Transcriptome- and genome-wide systematic characterization of bHLH transcription factor family identifies promising members that respond to abiotic stress in tomato

doi:10.21203/rs.3.rs-4866103/v1

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Transcriptome- and genome-wide systematic characterization of bHLH transcription factor family identifies promising members that respond to abiotic stress in tomato

https://doi.org/10.21203/rs.3.rs-4866103/v1

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The bHLH (basic helix-loop-helix) transcription factors function as crucial regulators in numerous biological processes including abiotic stress responses and plant development. According to our RNA-seq analysis of tomato seedlings under salt stress, we found that although bHLH gene family in tomato has been studied, there are still so many tomato bHLH genes have not been identified and named, which will hinder the later study of SlbHLHs. Therefore, the identification of tomato bHLH gene family is lagging and further genome-wide systematical identification and characterization is necessary for tomato bHLH genes. Here, numerous members of different gene families were identified to be the potential and significant candidates that respond to salt stress by RNA-seq analysis. 195 SlbHLHs that unevenly distributed onto 12 chromosomes were identified from tomato genome and were classified into 26 subfamilies based on their molecular features. The collinearity between SlbHLHs and interrelated orthologs from ten plants further revealed evolutionary insights into SlbHLHs. Cis-elements investigations of SlbHLHs promotors futhrer suggested the potential roles of SlbHLHs in tomato development and stress responses. 30 SlbHLHs were defined as the differentially expressed genes in response to salt stress by RNA-seq. The expression profiles of selected SlbHLHs were varyingly and markedly induced by multiple abiotic stresses and hormone treatments. These results provide valuable foundations for further exploring the salt resistance mechanism of tomato and functional characterizations of SlbHLHs, and numerous SlbHLHs may function as the key regulators to enhance plant tolerance to abiotic stress.

Biological sciences/Plant sciences/Plant stress responses/Abiotic

Biological sciences/Plant sciences/Plant molecular biology

RNA-seq

abiotic stress

bHLH transcription factors

systematic characterization

expression analysis

tomato

Transcription factors (TFs) are crucial regulators identified in eukaryotes that take part in the adjustments of different signal transduction pathways and modify the transcript levels of diverse and numerous genes that related to development and stress via the interaction with cis-elements, such as MADS-box, bHLH, NAC, bZIP, WRKY, ERF, and GRAS [1–4]. These TFs can help plants to mediate adaptations to different developmental stages and environmental stresses. Among them, the basic helix-loop-helix (bHLH) TFs have been widely studied in almost all eukaryotes with pleiotropic regulatory functions [5]. All the bHLH transcription factors share a conserved domain that comprises approximately 50 to 60 amino acids and form two linked subregions (the helix–loop–helix (HLH) region and the basic region) [6]. The basic region which is located in the N-terminus of bHLH domain contains 10 to 15 predominantly basic amino acids. It is a DNA-binding region that can bind to the E-box cis-element (5′-CANNTG-3′) of specific genes [7–9]. Combination of bHLH TFs with E-box has been reported to control gene expression levels in multiple biological processes. The HLH region exists in the C-terminus of bHLH domain and comprises around 40 to 50 amino acids. It is made up of two amphiphilic α-helices and hydrophobic residues connected by a variable length loop and acts as a dimerization domain [10, 11]. Structural studies of bHLH proteins in mammalian and yeast indicated that the basic region is the main interface where the DNA contaction occurs, whereas these two helices promote bHLH proteins to form homo- or heterodimers, which is a precondition for the DNA binding [12]. In addition, some bHLH proteins were found to have an additional Myc domain locating at the N-terminus of proteins. In plants, Myc-bHLH protein harbors an MYB interaction region, which interacts with the R2R3-MYB protein to control gene transcript levels and downstream processes [13].

The bHLH TFs are one of the largest TFs families in plant species, and they have been widely revealed to play significant roles in the processes of multiple biological and physiological processes, including diverse plant growth and development and various environmental stresses [9, 14, 15]. Previous studies have reported that bHLH TFs regulate different developmental processes of various vegetative and reproductive tissues. For instance, increased transcripts of an atypical bHLH gene SlPRE2 results in rolling tomato leaves with reduced content of chlorophyll and increased tomato internode length of stem, while upregulation of SlPRE2 leads to decreased fruits and placenta size and thin pericarp [16, 17]. Two bHLH gene AtRHD6 and AtRSL1 control root hair development in Arabidopsis thaliana [18]. The loss-of-function of a bHLH transcription factor MUTE in Arabidopsis generates small, pale and sterile plants with the complete absence of stomata [19]. The bHLH Factor GL3 regulates the initiation of leaf trichome in Arabidopsis [20]. Three HECATE genes, HECATE1 (HEC1), HECATE2 (HEC2) and HECATE3 (HEC3), which encodes the bHLH transcription factors participate in the female reproductive tract development [21]. Two functionally diverging, partially redundant bHLH genes SPATULA and ALCATRAZ are essential for the development of Arabidopsis gynoecium and fruit [22, 23]. Other three bHLH transcription factors, ABORTED MICROSPORES (AMS), BIGPETALp and SPATULA regulate the development of pollen [24], petal [25] and carpel [26], respectively. And GhPRE1A can promote the elongation of cotton fibre via activating the bHLH factor GhPAS1 [27].

