates using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). The specific primers of target genes were designed based on the full-length CDS of RsbHLH genes, and the EF1α gene was treated as reference gene (Table S1 ). Each of these biological replicates was run in three technical replicates. Relative expression was calculated using the 2 –∆∆CT method. Prediction of protein interaction network The OrthoVenn2 online tool ( https://orthovenn2.bioinfotoolkits.net/home ) was used to identifiy the orthologous gene pairs.between R. simsii and Arabidopsis . After screening, we obtained the homologous relationship pairs between RsbHLH genes and Arabidopsis genes. We then used the STRING database ( https://string-db.org/ ) to analyze the protein interaction network relationships between Arabidopsis genes in the homologous relationship pairs, and mapped out the protein interaction network relationships between RsbHLH genes (the minimum required interaction score was set to 0.650, and the maximum number of interactors of the 1st shell to show was set to 12)[ 37 ]. Results Identification of the RsbHLH gene family in R. simsii A total of 116 nonredundant RsbHLH family genes with conserved core bHLH domain were identified in the R. simsii genome (Table S2 ). The length of RsbHLH protein sequences ranged from 135 (RsbHLH016) to 1174 (RsbHLH034) amino acids, with an average of 391 amino acids. The molecular weights of the identified bHLH proteins ranged from 14.85 kDa to 130.67 kDa. The predicted isoelectric points of bHLH proteins ranged from 4.67 to 9.51, with an average of 6.65. Among them, the instability indexes of 111 RsbHLH family proteins were greater than 40, indicating that most of RsbHLH family proteins are unstable. The aliphatic indexes of RsbHLH proteins ranged from 46.34 to 97.40. The hydrophobicity indexes of RsbHLHs are negative, indicating that the RsbHLH family proteins are commonly used as hydrophilic proteins. Moreover, the prediction of subcellular localization for the RsbHLH family proteins was also performed in this study (Fig. 1 and Table S2 ). Our result showed that 90.51% (105/116) of RdbHLH family proteins was localized to the nucleus. In addition, 111 RdbHLH genes in the RdbHLH gene family were distributed on 13 R. simsii chromosomes, and the remaining five RsbHLH genes were anchored in the scaffolds (Fig. 1 and Table S3 ). The distribution of RsbHLH genes on the 13 chromosomes was uneven, with most RsbHLH genes located on chromosome 8 (14 genes, 12.07%), chromosomes 3 (11 genes, 9.48%) and 7(11 genes, 9.48%). In contrast, there were only 3 RsbHLH genes (2.59%) on chromosome 4. The percentage of RsbHLH genes on each chromosome ranged from 0.12% on chromosome 4 to 0.56% on chromosome 8. Phylogenetic analysis of the bHLH gene family among R. simsii , R. irroratum , R. williamsianum , and Arabidopsis To explore the evolutionary relationships of RsbHLH family genes, we constructed an unroot phylogenetic tree of bHLH family genes from R. simsii , R. irroratum , R. williamsianum , and Arabidopsis using the neighbor-joining (NJ) method in MEGA 11.0 software with default parameters. The RsbHLH family members of R. simsii were classified into 13 groups (I to XIII) and 25 subgroups based on the topology of phylogenetic tree and the classification of Arabidopsis AtbHLH proteins (Fig. 2 and Table S4 ). The number of RsbHLH genes varies among the different groups. Group Ⅷ, as the largest group among 13 groups, contained 21 RsbHLH genes, which was further classified into 5 subgroups (subgroup a-e). Groups I and IV contained 19 and 15 RsbHLH genes, respectively, which were divided into two subgroups (Ⅰa andⅠb) and four subgroups (IVa, IVb, IVc, and IVd). There is no RsbHLH genes distributed in group VI. Moreover, the groups VII and XII contained much more bHLH genes in Arabidopsis than those in R. simsii , R. irroratum , and R. williamsianum . Moreover, most groups were further clustered into two or more subgroups (Fig. 2 ). Subgroup IIId and VIIIb contained the bHLH gene clusters from R. simsii , indicating that the species-specific expansion of these genes occurred in R. simsii after the divergence of core eudicots. Interestingly, some subgroups only contained AtbHLH genes without RsbHLH genes or included RsbHLH genes from the Rhododendron species without Arabidopsis AtbHLH genes. Group VI only contained the Aranidopsis bHLH genes, and didn’t include the bHLH gene from the Rhododendron species. The group XIII only contained the bHLH genes from Rhododendron species, and didn’t contain the Arabidopsis AtbHLH genes. These results indicated that bHLH genes could have been either acquired and lost in Arabidopsis or the Rhododendron species. Gene structure and protein motif analysis of the RsbHLH gene family in R. simsii To further investigate the gene structural diversity and protein motif composition of RsbHLH family members, the exon-intron distribution was first analyzed and visualized using the Gene Structure Display Server 2.0 (GSDS, Fig. 3 a). Gene structure analysis showed that a total of 116 RsbHLH genes had exons varying from 1 to 15. Thirteen RsbHLH genes (11.21%) had only one exon or lacked intron, including RsbHLH74 , RsbHLH102 , RsbHLH050 , RsbHLH090 , RsbHLH032 , RsbHLH030 , RsbHLH040 , RsbHLH010 , RsbHLH053 , RsbHLH004 , RsbHLH059 , RsbHLH114 , and RsbHLH028. The 72.4% of RsbHLH family members (84 RsbHLH genes) contained 2 to 8 introns. Interestingly, the RsbHLH19 gene possessed 15 exons and 14 introns, which was the gene with the least number of introns among RsbHLH family members. In addition, gene structure analysis showed that most RsbHLH genes in the same subgroup possessed the similar intron/exon distribution, including the number and length of introns. For example, all RsbHLH genes in subgroup IV possessed more than 6 exons, and the members of subgroup XVI only contain 3 exons. Thus, the results of gene structure analysis demonstrated that RsbHLH genes in the same subgroup had similar gene structures, and further verified the reliability of the topology of phylogenetic tree of the RsbHLH family genes (Fig. 2 ). A total of 15 putative conserved motifs were identified in RsbHLH proteins using the MEME suite, namely Motifs 1–15 (Fig. 3 b and Table S5 ). Motif 1 and motif 2, located in bHLH domains, were distributed in almost all RsbHLH proteins. Six RsbHLH proteins, including RsbHLH071, RsbHLH078, RsbHLH002, RsbHLH011, RsbHLH062, and RsbHLH054, only contained one of motif 1 and motif 2. Moreover, the distribution of conserved motifs of all RsbHLH proteins in the same subgroup were similar. For example, motif 15, motif 4, motif 10, and motifs 7 were specifically distributed in most members of subgroup X, subgroup XVII, subgroup XVI, and subgroup XVII, which played important roles in biological regulatory functions of these subgroups genes. In addition, some motifs existed in members of multiple subgroups. The motif 8 was commonly distributed in four subgroups: subgroups V-VII and XI, and motif 3 was widely distributed in six subgroups (IV, VIII, IX, X, XI, and XIV). These results suggested that the RsbHLH genes in these subgroups might possess similar biological function. Cis -elements analysis of RsbHLH gene promoters Cis -regulatory elements, which are usually restricted to 5′ upstream areas of genes, are the binding sites of transcriptional factors. In the present study, the 1500 bp upstream regions of the transcription start site (TSS) of RsbHLH genes were applied for identifying cis -regulatory elements and investigate gene regulation patterns with PlantCARE (Fig. 4 and Table S6 ). A total of 37 functionally annotated cis -acting elements in the promoter of most RsbHLH genes, which were further clustered into four categories: hormone-responsive elements (TATC-box, TCA-element, CGTCA-motif, TGACG-motif, GARE-motif, P-box, GA-motif, TCCC-motif, and AuxRR-core), stress-responsive elements (ABRE, MBS, WUN-motif, LTR, ARE, and TC-rich repeats), light-responsive elements (G-box, CAT-box, GT1-box, I-box, ATC-motif, Box4, TCT-motif, AE-box, MRE, ACE, SP1, ATCT-motif, L-box, AAAC-motif, and 3-AF1 bind site), and the development-related cis -elements (O 2 -site, circadian, AT-rich element, GCN4-motif, AACA-motif, HD-Zip1, and MSA-LIKE). This result further suggested that the RsbHLH family genes played important roles in regulating the plant growth and development as well as in response to various environmental stress. GO enrichment analysis of the RsbHLH genes in R. simsii The bHLH transcription factors play pivotal roles in numerous biological process, especially in the regulation of secondary metabolism in plants. To explore the biological functions of the RsbHLH famiy genes in R. simsii , we conducted GO annotation and enrichment analysis of 116 RsbHLH genes (Fig. 5 and Table S7 ). A total of eighty-seven Go term were enriched, eleven molecular functions, two cellular components, and seventy-four biological processes in GO terms were enriched in the PdbHLH genes relative to the complete GO database. In the biological process category, RsbHLH genes were mainly enriched in positive regulation of transcription, DNA-templated (n = 21), positive regulation of RNA biosynthetic process (n = 21), positive regulation of nucleic acid-templated transcription (n = 21), positive regulation of RNA metabolic process (n = 21), positive regulation of transcription by RNA polymerase II(13), regulation of transcription by RNA polymerase II(18) and transcription by RNA polymerase II(18) and so on. In the cellular component category, the genes were enriched in RNA polymerase II transcription regulator complex (4) and transcription regulator complex (4). In the molecular function category, the genes were enriched in transcription regulatory region sequence-specific DNA binding (21), core promoter sequence-specific DNA binding (13), sequence-specific double-stranded DNA binding (21), double-stranded DNA binding (21), DNA-binding transcription activator activity(13), DNA-binding transcription activator activity, RNA polymerase II-specific(13) and regulatory region nucleic acid binding(22) and so on. GO enrichment results suggested that RsbHLH transcription factors mainly involved in transcription, RNA metabolic process, DNA-bing and floral organ development. Gene duplication, Ka/Ks, and Collinearity Analysis Gene duplication events are closely related to the plant evolution process and contribute to the expansion of the gene family. In the present study, the evolutionary patterns of the RsbHLH genes were surveyed using MCScanX software. Amazingly, 42 (36.21%) and 57 (49.14%) of the RsbHLH family genes in R. simsii were duplicated and retained from dispersed and whole genome duplication (WGD)/segmental duplication, respectively. Only seven RsbHLH genes originated from tandem duplication. To further investigate the potential role of segmental duplication in the expansion of the RsbHLH family genesly, we conducted the collinearity analysis of the RsbHLH family genes in R. simsii genome using the all- vs. -all local BLASTP algorithm-based search. A total of 28 segmental duplication gene pairs (52 RsbHLH genes) were detected in the R. simsii genome, which accounted for 45.69% of WGD-type RsbHLH genes (Fig. 6 a and Table S8 ). The RsbHLH genes in each synteny block were distributed on all chromosomes. In addition, the ratio of non-synonymous to synonymous substitutions (Ka/Ks) was evaluated using the PAML package (Table S8 ). The Ka/Ks ratio 1 (Hurst, 2002). Our results showed that the Ka/Ks ratios of 28 segmental duplication gene pairs were less than one, suggesting that the the 52 RsbHLH genes were under negative selection (Fig. 8 a and Table S8 ). The Ks values were commonly used to calculate the the occurrence time of segmental duplication events. The segmental duplication events of 28 gene pairs in R. simsii genome occurred from 25.35 (Ks = 0.7304) to122.77 mya (Ks = 3.683). The identification of orthology is central to comparative genomics, which has been employed in many studies using synteny analysis. According to the identified synteny relationships, we identified orthologous pairs of bHLH genes among four representative plants (Fig. 6 b, Table S9 ). There were 69 orthologous bHLH gene pairs between R. simsii and Arabidopsis , 130 orthologous bHLH gene pairs between R. simsii and R. irroratum , and 120 orthologous bHLH gene pairs between R. simsii and R. williamsianum. The number of orthologous events of RsbHLH - RibHLH was much greater than that of RsbHLH-RwbHLH . In addition, the Ka, Ks, and Ka/Ks values of orthologous RsbHLH gene pairs among four species were also investigated(Figure 7 ). This result showed that the Ka/Ks ratio of orthologous bHLH gene pairs among four species were less than 1, suggesting that the orthologous bHLH genes in four species were subjected to purifying selection during evolution. The Ka/Ks ratio of orthologous bHLH gene pairs in R. simsii and Arabidopsis was significantly lower than those in R. simsii and R. irroratum and R. simsii and R. williamsianum. This result further indicated that the specific expansion and sequence divergence occurred in Rhododendron species. The expression pattern of the RsbHLH family genes in different organs of R. simsii To investigate the potential regulatory mechanisms of the RsbHLH family genes in R. simsii , the expression patterns of RsbHLH genes in three organs (stems, leaves, and flowers) of R. simsii were evaluated using the released RNA-seq data (Table S10 ). A total of 49 RsbHLH genes, 64 RsbHLH genes, and 58 RsbHLH genes were highly expressed in flowers, leaves, and stems, respectively (Fig. 8 a). There are 46 RsbHLH genes simultaneously expressed in flowers and leaves, 45 simultaneously expressed in flowers and stems, and 54 simultaneously expressed in leaves and stems, respectively. The 46 RsbHLH genes are commonly expressed in all three organs. In addition, the expression levels of 23 RsbHLH genes in leaves and stems were higher than those in flowers, suggesting that these RsbHLH genes played important roles in regulating the growth and development of vegetative organs. The expression levels of 13 RsbHLH genes ( RsbHLH096 , RsbHLH075 , RsbHLH023 , RsbHLH059 , RsbHLH115 , RsbHLH004 , RsbHLH114 , RsbHLH077 , RsbHLH012 , RsbHLH072 , RsbHLH109 , RsbHLH013 , and RsbHLH037 ) in flowers were higher than those in leaves and stems, suggesting that these RsbHLH genes played essential roles in regulating the flower development. To further explore the potential regulatory mechanisms of the RsbHLH genes in flower development of R. simsii , the expression levels of RsbHLH genes in five different stages of flower development of R. simsii (T1-T5) were evaluated using the released RNA-seq data (Table S11 ). As shown in Fig. 8 b, a total of 64 RsbHLH genes were differentially expressed in five different stages of flower development, which can be divided into four types: Ⅰ) The expression levels of 21 RsbHLH genes in early stage (TI-T3) of flower development were higher than those in late stage (T4།T5) of flower development. Ⅱ) The expression levels of 13 RsbHLH genes in early stage (TI།T3) of flower development were less than those in late stage (T4།T5) of flower development. Type Ⅲ included 13 RsbHLH genes, which increased in the early stage and then sharply decreased in late stage of flower development. Type Ⅳ contained 2 RsbHLH genes, which decreased in the early stage and then increased the late stage of flower development. Protein interaction prediction Different bHLH proteins can generate homodimers or heterodimers with other proteins [ 9 , 10 ], which is a prerequisite for DNA recognition and DNA-binding specificity. In this study, we performed protein interaction networks of the RsbHLH family genes (Fig. 9 and Table S12 ). Our result showed that 32 RsbHLH genes were used to construct the protein interaction networks, 26 RsbHLH proteins of which were interacting with more than one RsbHLH protein. One RsbHLH protein (PsbHLH037) interacted with four RsbHLH proteins (RsbHLH045, RsbHLH057, RsbHLH097, and RsbHLH114), and RsbHLH045 protein interacts with RsbHLH066 and RsbHLH037, respectively. Moreover, 11 RsbHLH proteins could interact with genes from other families. For example, RsbHLH059 protein can interact with TIFY7 (Rs09G0091800)/JAZ1 (Rs05G0004700), and RsbHLH094 protein can interact with CRY2 (Rs02G0037100)/PHYB (Rs04G0239100). RsbHLH017 proteins interacts with six proteins such as JAZ1 (Rs05G0004700), MYB75 (Rs08G0087700), MYB0 (Rs08G0239000), MYB66 (Rs08G0171500), TTG1 (Rs09G0209900), and TT2 (Rs12G0209800). In particular, RsbHLH017 protein interacts with MYB75/MYB0/MYB66 and TTG1 to generate MBW complexes. In addition, five RsbHLH proteins (RsbHLH066, RsbHLH015, RsbHLH067, RsbHLH115, and RsbHLH053) can interact with PHYA (Rs11G0107300), JAZ1, JAZ1, TTG1, and TIFY7, respectively. Functional annotations further revealed that the orthologous genes of these RsbHLH genes had been reported to be involved in regulating the biosynthesis of flavonoids and anthocyanin biosynthesis and in responose to various abiotic stresses. Thus, it is very interesting to explore the regulation of bHLH proteins in diverse biological processes of R. simsii . Validation of RNA-Seq experiment by RT-qPCR To further assess the reliability and vilidity of RNA-seq experiments, RT-qPCR was conducted on 12 RsbHLH genes with high expression levels in different stages of flower development. Comparative analysis of expression patterns of these RsbHLH genes were performed in four different stages of flower development of R. simsii , and the result showed that the expression trends of 12 RsbHLH genes in RT-qPCR experiment were consistent with the RNA-Seq data (Fig. 10 ). Expression patterns of RsbHLH genes in R. simsii during extreme temperature treatments Temperature in particular has been shown to have a tremendous effect on the timing of flowering: the vernalization response and thermosensory pathway. In the present study, we further investigated the expression patterns of 12 candidate RsbHLH genes in R. simsii treated with high and low temperature (Fig. 11 and Fig. 12 ). Under high temperature treatment, nine RsbHLH genes, including RsbHLH033 , RsbHLH016 , RsbHLH048 , RsbHLH053 , RsbHLH058 , RsbHLH059 , RsbHLH107 , RsbHLH114 , and RsbHLH115 , were significantly upregulated following 3d after high temperature treatment, suggesting that these genes might play a positive role in response to heat stress. Only one RsbHLH gene ( RsbHLH075 ) were significantly downregulated following 3d after high temperature treatment, suggesting that this gene might play a negative role in response to heat stress. In addition, the expression levels of five RsbHLH genes ( RsbHLH053 , RsbHLH058 , RsbHLH059 , RsbHLH107 , and RsbHLH109 ) were significantly increased after 3-day low-temperature treatment in comparison with the control group, suggesting that these genes play positive roles in response to low temperature. Six RsbHLH genes ( RsbHLH033 , RsbHLH016 , RsbHLH048 , RsbHLH075 , RsbHLH114 , and RsbHLH115 ) were decreased following 3d after low temperature treatment, indicating that these genes might be involved in negative regulation response to low temperature. Interestingly, five RsbHLH genes ( RsbHLH033 , RsbHLH016 , RsbHLH048 , RsbHLH114 , and RsbHLH115 ) exhibited opposite expression patterns after high and low temperature treatment, while the other five RsbHLH genes ( RsbHLH053 , RsbHLH058 , RsbHLH059 , RsbHLH075 , and RsbHLH107 ) displayed the same expression patterns. Discussion Basic helix-loop-helix (bHLH) gene family, as one of the largest transcription factor families in plants, played essential roles in regulating plant growth and development as well as in response to various environmental stress[ 4 , 18 , 19 , 38 , 39 ]. However, comprehensive identification of the bHLH family genes in R. simsii has not been conducted. In the present study, we performed genome-wide characterization of the RsbHLH family genes in R. simsii . A total of 116 RsbHLH genes were identified and divided into 13 groups based on the phylogenetic tree topology, gene/protein structure, and the identified AtbHLH genes. Moreover, the numerical distribution of the bHLH family genes from the lower plants to flowering plants were investigated, found that the genome of flowering plants comprises an very large number of bHLH genes, while the genome of lower plants contain only a few bHLH genes. This result indicated that the bHLH family genes underwent drastic expansion during plant evolution. Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms, which mainly included five types such as WGD (whole-genome duplication)/segmental duplication, tandem duplication, dispersed duplication, proximal, and singleton[ 40 ]. Different duplication events contributed differently to gene expansion in different families. Segmental and tandem duplication played important roles in the expansion of WRKY and APETALA2/ethylene-responsive factor (AP2/ERF) family genes in plants[ 41 – 43 ]. Transposed duplication contributed to the expansion of MADS and NBS-LRR family genes[ 44 ]. Our results revealed that more than 85% of the RsbHLH family genes in R. simsii were duplicated and retained from dispersed and WGD/segmental duplication events, respectively, indicating that the WGD/segmental and dispersed duplication events played major roles in the expansion of RsbHLH family genes. Gene expansion of the RsbHLH family genes caused rapid functional and sequence differentiation of RsbHLH genes, which is accompanied by neofunctionalization and subfunctionalization as well as gene expression patterns. For example, two genes (RsbHLH010 and RsbHLH007) were derived and retained from segmental duplication. The RdbHLH007 was highly expressed in the stems and leaves of R. simsii , while the expression levels of RsbHLH010 was not detected. The cis -acting elements distributed in the gene promoters can reveal the potential function of this gene. Our result showed that the cis -acting elements in the promoters of RsbHLH genes in R. simsii included four types of cis -elements scch as including light-, stress-, hormone-, and the development-related elements, which was consistent with the previous studies. Transcription factors can selectively recognize and bind to the upstream promoters of the essential genes associated with various biological pathways, and regulate intrinsic cellular processes, such as differentiation and development, and of the cellular response to external perturbation through signaling pathways[ 45 – 47 ]. Flower development is regulated by a variety of transcription factors[ 48 – 50 ]. The bHLH gene family, as the second largest TF family, played essential roles in regulating flower development[ 51 – 53 ]. Our results showed that 64 RsbHLH genes were highly expressed in flowers, and the expression levels of 11 RsbHLH genes in flowers were higher than those in leaves and stems. These results suggested that these 13 RsbHLH genes played essential roles in specific regulation of the flower development. Moreover, the orthologous genes of 13 RsbHLH genes ( RsbHLH096 , RsbHLH075 , RsbHLH023 , RsbHLH059 , RsbHLH115 , RsbHLH004 , RsbHLH114 , RsbHLH077 , RsbHLH012 , RsbHLH072 , RsbHLH109 , RsbHLH013 , and RsbHLH037 ) in Arabidopsis were BPE , BHLH79 , BHLH48 , JAM2 , ILR3 , MYC4 , SACL3 , BIM2 , ATMYC1 , BIM1 , BHLH103 , JAM2 , and BHLH041 , respectively. Previous studies have revealed that most of these genes in Arabidopsis are involved in flower development [ 54 – 58 ]. Szecsi et al. (2006) revealed that BIGPETAL (BPE) was involved in controlling the Arabidopsis petal size[ 54 ], and overexpression of AtbHLH48 caused early flowering under long-day conditions by directly activating the transcription of FT gene[ 55 ]. Nakata and Ohme-Takagi (2013) showed that overexpression of JAM2 and JAM3 resulted in reduced male fertility[ 56 ]. Chen et al. (2016) reported that MYC4 could interact with the MYBs to form the bHLH-MYB complex, and JAZs repress the bHLH-MYB complex to regulate JA-mediated stamen development[ 57 ]. Hu et al. (2015) reported that the hypermethylated BIM2 gene may suppress downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S[ 58 ]. Temperature in particular has been shown to have a tremendous effect on the timing of flowering: the vernalization response and thermosensory pathway[ 59 ]. The qRT-PCR results showed that five RsbHLH genes ( RsbHLH033 , RsbHLH016 , RsbHLH048 , RsbHLH114 , and RsbHLH115 ) exhibited opposite expression patterns after extreme temperature treatment, while the other five RsbHLH genes ( RsbHLH053 , RsbHLH058 , RsbHLH059 , RsbHLH075 , and RsbHLH107 ) displayed the same expression patterns. Further gene function annotation displayed that the orthologous genes of five RsHLHs ( RsbHLH115 , RsbHLH48 , RsbHLH53 , RsbHLH75, and RsbHLH58 ) in Arabidopsis were ILR3 , PIF3 , MYC2 , bHLH79 , and FBH4 , respectively. Previous studies have revealed that these five genes in Arabidopsis are involved in regulating extreme temperature stress. Conclusions In the present study, 116 RsbHLH genes were identified in R. simsii at genome level, which were classified into 13 groups (including 25 subgroups) based on the sequence similarity and phylogenetic relationships. Analysis of chromosomal distribution, gene structure, protein motif discovery, phylogenetic relationship, and cis-acting elements in the promoters of RsbHLH genes were performed. Comprehensive analysis revealed that 13 RsbHLH genes might be involved in regulating the flower development of R. simsii. The qRT-PCR results showed that servral RsbHLH genes were confirmed to involve in flower development and responsive to extreme temperature. The above results could provide a basis for the functional characterization of bHLH genes, and also provide candidate genes for the future improvement of flower development of R. simsii . Declarations Acknowledgments Not applicable. Authors’ Contributions X.Z. and X.W. designed the experiment; S.T. and X.W. conducted the experiment and analyzed the data; X.W. wrote the manuscript; X.W. and X.Z revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the National Natural Science Foundation of China Project (32260097) and the National Guidance of Local Science and Technology Development Fund of China [2023]009. This study was also supported by the Science and Technology Project of Guizhou Province, China (Qiankehe Foundation-ZK [2022]) and The Science and Technology Department of Guizhou Province (Qian Ke He Zhicheng [2021]Yiban503). Availability of data and materials Not applicable. Ethics approval and consent to participate There is no ethics approval and consent to participate in this manuscript. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Ogbourne S, Antalis TM. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem J. 1998;331(1):1–14. Zuo Z-F, Lee H-Y, Kang H-G. Basic helix-loop-helix transcription factors: Regulators for plant growth development and abiotic stress responses. Int J Mol Sci. 2023;24(2):1419. Gao M, Zhu Y, Yang J, Zhang H, Cheng C, Zhang Y, Wan R, Fei Z, Wang X. 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BMC Genomics. 2015;16:1–14. Capovilla G, Schmid M, Posé D. Control of flowering by ambient temperature. J Exp Bot. 2015;66(1):59–69. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx TableS2.docx TableS3.docx TableS4.docx TableS5.docx TableS6.docx TableS7.docx TableS8.docx TableS9.docx TableS10.docx TableS11.docx TableS12.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4524787","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315161644,"identity":"f939bbe4-ff7e-4287-8652-880a216cbada","order_by":0,"name":"Shenghui Tu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shenghui","middleName":"","lastName":"Tu","suffix":""},{"id":315161645,"identity":"2a998325-8615-4d6e-9640-6cd30cec98f1","order_by":1,"name":"Xuechun zhao","email":"","orcid":"","institution":"Guizhou university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuechun","middleName":"","lastName":"zhao","suffix":""},{"id":315161646,"identity":"1c14d066-f541-4b11-a1d8-619fe6d507b3","order_by":2,"name":"Xiaojing wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFCCAyDCRg7CYSNeS5oxKVrA4HBiA9FaDA6eMfxc8Cstfb7/GQOGD2WHGfhnN+DXItlwxlh6Zp9N7sYDZwwYZ5w7zCBx5wB+LfwMZzdI8/ak5W5s7DFg5m07zGAgkYBfCxvD2c2/eXsOpxs28xgw/yVGC9CWbdI8Pw4nyLMBtTASo0Wy4fw3a96GNMMNPGwFB3vOpfNI3CCgxeDGseTbPH9s5OX7D2988KPMWo5/BgEtDBIHGBgY24B6D0BilYeAepBnGoDEHwYG+QbCakfBKBgFo2CEAgD4j0XvapuJ+gAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou university","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"wang","suffix":""}],"badges":[],"createdAt":"2024-06-04 03:06:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4524787/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4524787/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58758579,"identity":"2e05d109-5c6e-4e90-9c68-e904886d7d1b","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1453138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRsbHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003eThe position of each \u003cem\u003eRsbHLH\u003c/em\u003e was labled on the right side of each chromosome (Chr). The size of a chromosome is indicated by its relative length.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/6a85723ef9d494d6c3488f53.png"},{"id":58758798,"identity":"3a34b650-0e6e-432b-b16f-17c9f2b2268a","added_by":"auto","created_at":"2024-06-20 18:17:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9508953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of bHLH gene family in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. simsii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. irroratum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. williamsianum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eAn unroot phylogenetic tree of bHLH family genes from \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e and \u003cem\u003eR. williamsianum\u003c/em\u003e was constructed using the neighbor-joining method in MEGA 11.0.13 software with a bootstrap test (replicated 1000 times). The bHLH gene families in \u003cem\u003eR. simsii, R. irroratum, R. williamsianum, and Arabidopsis \u003c/em\u003ewere marked by different symbols, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/609f35b53cfca7dc7d358ddd.png"},{"id":58758933,"identity":"9f5a1ac5-0408-4e0d-9943-b03ef2cea17a","added_by":"auto","created_at":"2024-06-20 18:25:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1206235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of gene structure and conserved motifs of the RsbHLH family genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. simsii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) Exon/intron distribution of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e. The exons are represented by green round-cornered rectangles. The black lines connecting two exons represent introns. (b) Distribution of conserved motifs in each RsbHLH proteins. Schematic diagram of motif distribution of RsbHLH proteins in \u003cem\u003eR. simsii\u003c/em\u003e using MEME. The relative positions of each conserved motif within the RsbHLH proteins are shown in color. The black lines represent the non-conserved sequences.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/8e680967a38bee4ab9810aff.png"},{"id":58758591,"identity":"bb389d3b-1ad7-4926-89a3-f696763dcb3a","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4106524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-element analysis of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e RsbHLH \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes from upstream 1500 bp sequence to the transcription start site. \u003c/strong\u003e(\u003cstrong\u003ea) \u003c/strong\u003eIdentification and classification of \u003cem\u003ecis-\u003c/em\u003eacting elements in the promoter regions of \u003cem\u003eRsbHLH \u003c/em\u003egenes in \u003cem\u003eR. simsii\u003c/em\u003e. (\u003cstrong\u003eb)\u003c/strong\u003e \u003cem\u003eCis\u003c/em\u003e-acting elements analysis of the promoter regions of \u003cem\u003eRsbHLH\u003c/em\u003egenes, the micro-parts in diverse colors are the sequence of the putative elements.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/0a49c21c45aa2f35cbec41b4.png"},{"id":58758586,"identity":"5e556ab4-88d9-485a-8baf-959ed1b37506","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":65073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO enrichment analysis of the RsbHLH proteins relative to the GO database.\u003c/strong\u003e The vertical axis indicates the enrichment factor, and the size of the circle indicates the number of genes annotated with a given GO term. GOATOOLS (http://github.com/tanghaibao/GOatools) was used to assign GO annotations to RsbHLHs, and Fisher’s exact test was used to identify biological functions enriched in the RsbHLHs relative to the full GO database. Visualization was performed using the Majorbio online platform (https://cloud.majorbio.com)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/96221f5cb7f955fc5bb2d245.png"},{"id":58758598,"identity":"2aab0cf6-fe2d-48f2-b9dd-6c8527a6e2b6","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6534992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization and synteny of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in Rhododendron genomes.\u003c/strong\u003e (a) Collinearity analysis of RsbHLH genes in \u0026nbsp;\u003cem\u003eR. simsii\u003c/em\u003e genome. Collinear blocks are shown with grey in \u003cem\u003eR. simsii\u003c/em\u003e genome and duplicated \u003cem\u003eRsbHLH \u003c/em\u003egene pairs are shown with orange color. (b) Synteny analyses of the \u003cem\u003ebHLH \u003c/em\u003egenes from \u003cem\u003eR.simsii\u003c/em\u003e with representative plants (\u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e and \u003cem\u003eR. williamsianum\u003c/em\u003e). The gray line represents the collinearity of the entire genome. The blue lines represents the collinearity between \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eArabidopsis bHLH\u003c/em\u003e genes; the green lines represents the collinearity between \u003cem\u003eR. simsii \u003c/em\u003eand \u003cem\u003eR. irroratum bHLH\u003c/em\u003e genes; the pink lines represents the collinearity between \u003cem\u003eR. simsii \u003c/em\u003eand \u003cem\u003eR. williamsianum bHLH\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/b8a8c701782d44fb32bb527b.png"},{"id":58758582,"identity":"935ff89c-0165-45b3-bbb2-a0e642d7d98e","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKa, Ks, and Ka/Ks distributions of orthologous \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene pairs.\u003c/strong\u003e (a) Ka, Ks, and Ka/Ks ratio of segmental duplicated gene pairs; (b) Ka, Ks, and Ka/Ks ratio of orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs between \u003cem\u003eR. simsii\u003c/em\u003e an\u003cem\u003ed Arabidopsis\u003c/em\u003e; (c) Ka, Ks, and Ka/Ks ratio of orthologous \u003cem\u003ebHLH\u003c/em\u003e HSP gene pairs between\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eR. simsii\u003c/em\u003e and\u003cem\u003eR. irroratum\u003c/em\u003e; (d) Ka, Ks, and Ka/Ks ratio of orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs between \u003cem\u003eR. simsii \u003c/em\u003eand \u003cem\u003eR. williamsianum\u003c/em\u003e. The box plots are exhibiting the distributions of Ka, Ks, and Ka/Ks values among paralogs and orthologs. The small square and the line in the box represent average and median values of the Ka, Ks, and Ka/Ks values, respectively.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/1e27a75f021459992f97c938.png"},{"id":58758584,"identity":"81f1c9ec-b2e3-44f2-8b0d-950f9295e598","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":849645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of the RsbHLH family genes in different tissues and different stages of flower development.\u003c/strong\u003e (a) Expression profle of the RsbHLH family genes in different tissues of \u003cem\u003eR.simsii\u003c/em\u003e. (b) The expression profle of the RsbHLH family genes in different stages of \u003cem\u003eR.simsii\u003c/em\u003e flower development (T1-T5). The RPKM values obtained were normalized by log\u003csub\u003e2\u003c/sub\u003e transformation and the heatmap constructed using the MeV software. The block colors represent the expression levels, like green, yellow and red correspond to low, medium and high expression values.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/b97741b0828721748048abf2.png"},{"id":58758599,"identity":"5711dc2e-1a6a-4913-aced-bb09ea9f72ad","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":515159,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction and analysis of the protein interaction network between\u003cem\u003e RsbHLH\u003c/em\u003e genes and other closely associated genes. The online tool STRING was used to predict the network.