Meanwhile, some bHLH TFs also take part in the regulation of multifarious anabolic pathways and signal transductions, including flavonoid, anthocyanin and tryptophan synthesis, light and hormones signal transduction. For instance, the bHLH protein MdbHLH3 in apples promotes the fruit colouration and anthocyanin accumulation in response to low temperature [28]. AtbHLH42 (TT8) regulates the biosynthesis of anthocyanin and procyanidin in vegetative organs in Arabidopsis thaliana [29]. Arabidopsis bHLH protein ATR2 activate the tryptophan pathway [30]. Two Arabidopsis bHLH proteins AtPIF3 and AtPIF4 interact with phytochrome to regulate the transcripts of genes which were involved in regulating light response [31, 32]. An atypical bHLH transcription factor HFR1 can fuction in the signal transduction of phytochrome A via heterodimerization with PIF3 in Arabidopsis thaliana [33]. Three closely related bHLH proteins, BEE1, BEE2, and BEE3 functional redundancy positively regulates brassinosteroids signaling in Arabidopsis [34]. The bHLH proteins INDEHISCENT (IND) in Arabidopsis control gibberellin signal transduction via directly activates GA3ox1 which is a key gene in gibberellin biosynthesis [35].

The bHLH transcription factors also participate in various abiotic stress response [9]. For example, the bHLH protein MYC2 in Arabidopsis and CabHLH035 in pepper are involved in salt response [36, 37]. The transcription factor AtbHLH68 had crucial functions in regulating the resistance to drought stress in Arabidopsis [38]. Two bHLH genes MdbHLH3 and MdCIbHLH1 play significant roles in regulating cold stress response in apple [28, 39]. Arabidopsis bHLH transcription factor PIF4 play crucial roles in stomatal development and plant architecture in high-temperature response [40, 41]. Furthermore, numerous bHLH transcription factors function as crucial regulators in multiple biological processes. For instance, rice OsbHLH148 takes part in trauma response and drought stress and is involved in jasmonic acid (JA) signaling transduction [42]. The transcripts of AtbHLH92 is dramatically induced in response to salt, drought and cold stresses [43]. The Arabidopsis bHLH protein AtbHLH6 serves as the transcriptional activator in the abscisic acid signaling [44], and also play a negative role in blue light-mediated photomorphogenic growth [45].

The diversity of bHLH proteins have been classified as numerous distinct groups according to phylogenetic analyses. Based on the functional properties, DNA-binding specificity and phylogenetic relationships, plant bHLHs are classified into 15–25 subgroups at the beginning [46–49]. Then 26 subfamilies are defined to represent deep evolutionary relationships among plant bHLH proteins using a more scientific classification method [6]. Although the members of tomato bHLH transcription factor family had been identified in previous studies [50, 51], some new tomato bHLH genes which had not been identified in previous studies have been studied and reported to play significant roles in tomato deveolpment, such as SlPRE2 (Solyc02g067380) [16, 17, 52], SlPRE3 (Solyc06g070910) [53] and SlPRE5 (Solyc04g005660) [54], and so on. Therefor, the genome-wide identification and characterization of bHLH TFs in tomato is lagging. Systematical and in-depth genome-wide identification of tomato bHLHs are needed to be studied to identify more bHLHs genes in tomato.

In the study, 195 bHLH genes were identified from tomato genome using more new softwares and websites, and these SlbHLHs were scientifically and systematically named and classified into 26 subfamilies following the method described by Pires and Dolan [6]. In addition, the RNA-seq was performed to screen genes that response to salt stress in tomato roots. 30 tomato SlbHLHs which expression were significantly up- or down-regulated were defined as the differentially expressed genes (DEGs). The molecular characterization, chromosomal location, phylogenetic relationships, conserved domain, structural feature, motif and collinearity investigation of SlbHLHs were studied. Moreover, the expression patterns of multiple SlbHLHs under various abiotic stresses and phytohormones were surveyed. The comprehensive characterization of tomato SlbHLHs provides empirical foundation for further functional study of SlbHLHs in response to abiotic stress and identification of important bHLH protein family can provide understanding for plant adaptive mechanism to environmental stresses.

2.1 Identification and characterization of tomato SlbHLH genes

In this study, the known bHLH proteins in rice and Arabidopsis were used as inquire sequences to screen all the possible bHLH TFs via the BLASTP program. Finally, a total of 195 putative non-redundant bHLH family members were identified from the known tomato genome database (SL4.0). Subsequently, these genes were named bHLH001-bHLH195 following their position on tomato 12 chromosomes (Fig. 1). After that, the molecular weight, protein size (aa), phosphorylation site and theoretical isoelectric point (pI) of 195 SlbHLH proteins were studied. The detailed results were listed in Table S1. The molecular weight and length of SlbHLHs varied widely, with molecular weight ranging from 9.80008 KDa (SlbHLH091) to 102.05852 KDa (SlbHLH182), and length varied from 86 aa (SlbHLH091) to 930 aa (SlbHLH182). The theoretical PI ranged from 4.65 (SlbHLH079) to 11.45 (SlbLHL007). The subcellular location prediction displayed that most of SlbLHLs were located in nucleus, and very few SlbLHLs were located in chloroplast and/or golgi apparatus. The prediction of phosphorylation site of SlbHLHs showed significant variations from 13 (SlbLHL127) to 190 (SlbLHL182), and most SlbHLHs contain more number of Ser sites than that of Thr and Tyr sites. In addition, over 90.8% of the SlbHLHs contain at least 30 phosphorylation sites.

2.2 Chromosome distribution of tomato SlbHLH genes

The chromosome localization investigation exhibited that the 195 SlbHLHs were mapped on all 12 chromosomes of tomato. Among these chromosomes, the Chr 1 contain the greatest number of SlbHLHs (29 genes), and the Chr 2 containd the second number of SlbHLHs (20 genes), the third (Chr 3, Chr 4, Chr 6) and the fourth (Chr 9, Chr 10) also cantian more SlbHLHs (19 and 18 genes, respectively). However, Chr 11 only contain four SlbHLHs, and the rest of chromosomes contain 11–14 SlbHLHs (Fig. 1). The interesting thing was that the chromosomes Chr 6 contain 19 SlbHLHs, while part of larger chromosomes (Chr 5, Chr8, Chr 11) only contain fewer SlbHLHs (11, 11, 4, respectively). In addition, most of the SlbHLHs were located at the ends of tomato chromosomes (Fig. 1). These date indicated that the distribution of SlbHLHs was relatively clustered and uneven among tomato chromosomes and was also disproportionate to chromosome size.

2.3 Phylogenetic relationship of SlbHLH proteins in tomato

To study the phylogenetic relationships of tomato SlbHLHs, the complete protein sequences of 169 Arabidopsis AtbHLHs were tested together with identified 195 tomato SlbHLHs in this investigation (Supplementary file 1, Supplementary file 2, Table S2). Then the MEGA-X software using the Maximum Likelihood method was performed to construct the unrooted phylogenetic tree. According to the topology of the tree, clades support values, and the classification of Arabidopsis AtbHLHs in previous study [6], the phylogenetic analysis exhibited that these 195 SlbHLH proteins were divided into 26 subfamilies (Fig. 2). The number of SlbHLH members included in the 26 subfamilies ranged from 1 to 20, with subfamily XII containing the most SlbHLHs (20) and subfamilies VIIIc (1) with only one SlbHLH. As to certain subfamilies, the bHLH TFs family displayed obvious species bias in the time of evolution, suggesting specific gene duplication happened after species differentiation [55]. Simultaneously, some genes exhibited one-to-one homology, for instance, the subfamily IVb (AtbHLH011 and SlbHLH129, AtbHLH047 and SlbHLH064, AtbHLH121 and SlbHLH028) and subfamily III (a + c) (AtbHLH029 and SlbHLH104), and subfamily IIIf (AtbHLH042 and SlbHLH151), and so on (Fig. 2). The member differences between AtbHLHs and SlbHLHs clustered in the same subfamily suggested that the bHLH gene family had apparent interspecific divergence between Arabidopsis and tomato.