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/a08761d190660da466bdf142.png"},{"id":58758600,"identity":"e3944f7d-c356-4056-a647-168d1e61326f","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":7411693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative expression levels of 12 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRsbHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in four stages of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. simsii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eflower development\u003c/strong\u003e. (a) Four stages of \u003cem\u003eR. simsii\u003c/em\u003e flower development were sampled and used in this study. (b) The relative expressions of 12 \u003cem\u003eRsbHLH\u003c/em\u003e genes analyzed by qRT-PCR. Gene expression level was normalized with EF1a, and the relative expression level was calculated by 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e. The data are the mean ± SE of three independent biological samples. Lowercase letters indicate significant differences between stages (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/5c7204d2a9fbe5a187b2564c.png"},{"id":58758601,"identity":"edf1de9e-2033-44f0-b9c8-94680749db6c","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":549107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of 12 candidate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRsbHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. simsii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treated with high-temperature. \u003c/strong\u003eTotal RNA was extracted from young leaves at different time points after high-temperature treatment (3d and 6d) and without high-temperature treatment (0d) and subjected to quantitative RT-PCR analysis.\u0026nbsp;The \u003cem\u003eEF1a\u003c/em\u003e gene was used as an internal reference gene, the relative expression level was calculated by 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e. The data are the mean ± SE of three independent biological samples. Lowercase letters indicate significant differences between treatments (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/c604965a0cd2eb5236e9b06a.png"},{"id":58758594,"identity":"2b3df487-b2fc-4a41-ab97-4805b1524cab","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":416950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of 12 candidate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRsbHLH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. simsii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treated with low-temperature. \u003c/strong\u003eTotal RNA was extracted from young leaves at different time points after low-temperature treatment (3d and 6d) and without low-temperature treatment (0d) and subjected to quantitative RT-PCR analysis.\u0026nbsp;The \u003cem\u003eEF1a\u003c/em\u003e gene was used as an internal reference gene, the relative expression level was calculated by 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e. The data are the mean ± SE of three independent biological samples. Lowercase letters indicate significant differences between treatments (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/bc7bd6ecbd35e4c801653a70.png"},{"id":74221848,"identity":"47f81b8e-0bb7-4fc4-9584-0a9e7901703d","added_by":"auto","created_at":"2025-01-20 06:47:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33799608,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/bec74306-15ca-4391-bb7d-096c391306e8.pdf"},{"id":58758577,"identity":"5c95aa14-d2a2-45ab-b375-069e5e68216f","added_by":"auto","created_at":"2024-06-20 18:09:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12238,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/a9be84c43d453fe17d402e84.docx"},{"id":58758585,"identity":"8ae2f2fa-2c44-4082-9d2a-bb20ec60d908","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29368,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/8923706cb0ffaafbf56e03f7.docx"},{"id":58758595,"identity":"5dc87429-b10d-4002-a34b-51a4090c6913","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12060,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/0e058a8b41c29d28e0054f8c.docx"},{"id":58758583,"identity":"55e0ce1f-7d91-4325-969d-3d300d6d5342","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14172,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/a8f3a8cf1e859edefaf5055f.docx"},{"id":58758596,"identity":"98f47777-4205-40ca-8707-6f6e95b4dc5a","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15660,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/1e22a921e137ed408da19afa.docx"},{"id":58758588,"identity":"5749f576-ec3c-4fa5-90e6-dc82cb5d1bea","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14698,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/aff8dd6c06426ac68427b19c.docx"},{"id":58758592,"identity":"fbbd8d85-198d-4151-bfad-9135d3c82e3c","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":23057,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/6e4c07bd61eccc11f8930dec.docx"},{"id":58758593,"identity":"1db4fe6e-4a86-4b2c-bd7f-3c34b08fe49a","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15182,"visible":true,"origin":"","legend":"","description":"","filename":"TableS8.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/271b5d2840f21512d5738354.docx"},{"id":58758800,"identity":"00fd191f-b6a2-4474-8c4a-f81810b9049b","added_by":"auto","created_at":"2024-06-20 18:17:45","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":28827,"visible":true,"origin":"","legend":"","description":"","filename":"TableS9.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/9348a4a3d5ca06581d2e5783.docx"},{"id":58758590,"identity":"06077a6f-1ef4-4945-bbac-a3f336518152","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":18162,"visible":true,"origin":"","legend":"","description":"","filename":"TableS10.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/e28c96e17f09a8a1e8b2a77c.docx"},{"id":58758597,"identity":"0d989973-c95f-4f04-aef6-3ff2d40e0c5e","added_by":"auto","created_at":"2024-06-20 18:09:46","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":20168,"visible":true,"origin":"","legend":"","description":"","filename":"TableS11.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/4207e2fa9c21937431381ea6.docx"},{"id":58758589,"identity":"682b6f92-e635-4bb5-aecf-c696b8aba7b4","added_by":"auto","created_at":"2024-06-20 18:09:45","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":13501,"visible":true,"origin":"","legend":"","description":"","filename":"TableS12.docx","url":"https://assets-eu.researchsquare.com/files/rs-4524787/v1/518b3c7bdb39fb67dc5f4749.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identifification and Characterization of RsbHLH Transcription Factors Involved in Flower development of R. simsii","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTranscription factors (TFs), as the key transcriptional regulation factors in Eukaryote, control the expression of specific genes by binding specific DNA sequences[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The Basic helix-loop-helix (bHLH) gene family, as one of the largest TF families, plays important roles in regulating plant growth and development as well as in response to various environmental stress[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The bHLH protein contains the conserved bHLH domain of approximately 60 amino acids, which has two functionally regions: a basic region and a HLH region[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The basic region of approximately 10\u0026ndash;15 amino acids was located at the N-terminus, which is used for recognizing and binding to the specific E-box motif (CANNTG)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The HLH domain of ~\u0026thinsp;40 amino acids located at the C-terminus acts as a dimerization domain, which is used for promoting the dimerization between proteins[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the sequencing and release of plant genomes, the bHLH gene family has been widely identified and characterized in many plants, such as \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003ePyrus ussuriensis\u003c/em\u003e, grape, potato, apple, mango, cabbage, and cotton[\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Numerous studies have revealed that the bHLH family genes not only play important roles in regulating plants growth and metabolism such as light signal transduction, photomorphogenesis, and secondary metabolism, but also participate in responding to various stresses[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For example, GhPAS1, encoding a bHLH transcription factor, was involved in regulates plant development and architecture in cotton through mediating BR signaling[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Makkena and Lamb (2013) reported that the \u003cem\u003eArabidopsis SPT\u003c/em\u003e gene encoding a bHLH transcription factor participated in regulating root growth by controlling the size of the root meristem[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The tomato bHLH transcription factor (SlPRE2) was involved in regulating the fruit development by responding to gibberellin signals, and a rice bHLH transcription factor (DTD) functioned coordinately with TDR to control tapetum function and pollen development[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, the bHLH family genes play important roles in regulating the biosynthesis of various secondary metabolites. In \u003cem\u003eActinidia chinensis\u003c/em\u003e, AcbHLH42 interacting with AcMYB123 can specifically regulate the biosynthesis of anthocyanins in the endocarp of kiwifruit by binding and activating the promoters of \u003cem\u003eAcF3GT1\u003c/em\u003e and \u003cem\u003eAcANS\u003c/em\u003e genes[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Li et al. (2017) reported that the DhbHLH1 promoted the accumulation of anthocyanins in lip tissue of \u003cem\u003eDendrobium hybrid orchids\u003c/em\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, the bHLH transcription factors are involved in response to various environmental stresses. In apple, MdbHLH104 regulates activity of the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase by the \u003cem\u003eMdAHA8\u003c/em\u003e gene, thereby enhancing the tolerance of apple to Fe deficiency[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Overexpression of SlICE1 (a MYC-type bHLH transcription factor) in transgenic tomato increased the expression of cold-responsive genes and enhanced cold tolerance in tomatoes[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The \u003cem\u003eArabidopsis\u003c/em\u003e AtbHLH92 transcription factor was involved in plant response to osmotic stresses[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and AtbHLH17 (AtAIB) had been reported to confer the drought tolerance of transgenic plants via regulating of ABA signaling[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eRhododendron simsii\u003c/em\u003e, also called the Indian Azalea, belongs to the \u003cem\u003eRhododendron\u003c/em\u003e genus of the Ericaceae family, wihich is a valuable horticultural and medicinal plant species due to the flowers with brilliant red corollas. However, several questions are still needed to be explored. Are there bHLH transcription factors involved in regulating flower development of \u003cem\u003eR. simsii\u003c/em\u003e? Which bHLH transcription factors are involved in regulating anthocyanin biosynthesis in \u003cem\u003eR. simsii\u003c/em\u003e flower. What\u0026rsquo;s the difference among the different RsbHLH transcription factors involving the anthocyanin biosynthesis in \u003cem\u003eR. simsii\u003c/em\u003e? The genome-wide identification and characterization of RsbHLH family genes can help us answers these questions. In the present study, we comprehensively evaluated the RsbHLH gene family and performed phylogenetic analysis, gene/protein structure analysis, gene duplication, and chromosome location. Moreover, the expression pattern of \u003cem\u003eRsbHLH\u003c/em\u003e genes in different tissue and the different stages of flower development was evaluated with the released RNA-seq data. The expression patterns of candidate \u003cem\u003eRsbHLH\u003c/em\u003e genes associated with flower development in \u003cem\u003eR. simsii\u003c/em\u003e under extreme temperature treatment was further investigated. Our study provides a reference for further functional studies of the RsbHLH family and offers the targeted gene resources for cultivating superior \u003cem\u003eR. simsii\u003c/em\u003e ornamental varieties via genetic engineering techniques.