2.4 Collinearity analysis of tomato SlbHLH genes

Genome duplication play significant roles in the process of evolutions and expansions of plant gene families [56]. To investigate the potential gene duplications amone the 195 SlbHLHs, collinear analysis of SlbHLHs was carried out. The results displayed that 13 tandem duplication among 195 SlbHLHs were found, including SlbHLH004-SlbHLH005, SlbHLH011-SlbHLH012, SlbHLH024-SlbHLH025, SlbHLH025-SlbHLH026, SlbHLH046-SlbHLH047, SlbHLH047-SlbHLH048, SlbHLH059-SlbHLH060, SlbHLH060-SlbHLH061, SlbHLH106-SlbHLH107, SlbHLH163-SlbHLH164, SlbHLH164-SlbHLH165, SlbHLH165-SlbHLH166, SlbHLH176-SlbHLH177 (Fig. 1, Table S3-1). And these SlbHLH genes displaying tandem repeats come from the same subfamily. Moreover, the MCScanX programs and BlastP were performed to find the segmental duplications and twenty-one gene pairs were found on 10 of 12 chromosomes as follows: SlbHLH016/168, SlbHLH030/054, SlbHLH032/042, SlbHLH040/087, SlbHLH045/081, SlbHLH049/085, SlbHLH057/114, SlbHLH062/111, SlbHLH063/112, SlbHLH065/110, SlbHLH068/105, SlbHLH078/189, SlbHLH079/190, SlbHLH081/191, SlbHLH083/192, SlbHLH086/193, SlbHLH115/182, SlbHLH130/162, SlbHLH131/161, SlbHLH133/142, SlbHLH132/139 (Fig. 3, Table S3-2). These results exhibited that some chromosomes (Chr02, Chr04, Chr06 and Chr12) harbored more linkage groups than others. Analogously, these linked SlbHLHs were linked inside their subfamilies. However, no fragment duplication of SlbHLH gene pairs were detected on Chr05 and Chr09 (Fig. 3). These results indicated that gene duplication events contributed to the SlbHLHs expansion.

2.5 Collinearity analysis of SlbHLHs between tomato and other plants

To further deduce the evolutionary relationships and origin of tomato SlbHLHs, the collinearity relationships between SlbHLHs and the orthologous genes of ten representative plants was investigated, including two model plants (Oryza sativa and Arabidopsis), two Solanaceae plants (Capsicum annuum and Solanum tuberosum), two Brassica plants (Brassica oleracea and Brassica rape), two Cucurbitaceous plants (Cucumis sativus and Citrullus lanatus), and two cereal plants (Zea mays and Triticum aestivum). The results showed that a total of 141 (72.3%) SlbHLHs shared collinearity relations with those in Solanum tuberosum, following by Capsicum annuum (68), Cucumis sativus (63), Citrullus lanatus (61), Arabidopsis (20), Brassica rapa (12), Brassica oleracea (11), Zea mays (1), Triticum aestivum (1), Oryza sativa (1) (Fig. 4). Therefore, the most collinearity relationship exists between tomato SlbHLHs and Solanum tuberosum genes, indicating a closer relationship between tomato and potato. Moreover, our results also exhibited that many SlbHLH genes had syntenic relationships with 2 to 3 genes of other plants, especially with Solanum tuberosum (Table S4). Analogously, the gene pairs where 2 to 3 SlbHLHs had the collinearity with the same one orthologous gene of tested twelve plants were also observed (Table S4). These results suggested that some orthologous genes may come from a common ancestor of these plants.

2.6 Motif composition analysis of SlbHLH proteins in tomato

To further evaluate the sequence diversities of SlbHLHs, we investigated the motif compositions. A total of 30 conserved motifs were identified from 195 SlbHLHs using the MEME tool on the basis of settings in Arabidopsis (motifs 1–30; Figure S1). In general, the SlbHLHs in the same subfamily exhibited similar motif compositions, which further consolidates the subfamily classification. The results exhibited that the motif number of each SlbHLH ranged from 1–9, which also showed differences in the composition of motifs. Motifs 1 and 2 were widely distributed in the most SlbHLHs, and other motifs were only exist in certain members. Universally, members in the same subfamily harbored similar motif compositions. For instance, all members in subfamilies VIIIc, XI and IVc cantain motifs 1, 2, 3, motifs 1, 2, 3, 9 and motifs 1, 2, 15, 19, respectively. And, some members in the same subfamily also had specific motifs in addition to their common motifs. Moreover, family members in different subfamilies also shared different specific motifs in number and compositions (Figure S1). These data indicated that the number and compositions vary significantly different bHLH subfamilies, and the specific motifs may indicate diverse and distinct functions of SlbHLHs in tomato.

2.7 Conserved domain and gene structure analyses of SlbHLHs

To analyze the sequence diversity of tomato SlbHLHs, the conserved domains and exon-intron structures of each SlbHLH were detected. Conserved domain detection by Batch CD-Search displayed that 20 different forms of bHLH domains with the higher diversity were detected and all SlbHLHs share one or two conserved bHLH domains. In the same subfamilies, the conserved bHLH domain containing in most SlbHLHs had a similar location distribution and gene members. And these SlbHLHs harbored the similar bHLH domain types with few exceptions. This results suggested that only bHLH domain can distinctly construct the phylogenetic relations among bHLH subfamilies. In addition, part of members of Vb, IIIb and Ib (1) contain additional ATG domain (Fig. 5A).

Gene structure analysis by Tbtools software showed that the numbers of SlbHLHs exon markedly varied from 1 to 13, with 25 SlbHLHs having no introns and 25 SlbHLHs only containing 1 intron. SlbHLH177 contained the biggest exon number (13), followed by SlbHLH177 with 12 exons. Furthermore, our results exhibited that the majority of SlbHLHs in the same subfamily generally had similar gene structures and displayed similar exon length and intron number. For example, most SlbHLHs had three or four exons, five or six exons and four exons in subgroups VIIIc (2), XI and IVc, respectively. However, some SlbHLHs exhibited exceptions with differential gene structures in the same subfamilies, such as SlbHLH177 in subfamily Ib (2) and SlbHLH136 in subfamily VIIIa (Fig. 5B). These results suggested that phylogenetic relationships of gene family members were mainly associated with conserved bHLH domains and gene structure.

2.8 Cis-element analysis in the SlbHLHs promoter regions

The cis-elements which are the non-coding sequences in the promoter region of genes are crucial for gene transcription and are universally participated in regulating multiple biological processes [57]. To study the potential regulatory mechanism of SlbHLHs responding to development, abiotic stresses and hormones, the cis-elements within the 2000bp upstream promoter regions of each SlbHLH gene were detected. The data displayed that a total of 4799 CREs in the pormoter sequence of SlbHLHs (Table S5-1) were predicted and 21 types of cis-elements were found (Fig. 6A, Table S5-1). Among them, SlbHLH096 which harbored a total of 44 cis-elements was the gene with the largest number of elements. The number of cis-elements varied in diverse subfamilies (Fig. 6B) and all cis-elements can be categorized as three broad categories (Fig. 6C) :

The first category of cis-elements were relevant to hormone response (1554), including abscisic acid responsive element (361, 7.52%), auxin responsive element (93, 1.95%), gibberellin responsive element (168, 3.52%), MeJA responsive element (420, 8.79%), ethylene responsive element (406, 8.50%) and salicylic acid responsive element (106, 2.22%). The promoter sequences of all subfamilies of SlbHLHs abundantly harbored the elements that in connection with ethylene responsive (ERE), salicylic acid responsive (TCA-element) and abscisic acid responsive (ABRE). Cis-elements (P-box, TATC-box and GARE-motif) associated with gibberellin response were nearly enriched in all subfamilies but absent from subfamily VIIIc (1) and III (a + c). Cis-elements associated with auxin response (TGA-element, AuxRR-core, AuxRE and TGA-box) nearly enriched in all subfamilies but absent from subfamily VIIIc (1) and VIIIa. Cis-elements associated with MeJA response (CGTCA-motif and TGACG-motif) early enriched in all subfamilies expect for the subfamily VIIIc (1) and IIIf. In addition, in the promoter regions of some SlbHLHs, such as SlbHLH015/017/023/029/070/096/110/147/148/175/195 shared multiple identical hormone responsive elements, indicating the possible that they had more intense and rapid responses to specific hormones. In the meantime, in the promoter regions of some SlbHLHs, such as SlbHLH010/027/028/030/073/110/186/ shared diverse hormone response elements indicating their potential functions in numerous hormone regulatory networks (Fig. 6B, Table S5-2).

The second category of cis-elements were associated with growth and development, containing light responsive element (2589, 53.95%), circadian control element (98, 2.04%), seed specific regulatory element (7, 0.15%), meristem expression related element (73, 1.52%), endosperm expression related element (49, 1.02%), cell cycle regulation element (3, 0.06%), root specific regulatory element (2, 0.04%), palisade mesophyll cell differentiation related element (25, 0.52%) and endosperm expression related element (49, 1.02%). Cis-elements related to light responsiveness (such as GA-motif, Box 4, G-box, chs-CMA1a, AE-box, TCT-motif, AT1-motif, GATA-motif et al.) were presented in all SlbHLHs promoters. Analogously, the cis-element related to meristem expression element (CAT-box) also existed in nearly all SlbHLHs except for 4 genes in subfamily IIIf and II. While other cis-elements involved in controlling growth and development were relatively insufficient, especially those elements associated with seed specific regulation, palisade mesophyll cell differentiation, cell cycle regulation, root specific regulation and endosperm specific negative expression. They were absent from numerous subfamilies such as RY-element for seed, MSA-like for cell cycle, motif I for Root specific regulation, HD-Zip 1 for Palisade mesophyll cell differentiation and AACA_motif for endosperm specific negative expression(Fig. 6B, Table S5-2).

The third category of cis-elements were related to stress responsiveness, including wound responsive element (151, 3.15%), drought responsive element (112, 2.33%), low-temperature responsive element (87, 1.81%), defense and stress responsive element (79, 1.65%), dehydration, low-temp, salt stresses related element (1, 0.02%) and flavonoid biosynthetic genes regulation element (18, 0.38%). Among them, cis-elements associated with stress and defense response (TC-rich repeats), low-temperature (LTR), wound response (WUN-motif) and drought response (MBS) presented in almost all subfamilies, while other two elements (dehydration and flavonoid biosynthetic genes regulation) were only existed in a few subfamilies (Fig. 6B, Table S5-2).