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials\u003c/h2\u003e \u003cp\u003eIn the present study, two-year-old \u003cem\u003eR. simsii\u003c/em\u003e seedlings were grown in a single growth chamber, which was programmed a photoperiod of 12 h of light at a temperature of 22\u0026deg;C, followed by 12 h of darkness at 18\u0026deg;C, relative humidity 70%, and light intensity 4000 Lux. 12 \u003cem\u003eR. simsii\u003c/em\u003e seedlings were moved into the incubator at 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for 0, 3, and 6 d for cold stress treatment, while another 12 seedlings were moved into the incubator at 40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for 0, 3, and 6 d for heat stress treatment. The leaves were quickly cut into pieces, frozen in liquid nitrogen, and then stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of RsbHLH family genes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genome sequences and corresponding annotations of three \u003cem\u003eRhododendron\u003c/em\u003e species (\u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e, and \u003cem\u003eR. williamsianum\u003c/em\u003e) were obtained from the Ericaceae Genome Resource database (TEGR, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.tegr.com.cn\u003c/span\u003e\u003cspan address=\"http://www.tegr.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The HMM file of bHLH domain (PF00010) downloaded from the Pfam database was applied for identifying the RsbHLH family genes from the genomes of three \u003cem\u003eRhododendron\u003c/em\u003e species through the HMMER software. Furthermore, the redundant sequences located at the same chromosome and short proteins (less than 100 aa) were removed based on the physical localizations. We further verify the presence of core domain for all candidate proteins using the SMART and Pfam databases[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Finally, the identified proteins with the conserved domain (bHLH) were regarded as the RsbHLH family genes and used in this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSequence features, subcellular localization prodiction and structural analysis of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe ProtParam online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the sequence features of \u003cem\u003eRsbHLH\u003c/em\u003e family genes in \u003cem\u003eR. simsii\u003c/em\u003e, including the number of amino acids, molecular weight, isoelectric point, instability index, aliphatic index, and hydrophilicity. Moreover, the subcellular localization of RsbHLHs was predicted using the Protein Subcellular Localization Prediction Tool[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] (PSORT, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genscript.com/psort.html\u003c/span\u003e\u003cspan address=\"https://www.genscript.com/psort.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe gene structure of \u003cem\u003eRsbHLH\u003c/em\u003e genes was graphically displayed by the GSDS 2.0 based on the \u003cem\u003eR. simsii\u003c/em\u003e genome annotation. The MEME software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was applied for identification the conserved motifs of RsbHLH proteins[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The parameters were set as follows: maximum numbers of motifs, 20; minimum motif width, 6 bp; maximum motif width, 50 bp. The RsbHLH family genes were renamed from RsbHLH001 to RsbHLH116 based on based on their chromosomal distribution,\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis of RsbHLH family genes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe bHLH protein sequences of four plant species, including RsbHLH, RibHLH, RwbHLH, and AtbHLH proteins from \u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e, \u003cem\u003eR. williamsianum\u003c/em\u003e, and \u003cem\u003eArabidopsis\u003c/em\u003e, were aligned using the clustal W algorithm with default parameters in MEGA 11 software[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The alignment results were used to construct a phylogenetic tree using the neighbor-joining (NJ) method, and the reliability of the tree nodes was evaluated using 1000 bootstrap replicates. The ChiPlot online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to draw the phylogenetic tree, and the RdbHLH gene family were furtehr classified into different groups based on the sequence suimilarity and identified \u003cem\u003eArabidopsis bHLH\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory elements in promoter of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 1500 bp upstream of the transcription start site (TSS) of each \u003cem\u003eRsbHLH\u003c/em\u003e gene was extracted and used to identify the conserved \u003cem\u003ecis\u003c/em\u003e-regulatory elements (CRE) in the promoter region of \u003cem\u003eRsbHLH\u003c/em\u003e genes using the PlantCARE tool[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal localization, gene duplication, and synteny analysis of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genome annotation information was used to extract the location information of \u003cem\u003eRsbHLH\u003c/em\u003e genes on chromosomes, and the chromosomal distribution of \u003cem\u003eRsbHLH\u003c/em\u003e genes was graphically visualized through MapChart software. Gene duplication analysis for \u003cem\u003eR. simsii\u003c/em\u003e was performed using the MCScanX software[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Firstly, the potential homologous gene pairs across the \u003cem\u003eR. simsii\u003c/em\u003e were detected using BLASTp software with e-value less than 1e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The MCScanX software was applied for identifying syntenic blocks based on the identified homologous gene pairs. Five genome duplication models, including whole-genome duplication (WGD)/segmental duplication, tandem duplication, dispersed, and proximal, were detected using the MCScanX program. The homologous gene pairs in syntenic blocks were used to estimate the Ka and Ks values using PAML package. In addition, the MCScanX program was used to perform collinearity analysis across four plant species (\u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e, and \u003cem\u003eR. williamsianum\u003c/em\u003e) to identified the orthologous gene pairs. The Dual Systeny Plot program was used to visualize the evolutionary relationships of \u003cem\u003ebHLH\u003c/em\u003e genes between \u003cem\u003eR. simsii\u003c/em\u003e and the other three plant species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGO (Gene Ontology) enrichment analysis\u003c/h2\u003e \u003cp\u003eGO annotations of the RsbHLH family genes were performed by the GOATOOLS software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://github.com/tanghaibao/GOatools\u003c/span\u003e\u003cspan address=\"http://github.com/tanghaibao/GOatools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Fisher\u0026rsquo;s exact test was applied for performing the GO term enrichment analysis for \u003cem\u003eRsbHLHs\u003c/em\u003e. The Bonferroni multiple testing correction was used to minimize false positives, and functions were considered to be significantly enriched when their Bonferroni-corrected P-values (Padjust) were \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analyses of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes in different organs and/or different stages of flower development of\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRNA-Seq data of different organs (stem, leaf, and flower) and the different stages of flower develoment (T1-T5) of \u003cem\u003eR. simsii\u003c/em\u003e was downloaded from the NCBI SAR database (SRP229032). After analysis and normalization, the expression patterns of \u003cem\u003eRsbHLH\u003c/em\u003e family genes were displayed and visualized in heat maps. The transcript abundance of \u003cem\u003eRsbHLH\u003c/em\u003e genes was calculated as fragments per kilobase of exon model per million mapped reads (FPKM). The log\u003csub\u003e2\u003c/sub\u003e (FPKM) from the RNA-seq data were subjected to hierarchical clustering with Cluster 3.0, and the results were graphically displayed using Java TreeView.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR analysis of\u003c/b\u003e \u003cb\u003eRsbHLHs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe PrimerScript\u0026reg; RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used to reverse transcribe the RNA into cDNA. RT-qPCR was conducted with three biological replicates and three technical replicates using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). The specific primers of target genes were designed based on the full-length CDS of \u003cem\u003eRsbHLH\u003c/em\u003e genes, and the \u003cem\u003eEF1α\u003c/em\u003e gene was treated as reference gene (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each of these biological replicates was run in three technical replicates. Relative expression was calculated using the 2\u003csup\u003e\u0026ndash;∆∆CT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of protein interaction network\u003c/h2\u003e \u003cp\u003eThe OrthoVenn2 online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://orthovenn2.bioinfotoolkits.net/home\u003c/span\u003e\u003cspan address=\"https://orthovenn2.bioinfotoolkits.net/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identifiy the orthologous gene pairs.between \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e. After screening, we obtained the homologous relationship pairs between \u003cem\u003eRsbHLH\u003c/em\u003e genes and \u003cem\u003eArabidopsis\u003c/em\u003e genes. We then used the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to analyze the protein interaction network relationships between \u003cem\u003eArabidopsis\u003c/em\u003e genes in the homologous relationship pairs, and mapped out the protein interaction network relationships between RsbHLH genes (the minimum required interaction score was set to 0.650, and the maximum number of interactors of the 1st shell to show was set to 12)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of the RsbHLH gene family in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 116 nonredundant RsbHLH family genes with conserved core bHLH domain were identified in the \u003cem\u003eR. simsii\u003c/em\u003e genome (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The length of RsbHLH protein sequences ranged from 135 (RsbHLH016) to 1174 (RsbHLH034) amino acids, with an average of 391 amino acids. The molecular weights of the identified bHLH proteins ranged from 14.85 kDa to 130.67 kDa. The predicted isoelectric points of bHLH proteins ranged from 4.67 to 9.51, with an average of 6.65. Among them, the instability indexes of 111 RsbHLH family proteins were greater than 40, indicating that most of RsbHLH family proteins are unstable. The aliphatic indexes of RsbHLH proteins ranged from 46.34 to 97.40. The hydrophobicity indexes of RsbHLHs are negative, indicating that the RsbHLH family proteins are commonly used as hydrophilic proteins. Moreover, the prediction of subcellular localization for the RsbHLH family proteins was also performed in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Our result showed that 90.51% (105/116) of RdbHLH family proteins was localized to the nucleus. In addition, 111 \u003cem\u003eRdbHLH\u003c/em\u003e genes in the RdbHLH gene family were distributed on 13 \u003cem\u003eR. simsii\u003c/em\u003e chromosomes, and the remaining five \u003cem\u003eRsbHLH\u003c/em\u003e genes were anchored in the scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The distribution of \u003cem\u003eRsbHLH\u003c/em\u003e genes on the 13 chromosomes was uneven, with most \u003cem\u003eRsbHLH\u003c/em\u003e genes located on chromosome 