2.9 Protein interaction network analysis of SlbHLHs in tomato

Investigating the functional relations of SlbHLH proteins is conducive to revealing their regulatory network. So the STRING software was employed to investigate the portein interacting network of tomato SlbHLHs protein based on the orthologous analysis of Arabidopsis AtbHLHs (Figure S2). In general, multiple members such as PRE1 (SlbHLH041), BIM2 (SlbHLH071/095), PYE (SlbHLH064), bHLH071 (SlbHLH138), bHLH093 (SlbHLH031/045/081/191) and AT5G50915 (SlbHLH057/114) had more interaction relationships than other members did. Among these proteins, GL3 (SlbHLH140), RHD6 (SlbHLH040), AT1G61660 (AtbHLH112, SlbHLH037/053), RSL2 (SlbHLH083/192), LHW (SlbHLH115/141/182), LRL1 (SlbHLH118/184) and UPB1 (SlbHLH009/100) participated in the regulation of root development via the pathway of brassinolide or jasmonic acid or auxin. BIM1 (SlbHLH062/111), BIM2 (SlbHLH071/095), PAR1 (SlbHLH127), MYC2 (SlbHLH132/139) and RGE1 (SlbHLH002/003/006/024/146) play significant roles in controlling embryonic development and/or photomorphogenesis. AT2G41130 (AtbHLH106, SlbHLH080/173), AT3G20640 (AtbHLH123, SlbHLH130/162), ICE1 (SlbHLH065/110), PIF4 (SlbHLH1063/123), and TT8 (SlbHLH151) were participate in controlling low temperture and/or salt stress responses. AT4G29100 (AtbHLH68, SlbHLH067/109/121) and MYC2 (SlbHLH132/139) could enhance the drought stress tolerance through regulating ABA-induced genes under the drought stress. Therefore, the examination of interactions of SlbHLHs indicated that many SlbHLHs tend to form complexes via protein interactions to play significant roles in controlling development and/or stress resistance of plants. These serviceable information will be helpful to investigate the regulatory mechanism of SlbHLHs in regulating stress tolerance and plant growth and development.

2.10 RNA-seq of tomato roots in response to salt stress

Numerous members of various transcription factor families have been reported to be involved in regulating various abiotic stresses. To explore the members of different transcription factor families in response to salt stress, the 8-week-old uniform WT tomato seedlings were treated using 300 mM NaCl for 24h. RNA-seq of salt treated and untreated tomato roots was carried out. The data exhibited that four cDNA libraries of each line were generated to be sequenced and more than 31 million sequence reads of each cDNA library were yielded, representing > 9 Gb sequence data of each sample. Finally, a total of 25274 genes were identified from the salt-treated and untreated tomato roots using FPKM>1 as the standard for gene expression (Fig. 7A). The RNA-seq data of two biological replicates exhibited good correlations and can be used for further investigation (Figure S3A). A summary of RNA-seq, assembly, annotation, and mapping was provided in Table S6-1. Finally, a total of 5548 genes with 2552 genes down-regulated and 2996 genes up-regulated were indentified as the differentially expressed genes (DEGs) between salt-treated and untreated tomato roots using the padj < 0.05 (a adjusted p-value) as the significance cut-off in DESeq2 (Fig. 7A-C, Table S6-2). Using the H-cluster method, these 5548 DEGs also were divided into 4 clusters (sub-clusters 1–4, Figure S3B) with the similar expression trends of these DEGs of each cluster, which was consistent with the hierarchical clustering analysis. Among these DEGs, 2089 DEGs were indentified as the members of different transcription factors families, such as the AP2/ERF, B3, bZIP, DEAD, GRAS, bHLH, Homeobox, MYB, NAC, WRKY, MADS-box, Dof family, and so on (Table S6-3).

To detect the potential functions of these DEGs in response to salt stress, GO (gene ontology) functional enrichment analysis was carried out to categorize them on the strength of their functions in cellular component (CC), biological process (BP), and molecular function (MF). The data displayed that these DEGs were enriched in 888 GO terms (114 CC, 459 BP, 315 MF; padj < 0.05 ). For MF, the DEGs were principally enriched in enzyme activity, transcription factor activity and DNA binding. For CC, the majority of DEGs were associated with cell wall components, protein complex, chromosome, cytoplasmic part, extracellular components and cell nucleus. For BP, most of DEGs were connected to cell wall modification and metabolic, response to stress, transmembrane transport and carbohydrate metabolic process (Fig. 7D, Table S6-4). KEGG analysis exhibited that 106 pathwas were screened and most of the DEGs were mainly enriched in multiple pathways associated with biosynthesis, metabolism and plant hormone signal transduction of numerous amino acids (Fig. 7E, Table S6-5). These data manifested that these DEGs were participate in response to salt stress may via regulating the expression of genes related to cell component modification, amino acid metabolism, enzyme activity, transcription and transmembrane transport.

2.11 Transcriptome‑wide identification of salt-responsive SlbHLHs and their expression patterns under multiple abiotic stresses

The expression level changes of SlbHLHs in WT and salt treated tomato seedlings were analysed based on our transcriptome data in this study. The results showed that almost one-sixth of the SlbHLHs were identified as the DEGs in response to salt stress (Fig. 8, Table S6-6). To verify the RNA-seq results, the expression of 12 SlbHLH genes that displayed obviously different expression change in RNA-seq data were further detected under four kinds of abiotic stresses: drought, salt, cold and heat by qRT-PCR (Fig. 9). The data displayed that 12 SlbHLHs exhibited varied and remarkable expression levels under four abiotic stresses particularly in response to NaCl and PEG6000 stress (Fig. 9). Notably, SlbHLH083 and SlbHLH191 showed the highest induced expression levels under NaCl stress with about 67.4-105.3-fold changes. Higher and similar expression fold changes (8.8–20.3) were examined in the transcript levels of SlbHLH031/053/123/147, while while low induction levels (2.3-4.8-fold) were detected in SlbHLH017/018/089/160 expression under salt stress. The qRT-PCR data displayed good agreement with our RNA-seq data, but no remarkable induction expression of SlbHLH078 was observed since the its induced level showed no significant expression change compared with the 0h data (Fig. 9).

For drought (PEG6000) stress, the expression levels of SlbHLH083/191 were singnificantly upregulated with about 109.1-142.8-fold changes, following by the increased transcripts of SlbHLH031/123/147 with 12.8-35.8-fold. The expression of SlbHLH017/018/053/078/089/160 were increased with about 2.2–4.4 fold, while the SlbHLH010 transcripts showed no obvious changes. For heat stress, the expression levels of SlbHLH017/053/191 were dramatically upregulated with about 7.4-10.4-fold changes, followed by the increased transcripts of SlbHLH010/018/123/147 with 2.6-3.4-fold changes, and SlbHLH031/078/083/089/160 transcripts showed no obvious changes (Fig. 9). For the cold stress, the transcripts of SlbHLH017/018/053/191 were markedly increased with about 4.3-6.7-fold changes, followed by a little increased expression of SlbHLH078/123/160 with 1.2-2.0-fold changes. Two bHLH genes (SlbHLH083/089/134) showed no obvious expression changes, while the expression levels of SlbHLH010/031/147 were downregulated by cold stress (Fig. 9). In conclusion, the diverse and significant transcript levels of SlbHLHs may function as differential and important regulatory genes responding to multiple abiotic stress.

2.12 Expression profiles of SlbHLHs under different hormone treatments

The expression pattern detections of 12 SlbHLHs under various hormone treatments including ABA, IAA, ACC, MeJA and SA were further performed by qRT-PCR. These hormone had been shown that they play critical roles in response to various environmental conditions during plant development [58, 59]. These results displayed that the transcriptional accumulations of these SlbHLHs were increased or reduced to varying degrees after hormone treatments (Fig. 10). Wherein, the expression levels of SlbHLH017/031/053/078/089 could be induced to various degrees by all the hormones treatements (ABA, ACC, IAA, MeJA and SA). Contrarily, the trancscripts of SlbHLH010/018/123/147/160/191 were decreased under several hormone treatments. As a kind of stress hormone, ABA can induce the transcripts of almost all detected SlbHLHs to varying degrees except SlbHLH147. The transcripts of SlbHLH017/018/078/089 exhibited higher fold changes with 4.1-6.5-fold compared with the 0h data under ABA treatment. Notably, the transcripts of SlbHLH017/053/089/191 were significantly upregulated with about 12.01-70.7-fold changes, following by the induced expression of SlbHLH010/031/078/160 with 2.1-4.7-fold changes. The transcripts of SlbHLH017/089 were slightly induced by IAA (3.1-3.6-fold) and the accumulations of SlbHLH017/018/078/083/089/191 were also increased to a similar degree (2.1-3.2-fold) after treated by MeJA. Furthermore, SA treatment could markedly induce the transcripts of SlbHLH078/089/123 to 12.8-45.1-fold, followed by the upregulated rexpression of SlbHLH010/017/018/031/147/160 with 2.35-7.5-fold changes. In addition, the transcript accumulations of multiple SlbHLHs were decreased after treated by different hormone at all or some time points (Fig. 10). The data indicated that many SlbHLH genes may play crucial functions in response to multiple hormone (especially ACC and SA) and/or signal transduction.