8 (14 genes, 12.07%), chromosomes 3 (11 genes, 9.48%) and 7(11 genes, 9.48%). In contrast, there were only 3 \u003cem\u003eRsbHLH\u003c/em\u003e genes (2.59%) on chromosome 4. The percentage of \u003cem\u003eRsbHLH\u003c/em\u003e genes on each chromosome ranged from 0.12% on chromosome 4 to 0.56% on chromosome 8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis of the bHLH gene family among\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e, \u003cb\u003eR. irroratum\u003c/b\u003e, \u003cb\u003eR. williamsianum\u003c/b\u003e, \u003cb\u003eand\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the evolutionary relationships of RsbHLH family genes, we constructed an unroot phylogenetic tree of bHLH family genes from \u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e, \u003cem\u003eR. williamsianum\u003c/em\u003e, and \u003cem\u003eArabidopsis\u003c/em\u003e using the neighbor-joining (NJ) method in MEGA 11.0 software with default parameters. The RsbHLH family members of \u003cem\u003eR. simsii\u003c/em\u003e were classified into 13 groups (I to XIII) and 25 subgroups based on the topology of phylogenetic tree and the classification of \u003cem\u003eArabidopsis\u003c/em\u003e AtbHLH proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The number of \u003cem\u003eRsbHLH\u003c/em\u003e genes varies among the different groups. Group Ⅷ, as the largest group among 13 groups, contained 21 \u003cem\u003eRsbHLH\u003c/em\u003e genes, which was further classified into 5 subgroups (subgroup a-e). Groups I and IV contained 19 and 15 \u003cem\u003eRsbHLH\u003c/em\u003e genes, respectively, which were divided into two subgroups (Ⅰa andⅠb) and four subgroups (IVa, IVb, IVc, and IVd). There is no RsbHLH genes distributed in group VI. Moreover, the groups VII and XII contained much more \u003cem\u003ebHLH\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e than those in \u003cem\u003eR. simsii\u003c/em\u003e, \u003cem\u003eR. irroratum\u003c/em\u003e, and \u003cem\u003eR. williamsianum\u003c/em\u003e. Moreover, most groups were further clustered into two or more subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Subgroup IIId and VIIIb contained the bHLH gene clusters from \u003cem\u003eR. simsii\u003c/em\u003e, indicating that the species-specific expansion of these genes occurred in \u003cem\u003eR. simsii\u003c/em\u003e after the divergence of core eudicots. Interestingly, some subgroups only contained \u003cem\u003eAtbHLH\u003c/em\u003e genes without \u003cem\u003eRsbHLH\u003c/em\u003e genes or included \u003cem\u003eRsbHLH\u003c/em\u003e genes from the \u003cem\u003eRhododendron\u003c/em\u003e species without \u003cem\u003eArabidopsis AtbHLH\u003c/em\u003e genes. Group VI only contained the \u003cem\u003eAranidopsis bHLH\u003c/em\u003e genes, and didn\u0026rsquo;t include the \u003cem\u003ebHLH\u003c/em\u003e gene from the \u003cem\u003eRhododendron\u003c/em\u003e species. The group XIII only contained the \u003cem\u003ebHLH\u003c/em\u003e genes from \u003cem\u003eRhododendron\u003c/em\u003e species, and didn\u0026rsquo;t contain the \u003cem\u003eArabidopsis AtbHLH\u003c/em\u003e genes. These results indicated that \u003cem\u003ebHLH\u003c/em\u003e genes could have been either acquired and lost in \u003cem\u003eArabidopsis\u003c/em\u003e or the \u003cem\u003eRhododendron\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene structure and protein motif analysis of the RsbHLH gene family in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the gene structural diversity and protein motif composition of RsbHLH family members, the exon-intron distribution was first analyzed and visualized using the Gene Structure Display Server 2.0 (GSDS, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Gene structure analysis showed that a total of 116 \u003cem\u003eRsbHLH\u003c/em\u003e genes had exons varying from 1 to 15. Thirteen \u003cem\u003eRsbHLH\u003c/em\u003e genes (11.21%) had only one exon or lacked intron, including \u003cem\u003eRsbHLH74\u003c/em\u003e, \u003cem\u003eRsbHLH102\u003c/em\u003e, \u003cem\u003eRsbHLH050\u003c/em\u003e, \u003cem\u003eRsbHLH090\u003c/em\u003e, \u003cem\u003eRsbHLH032\u003c/em\u003e, \u003cem\u003eRsbHLH030\u003c/em\u003e, \u003cem\u003eRsbHLH040\u003c/em\u003e, \u003cem\u003eRsbHLH010\u003c/em\u003e, \u003cem\u003eRsbHLH053\u003c/em\u003e, \u003cem\u003eRsbHLH004\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH028.\u003c/em\u003e The 72.4% of RsbHLH family members (84 \u003cem\u003eRsbHLH\u003c/em\u003e genes) contained 2 to 8 introns. Interestingly, the \u003cem\u003eRsbHLH19\u003c/em\u003e gene possessed 15 exons and 14 introns, which was the gene with the least number of introns among RsbHLH family members. In addition, gene structure analysis showed that most \u003cem\u003eRsbHLH\u003c/em\u003e genes in the same subgroup possessed the similar intron/exon distribution, including the number and length of introns. For example, all \u003cem\u003eRsbHLH\u003c/em\u003e genes in subgroup IV possessed more than 6 exons, and the members of subgroup XVI only contain 3 exons. Thus, the results of gene structure analysis demonstrated that \u003cem\u003eRsbHLH\u003c/em\u003e genes in the same subgroup had similar gene structures, and further verified the reliability of the topology of phylogenetic tree of the RsbHLH family genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA total of 15 putative conserved motifs were identified in RsbHLH proteins using the MEME suite, namely Motifs 1\u0026ndash;15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Motif 1 and motif 2, located in bHLH domains, were distributed in almost all RsbHLH proteins. Six RsbHLH proteins, including RsbHLH071, RsbHLH078, RsbHLH002, RsbHLH011, RsbHLH062, and RsbHLH054, only contained one of motif 1 and motif 2. Moreover, the distribution of conserved motifs of all RsbHLH proteins in the same subgroup were similar. For example, motif 15, motif 4, motif 10, and motifs 7 were specifically distributed in most members of subgroup X, subgroup XVII, subgroup XVI, and subgroup XVII, which played important roles in biological regulatory functions of these subgroups genes. In addition, some motifs existed in members of multiple subgroups. The motif 8 was commonly distributed in four subgroups: subgroups V-VII and XI, and motif 3 was widely distributed in six subgroups (IV, VIII, IX, X, XI, and XIV). These results suggested that the \u003cem\u003eRsbHLH\u003c/em\u003e genes in these subgroups might possess similar biological function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCis\u003c/b\u003e \u003cb\u003e-elements analysis of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egene promoters\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eCis\u003c/em\u003e-regulatory elements, which are usually restricted to 5\u0026prime; upstream areas of genes, are the binding sites of transcriptional factors. In the present study, the 1500 bp upstream regions of the transcription start site (TSS) of \u003cem\u003eRsbHLH\u003c/em\u003e genes were applied for identifying \u003cem\u003ecis\u003c/em\u003e-regulatory elements and investigate gene regulation patterns with PlantCARE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). A total of 37 functionally annotated \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter of most \u003cem\u003eRsbHLH\u003c/em\u003e genes, which were further clustered into four categories: hormone-responsive elements (TATC-box, TCA-element, CGTCA-motif, TGACG-motif, GARE-motif, P-box, GA-motif, TCCC-motif, and AuxRR-core), stress-responsive elements (ABRE, MBS, WUN-motif, LTR, ARE, and TC-rich repeats), light-responsive elements (G-box, CAT-box, GT1-box, I-box, ATC-motif, Box4, TCT-motif, AE-box, MRE, ACE, SP1, ATCT-motif, L-box, AAAC-motif, and 3-AF1 bind site), and the development-related \u003cem\u003ecis\u003c/em\u003e-elements (O\u003csub\u003e2\u003c/sub\u003e-site, circadian, AT-rich element, GCN4-motif, AACA-motif, HD-Zip1, and MSA-LIKE). This result further suggested that the \u003cem\u003eRsbHLH\u003c/em\u003e family genes played important roles in regulating the plant growth and development as well as in response to various environmental stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGO enrichment analysis of the\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe bHLH transcription factors play pivotal roles in numerous biological process, especially in the regulation of secondary metabolism in plants. To explore the biological functions of the RsbHLH famiy genes in \u003cem\u003eR. simsii\u003c/em\u003e, we conducted GO annotation and enrichment analysis of 116 \u003cem\u003eRsbHLH\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). A total of eighty-seven Go term were enriched, eleven molecular functions, two cellular components, and seventy-four biological processes in GO terms were enriched in the PdbHLH genes relative to the complete GO database. In the biological process category, RsbHLH genes were mainly enriched in positive regulation of transcription, DNA-templated (n\u0026thinsp;=\u0026thinsp;21), positive regulation of RNA biosynthetic process (n\u0026thinsp;=\u0026thinsp;21), positive regulation of nucleic acid-templated transcription (n\u0026thinsp;=\u0026thinsp;21), positive regulation of RNA metabolic process (n\u0026thinsp;=\u0026thinsp;21), positive regulation of transcription by RNA polymerase II(13), regulation of transcription by RNA polymerase II(18) and transcription by RNA polymerase II(18) and so on. In the cellular component category, the genes were enriched in RNA polymerase II transcription regulator complex (4) and transcription regulator complex (4). In the molecular function category, the genes were enriched in transcription regulatory region sequence-specific DNA binding (21), core promoter sequence-specific DNA binding (13), sequence-specific double-stranded DNA binding (21), double-stranded DNA binding (21), DNA-binding transcription activator activity(13), DNA-binding transcription activator activity, RNA polymerase II-specific(13) and regulatory region nucleic acid binding(22) and so on. GO enrichment results suggested that RsbHLH transcription factors mainly involved in transcription, RNA metabolic process, DNA-bing and floral organ development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGene duplication, Ka/Ks, and Collinearity Analysis\u003c/h2\u003e \u003cp\u003eGene duplication events are closely related to the plant evolution process and contribute to the expansion of the gene family. In the present study, the evolutionary patterns of the \u003cem\u003eRsbHLH\u003c/em\u003e genes were surveyed using MCScanX software. Amazingly, 42 (36.21%) and 57 (49.14%) of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e were duplicated and retained from dispersed and whole genome duplication (WGD)/segmental duplication, respectively. Only seven \u003cem\u003eRsbHLH\u003c/em\u003e genes originated from tandem duplication. To further investigate the potential role of segmental duplication in the expansion of the RsbHLH family genesly, we conducted the collinearity analysis of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e genome using the all-\u003cem\u003evs.\u003c/em\u003e-all local BLASTP algorithm-based search. A total of 28 segmental duplication gene pairs (52 \u003cem\u003eRsbHLH\u003c/em\u003e genes) were detected in the \u003cem\u003eR. simsii\u003c/em\u003e genome, which accounted for 45.69% of WGD-type \u003cem\u003eRsbHLH\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). The \u003cem\u003eRsbHLH\u003c/em\u003e genes in each synteny block were distributed on all chromosomes. In addition, the ratio of non-synonymous to synonymous substitutions (Ka/Ks) was evaluated using the PAML package (Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). The Ka/Ks ratio\u0026thinsp;\u0026lt;\u0026thinsp;1 indicates negative (purifying) selection. The criterion for positive (adaptive) selection is Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1 (Hurst, 2002). Our results showed that the Ka/Ks ratios of 28 segmental duplication gene pairs were less than one, suggesting that the the 52 \u003cem\u003eRsbHLH\u003c/em\u003e genes were under negative selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). The Ks values were commonly used to calculate the the occurrence time of segmental duplication events. The segmental duplication events of 28 gene pairs in \u003cem\u003eR. simsii\u003c/em\u003e genome occurred from 25.35 (Ks\u0026thinsp;=\u0026thinsp;0.7304) to122.77 mya (Ks\u0026thinsp;=\u0026thinsp;3.683).\u003c/p\u003e \u003cp\u003eThe identification of orthology is central to comparative genomics, which has been employed in many studies using synteny analysis. According to the identified synteny relationships, we identified orthologous pairs of \u003cem\u003ebHLH\u003c/em\u003e genes among four representative plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, Table \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). There were 69 orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs between \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e, 130 orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs between \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eR. irroratum\u003c/em\u003e, and 120 orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs between \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eR. williamsianum.\u003c/em\u003e The number of orthologous events of \u003cem\u003eRsbHLH\u003c/em\u003e-\u003cem\u003eRibHLH\u003c/em\u003e was much greater than that of \u003cem\u003eRsbHLH-RwbHLH\u003c/em\u003e. In addition, the Ka, Ks, and Ka/Ks values of orthologous \u003cem\u003eRsbHLH\u003c/em\u003e gene pairs among four species were also investigated(Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This result showed that the Ka/Ks ratio of orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs among four species were less than 1, suggesting that the orthologous \u003cem\u003ebHLH\u003c/em\u003e genes in four species were subjected to purifying selection during evolution. The Ka/Ks ratio of orthologous \u003cem\u003ebHLH\u003c/em\u003e gene pairs in \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e was significantly lower than those in \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eR. irroratum\u003c/em\u003e and \u003cem\u003eR. simsii\u003c/em\u003e and \u003cem\u003eR. williamsianum.\u003c/em\u003e This result further indicated that the specific expansion and sequence divergence occurred in \u003cem\u003eRhododendron\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression pattern of the RsbHLH family genes in different organs of\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the potential regulatory mechanisms of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e, the expression patterns of \u003cem\u003eRsbHLH\u003c/em\u003e genes in three organs (stems, leaves, and flowers) of \u003cem\u003eR. simsii\u003c/em\u003e were evaluated using the released RNA-seq data (Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). A total of 49 \u003cem\u003eRsbHLH\u003c/em\u003e genes, 64 \u003cem\u003eRsbHLH\u003c/em\u003e genes, and 58 \u003cem\u003eRsbHLH\u003c/em\u003e genes were highly expressed in flowers, leaves, and stems, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). There are 46 \u003cem\u003eRsbHLH\u003c/em\u003e genes simultaneously expressed in flowers and leaves, 45 simultaneously expressed in flowers and stems, and 54 simultaneously expressed in leaves and stems, respectively. The 46 \u003cem\u003eRsbHLH\u003c/em\u003e genes are commonly expressed in all three organs. In addition, the expression levels of 23 \u003cem\u003eRsbHLH\u003c/em\u003e genes in leaves and stems were higher than those in flowers, suggesting that these \u003cem\u003eRsbHLH\u003c/em\u003e genes played important roles in regulating the growth and development of vegetative organs. The expression levels of 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH096\u003c/em\u003e, \u003cem\u003eRsbHLH075\u003c/em\u003e, \u003cem\u003eRsbHLH023\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH115\u003c/em\u003e, \u003cem\u003eRsbHLH004\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, \u003cem\u003eRsbHLH077\u003c/em\u003e, \u003cem\u003eRsbHLH012\u003c/em\u003e, \u003cem\u003eRsbHLH072\u003c/em\u003e, \u003cem\u003eRsbHLH109\u003c/em\u003e, \u003cem\u003eRsbHLH013\u003c/em\u003e, and \u003cem\u003eRsbHLH037\u003c/em\u003e) in flowers were higher than those in leaves and stems, suggesting that these \u003cem\u003eRsbHLH\u003c/em\u003e genes played essential roles in regulating the flower development.\u003c/p\u003e \u003cp\u003eTo further explore the potential regulatory mechanisms of the RsbHLH genes in flower development of \u003cem\u003eR. simsii\u003c/em\u003e, the expression levels of \u003cem\u003eRsbHLH\u003c/em\u003e genes in five different stages of flower development of \u003cem\u003eR. simsii\u003c/em\u003e (T1-T5) were evaluated using the released RNA-seq data (Table \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, a total of 64 \u003cem\u003eRsbHLH\u003c/em\u003e genes were differentially expressed in five different stages of flower development, which can be divided into four types: Ⅰ) The expression levels of 21 \u003cem\u003eRsbHLH\u003c/em\u003e genes in early stage (TI-T3) of flower development were higher than those in late stage (T4།T5) of flower development. Ⅱ) The expression levels of 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes in early stage (TI།T3) of flower development were less than those in late stage (T4།T5) of flower development. Type Ⅲ included 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes, which increased in the early stage and then sharply decreased in late stage of flower development. Type Ⅳ contained 2 \u003cem\u003eRsbHLH\u003c/em\u003e genes, which decreased in the early stage and then increased the late stage of flower development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein interaction prediction\u003c/h2\u003e \u003cp\u003eDifferent bHLH proteins can generate homodimers or heterodimers with other proteins [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which is a prerequisite for DNA recognition and DNA-binding specificity. In this study, we performed protein interaction networks of the RsbHLH family genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Table \u003cspan refid=\"MOESM12\" class=\"InternalRef\"\u003eS12\u003c/span\u003e). Our result showed that 32 \u003cem\u003eRsbHLH\u003c/em\u003e genes were used to construct the protein interaction networks, 26 RsbHLH proteins of which were interacting with more than one RsbHLH protein. One RsbHLH protein (PsbHLH037) interacted with four RsbHLH proteins (RsbHLH045, RsbHLH057, RsbHLH097, and RsbHLH114), and RsbHLH045 protein interacts with RsbHLH066 and RsbHLH037, respectively. Moreover, 11 RsbHLH proteins could interact with genes from other families. For example, RsbHLH059 protein can interact with TIFY7 (Rs09G0091800)/JAZ1 (Rs05G0004700), and RsbHLH094 protein can interact with CRY2 (Rs02G0037100)/PHYB (Rs04G0239100). RsbHLH017 proteins interacts with six proteins such as JAZ1 (Rs05G0004700), MYB75 (Rs08G0087700), MYB0 (Rs08G0239000), MYB66 (Rs08G0171500), TTG1 (Rs09G0209900), and TT2 (Rs12G0209800). In particular, RsbHLH017 protein interacts with MYB75/MYB0/MYB66 and TTG1 to generate MBW complexes. In addition, five RsbHLH proteins (RsbHLH066, RsbHLH015, RsbHLH067, RsbHLH115, and RsbHLH053) can interact with PHYA (Rs11G0107300), JAZ1, JAZ1, TTG1, and TIFY7, respectively. Functional annotations further revealed that the orthologous genes of these \u003cem\u003eRsbHLH\u003c/em\u003e genes had been reported to be involved in regulating the biosynthesis of flavonoids and anthocyanin biosynthesis and in responose to various abiotic stresses. Thus, it is very interesting to explore the regulation of bHLH proteins in diverse biological processes of \u003cem\u003eR. simsii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eValidation of RNA-Seq experiment by RT-qPCR\u003c/h2\u003e \u003cp\u003eTo further assess the reliability and vilidity of RNA-seq experiments, RT-qPCR was conducted on 12 \u003cem\u003eRsbHLH\u003c/em\u003e genes with high expression levels in different stages of flower development. Comparative analysis of expression patterns of these \u003cem\u003eRsbHLH\u003c/em\u003e genes were performed in four different stages of flower development of \u003cem\u003eR. simsii\u003c/em\u003e, and the result showed that the expression trends of 12 \u003cem\u003eRsbHLH\u003c/em\u003e genes in RT-qPCR experiment were consistent with the RNA-Seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression patterns of\u003c/b\u003e \u003cb\u003eRsbHLH\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eR. simsii\u003c/b\u003e \u003cb\u003eduring extreme temperature treatments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTemperature in particular has been shown to have a tremendous effect on the timing of flowering: the vernalization response and thermosensory pathway. In the present study, we further investigated the expression patterns of 12 candidate \u003cem\u003eRsbHLH\u003c/em\u003e genes in \u003cem\u003eR. simsii\u003c/em\u003e treated with high and low temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Under high temperature treatment, nine RsbHLH genes, including \u003cem\u003eRsbHLH033\u003c/em\u003e, \u003cem\u003eRsbHLH016\u003c/em\u003e, \u003cem\u003eRsbHLH048\u003c/em\u003e, \u003cem\u003eRsbHLH053\u003c/em\u003e, \u003cem\u003eRsbHLH058\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH107\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH115\u003c/em\u003e, were significantly upregulated following 3d after high temperature treatment, suggesting that these genes might play a positive role in response to heat stress. Only one \u003cem\u003eRsbHLH\u003c/em\u003e gene (\u003cem\u003eRsbHLH075\u003c/em\u003e) were significantly downregulated following 3d after high temperature treatment, suggesting that this gene might play a negative role in response to heat stress. In addition, the expression levels of five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH053\u003c/em\u003e, \u003cem\u003eRsbHLH058\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH107\u003c/em\u003e, and \u003cem\u003eRsbHLH109\u003c/em\u003e) were significantly increased after 3-day low-temperature treatment in comparison with the control group, suggesting that these genes play positive roles in response to low temperature. Six \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH033\u003c/em\u003e, \u003cem\u003eRsbHLH016\u003c/em\u003e, \u003cem\u003eRsbHLH048\u003c/em\u003e, \u003cem\u003eRsbHLH075\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH115\u003c/em\u003e) were decreased following 3d after low temperature treatment, indicating that these genes might be involved in negative regulation response to low temperature. Interestingly, five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH033\u003c/em\u003e, \u003cem\u003eRsbHLH016\u003c/em\u003e, \u003cem\u003eRsbHLH048\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH115\u003c/em\u003e) exhibited opposite expression patterns after high and low temperature treatment, while the other five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH053\u003c/em\u003e, \u003cem\u003eRsbHLH058\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH075\u003c/em\u003e, and \u003cem\u003eRsbHLH107\u003c/em\u003e) displayed the same expression patterns.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBasic helix-loop-helix (bHLH) gene family, as one of the largest transcription factor families in plants, played essential roles in regulating plant growth and development as well as in response to various environmental stress[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, comprehensive identification of the bHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e has not been conducted. In the present study, we performed genome-wide characterization of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e. A total of 116 \u003cem\u003eRsbHLH\u003c/em\u003e genes were identified and divided into 13 groups based on the phylogenetic tree topology, gene/protein structure, and the identified \u003cem\u003eAtbHLH\u003c/em\u003e genes. Moreover, the numerical distribution of the bHLH family genes from the lower plants to flowering plants were investigated, found that the genome of flowering plants comprises an very large number of \u003cem\u003ebHLH\u003c/em\u003e genes, while the genome of lower plants contain only a few \u003cem\u003ebHLH\u003c/em\u003e genes. This result indicated that the bHLH family genes underwent drastic expansion during plant evolution. Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms, which mainly included five types such as WGD (whole-genome duplication)/segmental duplication, tandem duplication, dispersed duplication, proximal, and singleton[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Different duplication events contributed differently to gene expansion in different families. Segmental and tandem duplication played important roles in the expansion of WRKY and APETALA2/ethylene-responsive factor (AP2/ERF) family genes in plants[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Transposed duplication contributed to the expansion of MADS and NBS-LRR family genes[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our results revealed that more than 85% of the RsbHLH family genes in \u003cem\u003eR. simsii\u003c/em\u003e were duplicated and retained from dispersed and WGD/segmental duplication events, respectively, indicating that the WGD/segmental and dispersed duplication events played major roles in the expansion of RsbHLH family genes. Gene expansion of the RsbHLH family genes caused rapid functional and sequence differentiation of \u003cem\u003eRsbHLH\u003c/em\u003e genes, which is accompanied by neofunctionalization and subfunctionalization as well as gene expression patterns. For example, two genes (RsbHLH010 and RsbHLH007) were derived and retained from segmental duplication. The RdbHLH007 was highly expressed in the stems and leaves of \u003cem\u003eR. simsii\u003c/em\u003e, while the expression levels of RsbHLH010 was not detected. The \u003cem\u003ecis\u003c/em\u003e-acting elements distributed in the gene promoters can reveal the potential function of this gene. Our result showed that the \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoters of \u003cem\u003eRsbHLH\u003c/em\u003e genes in \u003cem\u003eR. simsii\u003c/em\u003e included four types of \u003cem\u003ecis\u003c/em\u003e-elements scch as including light-, stress-, hormone-, and the development-related elements, which was consistent with the previous studies.\u003c/p\u003e \u003cp\u003eTranscription factors can selectively recognize and bind to the upstream promoters of the essential genes associated with various biological pathways, and regulate intrinsic cellular processes, such as differentiation and development, and of the cellular response to external perturbation through signaling pathways[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Flower development is regulated by a variety of transcription factors[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The bHLH gene family, as the second largest TF family, played essential roles in regulating flower development[\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our results showed that 64 \u003cem\u003eRsbHLH\u003c/em\u003e genes were highly expressed in flowers, and the expression levels of 11 \u003cem\u003eRsbHLH\u003c/em\u003e genes in flowers were higher than those in leaves and stems. These results suggested that these 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes played essential roles in specific regulation of the flower development. Moreover, the orthologous genes of 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH096\u003c/em\u003e, \u003cem\u003eRsbHLH075\u003c/em\u003e, \u003cem\u003eRsbHLH023\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH115\u003c/em\u003e, \u003cem\u003eRsbHLH004\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, \u003cem\u003eRsbHLH077\u003c/em\u003e, \u003cem\u003eRsbHLH012\u003c/em\u003e, \u003cem\u003eRsbHLH072\u003c/em\u003e, \u003cem\u003eRsbHLH109\u003c/em\u003e, \u003cem\u003eRsbHLH013\u003c/em\u003e, and \u003cem\u003eRsbHLH037\u003c/em\u003e) in \u003cem\u003eArabidopsis\u003c/em\u003e were \u003cem\u003eBPE\u003c/em\u003e, \u003cem\u003eBHLH79\u003c/em\u003e, \u003cem\u003eBHLH48\u003c/em\u003e, \u003cem\u003eJAM2\u003c/em\u003e, \u003cem\u003eILR3\u003c/em\u003e, \u003cem\u003eMYC4\u003c/em\u003e, \u003cem\u003eSACL3\u003c/em\u003e, \u003cem\u003eBIM2\u003c/em\u003e, \u003cem\u003eATMYC1\u003c/em\u003e, \u003cem\u003eBIM1\u003c/em\u003e, \u003cem\u003eBHLH103\u003c/em\u003e, \u003cem\u003eJAM2\u003c/em\u003e, and \u003cem\u003eBHLH041\u003c/em\u003e, respectively. Previous studies have revealed that most of these genes in \u003cem\u003eArabidopsis\u003c/em\u003e are involved in flower development [\u003cspan additionalcitationids=\"CR55 CR56 CR57\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Szecsi et al. (2006) revealed that BIGPETAL (BPE) was involved in controlling the \u003cem\u003eArabidopsis\u003c/em\u003e petal size[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and overexpression of \u003cem\u003eAtbHLH48\u003c/em\u003e caused early flowering under long-day conditions by directly activating the transcription of \u003cem\u003eFT\u003c/em\u003e gene[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Nakata and Ohme-Takagi (2013) showed that overexpression of JAM2 and JAM3 resulted in reduced male fertility[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Chen et al. (2016) reported that MYC4 could interact with the MYBs to form the bHLH-MYB complex, and JAZs repress the bHLH-MYB complex to regulate JA-mediated stamen development[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Hu et al. (2015) reported that the hypermethylated BIM2 gene may suppress downstream genes in brassinosteroid signaling pathway, and thus affect the male fertility in PA64S[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Temperature in particular has been shown to have a tremendous effect on the timing of flowering: the vernalization response and thermosensory pathway[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The qRT-PCR results showed that five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH033\u003c/em\u003e, \u003cem\u003eRsbHLH016\u003c/em\u003e, \u003cem\u003eRsbHLH048\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH115\u003c/em\u003e) exhibited opposite expression patterns after extreme temperature treatment, while the other five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH053\u003c/em\u003e, \u003cem\u003eRsbHLH058\u003c/em\u003e, \u003cem\u003eRsbHLH059\u003c/em\u003e, \u003cem\u003eRsbHLH075\u003c/em\u003e, and \u003cem\u003eRsbHLH107\u003c/em\u003e) displayed the same expression patterns. Further gene function annotation displayed that the orthologous genes of five \u003cem\u003eRsHLHs\u003c/em\u003e (\u003cem\u003eRsbHLH115\u003c/em\u003e, \u003cem\u003eRsbHLH48\u003c/em\u003e, \u003cem\u003eRsbHLH53\u003c/em\u003e, \u003cem\u003eRsbHLH75, and RsbHLH58\u003c/em\u003e) in \u003cem\u003eArabidopsis\u003c/em\u003e were \u003cem\u003eILR3\u003c/em\u003e, \u003cem\u003ePIF3\u003c/em\u003e, \u003cem\u003eMYC2\u003c/em\u003e, \u003cem\u003ebHLH79\u003c/em\u003e, \u003cem\u003eand FBH4\u003c/em\u003e, respectively. Previous studies have revealed that these five genes in \u003cem\u003eArabidopsis\u003c/em\u003e are involved in regulating extreme temperature stress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, 116 \u003cem\u003eRsbHLH\u003c/em\u003e genes were identified in \u003cem\u003eR. simsii\u003c/em\u003e at genome level, which were classified into 13 groups (including 25 subgroups) based on the sequence similarity and phylogenetic relationships. Analysis of chromosomal distribution, gene structure, protein motif discovery, phylogenetic relationship, and cis-acting elements in the promoters of \u003cem\u003eRsbHLH\u003c/em\u003e genes were performed. Comprehensive analysis revealed that 13 \u003cem\u003eRsbHLH\u003c/em\u003e genes might be involved in regulating the flower development of \u003cem\u003eR. simsii.\u003c/em\u003e The qRT-PCR results showed that servral \u003cem\u003eRsbHLH\u003c/em\u003e genes were confirmed to involve in flower development and responsive to extreme temperature. The above results could provide a basis for the functional characterization of bHLH genes, and also provide candidate genes for the future improvement of flower development of \u003cem\u003eR. simsii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.Z. and X.W. designed the experiment; S.T. and X.W. conducted the experiment and analyzed the data; X.W. wrote the manuscript; X.W. and X.Z revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China Project (32260097)\u003c/p\u003e\n\u003cp\u003eand the National Guidance of Local Science and Technology Development Fund of China [2023]009. This study was also supported by the Science and Technology Project of Guizhou Province, China (Qiankehe Foundation-ZK [2022]) and The Science and Technology Department of Guizhou Province (Qian Ke He Zhicheng [2021]Yiban503).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no ethics approval and consent to participate in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOgbourne S, Antalis TM. 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J Exp Bot. 2015;66(1):59\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"R. simsii, Flower development, RsbHLH genes, Phylogenetic analysis, Structure analysis, Expression patterns","lastPublishedDoi":"10.21203/rs.3.rs-4524787/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4524787/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBasic helix-loop-helix (bHLH) gene family, as one of the largest transcription factor families in plants, played essential roles in regulating plant growth and development as well as in response to various environmental stress. However, the bHLH gene family in \u003cem\u003eR. simsii\u003c/em\u003e has not yet been reported. In this study, 116 \u003cem\u003eRsbHLH\u003c/em\u003e genes were identified in the \u003cem\u003eR. simsii\u003c/em\u003e genome, which were divided into 13 groups based on the sequence similarity and phylogenetic relationships. Analysis of gene/protein structure, chromosome location, \u003cem\u003ecis\u003c/em\u003e-acting elements and synteny of the RsbHLH family genes were conducted using the bioinformatic methods. Gene duplication analysis showed that 99 \u003cem\u003eRsbHLH\u003c/em\u003e genes were expanded and retained after dispersed and WGD/segmental duplication events. Investigation of \u003cem\u003ecis\u003c/em\u003e-acting elements within promoters of \u003cem\u003eRsbHLHs\u003c/em\u003e indicated that many \u003cem\u003eRsbHLH\u003c/em\u003e genes might be involved in regulating the diverse physiological processes in \u003cem\u003eR. simsii.\u003c/em\u003e The expression patterns of \u003cem\u003eRsbHLHs\u003c/em\u003e in different tissues and in different stages of flower development were investigated based on the RNA-seq data. Further analysis revealed that 13 candidate \u003cem\u003eRsbHLH\u003c/em\u003e genes might be involved in regulating the flower development of \u003cem\u003eR. simsii.\u003c/em\u003e In addition, the results of qRT-PCR showed that five \u003cem\u003eRsbHLH\u003c/em\u003e genes (\u003cem\u003eRsbHLH033\u003c/em\u003e, \u003cem\u003eRsbHLH016\u003c/em\u003e, \u003cem\u003eRsbHLH048\u003c/em\u003e, \u003cem\u003eRsbHLH114\u003c/em\u003e, and \u003cem\u003eRsbHLH115\u003c/em\u003e) may be involved in positively and negatively regulating the high- and low-temperature stress. These results provide a basis for the functional characterization of \u003cem\u003eRsbHLH\u003c/em\u003e genes and investigations on the molecular mechanisms of flower development of \u003cem\u003eR. simsii\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identifification and Characterization of RsbHLH Transcription Factors Involved in Flower development of R. simsii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 18:09:39","doi":"10.21203/rs.3.rs-4524787/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17dc15fe-4c13-4be6-a152-b41daad8b635","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T06:38:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 18:09:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4524787","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4524787","identity":"rs-4524787","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}