Nowadays, tomato has been used as an excellent kind of model plant to study gene function during plant growth and development. Genome-wide systematic characterization of gene families in tomato will provide insights for understanding the regulatory mechanism of crucial regulators during plant development and adaptation to adverse environmental conditions. The bHLH TFs are one of the largest transcription factor families that play crucial roles in controling multifarious biological processes during plant development and in regulating resistance to various adverse environmental stresses [9, 14, 15]. Although tomato bHLH transcription factors had been identified in previous studies [50, 51], some new tomato bHLH genes which had not been identified in previous studies have been reported to play significant roles in tomato deveolpment, such as SlPRE2 (Solyc02g067380) [16, 17, 52], SlPRE3 (Solyc06g070910) [53] and SlPRE5 (Solyc04g005660) [54], and so on. The genome-wide identification and characterization of bHLH TFs in tomato is lagging. Further genome-wide identification of tomato bHLHs are required to identify more bHLH genes in tomato. In this study, the DEGs that response to salt stress in root were analyzed and detected in RNA-seq of tomato root stress. Then the features of SlbHLHs in tomato were systematically and deeply characterized at the genome level, and the transcripts in response to various hormones and abiotic stresses were also analyzed.

3.1 Characterization of SlbHLHs in stomato

In this report, a total of 195 SlbHLHs were identified from tomato genomes, the protein lengths of these tomato bHLH TFs vary from 86 to 930 with the theoretical pI ranges from 4.65 to 11.45. The important variabilities and differences indicate the high levels of complicacy, which may be related to genome sizes or gene-duplication events. The number of 195 SlbHLH genes in tomato was more than Arabidopsis (162) [49], rice (167) [49], tobacco (100) [60], pepper (122) [61], potato (124) [62], foxtail millet (187) [63], and wheat (159) [64], but less than maize (208) [65], Chinese cabbage (230) [66], alfalfa (469) [67], Artemisia annua (226) [68], cauliflower (256) [69], and mango (212) [70]. These results indicated that the significant divergence of bHLH genes occurred among monocot and dicot plants. Notably, no obvious correlation between the of bHLH gene number and the genome size was observed. For instance, 162 bHLH genes were identified from Arabidopsis, while rice (373Mb), tomato (950 Mb), Chinese cabbage (278Mb), and wheat (13.7Gb), with a genome size nearly 3.2, 8.3, 2.4, 112.3 times larger than Arabidopsis (115 Mb), respectively, harboring more or less number of bHLH genes than Arabidopsis. Moreover, 195 SlbHLHs were located on all the chromosomes, but the number of SlbHLHs did not always correspond to the size of tomato chromosome. Analogously, this disproportionate distribution was also observed in rice [49], pepper [61], potato [62], maize [65]. So SlbHLHs distributions were relatively clustered on tomato chromosomes may on account of the uneven duplication events of chromosome fragments.

Gene structures, conserved domain and motifs of bHLH TFs can provided vital clues for investigating their evolutionary relationships. Though the protein properties of SlbHLHs showed significant differences, the domain, sequence motifs and gene structures of SlbHLHs in the same clade were relatively conserved, which can provide the basical reference for the functional relevance and phylogenetic analysis of tomato bHLH genes. Gene structure, domain and motif analysis displayed that closely related tomato SlbHLHs tend to exhibit similar domain, exon/intron structure and motif compositions, as found in other plants such as rice and Arabidopsis [49]. Gene structure study displayed that 85% of SlbHLH genes harbored 1–10 exons in their coding region, which were similar to the bHLH genes in pepper [61], foxtail millet [63], wheat [64], maize [65], cauliflower [69], and passion fruit [71]. Nonetheless, several SlbHLHs exhibited the obvious exceptions of exon number (more than 10 exons), indicating that the high divergences among the SlbHLH genes. These increases or decreases may be caused by chromosomal fusion and rearrangement, and might lead to functional diversifications of gene family members [72].

In all SlbHLH proteins, 20 different types of bHLH domains that harbored the high diversity were observed in tomato, and these gene members which blong to the same subfamily had the similar types of bHLH domains with few exceptions. The motif 1 and motif 2 which served as the components of the conserved bHLH domain were highly conserved within almost all tomato bHLH proteins and were crucial for the functional specificities of bHLH TFs [73]. Although most bHLH proteins had similar conserved domains and motifs, significant differences of molecular characteristics among bHLH proteins were observed, which may result from the differences of their non-conserved regions. The additional compositions and numbers of motifs vary distinctly among different SlbHLH subfamilies. A number of specific motifs were found only in a certain subfamilies, indicating that they may play diverse functions during tomato development and growth, because the widely reported crucial roles of bHLH genes in numerous biological processes [17, 36, 74–77].

3.2 Phylogenetic relationship and collinearity analysis of SlbHLHs tomato

Phylogenetic analysis diplayed that the 195 tomato bHLH TFs were divided into 26 subfamilies on the basis of classifications and sequence homologies from Arabidopsis AtbHLH proteins and known bHLH family members of other plants [5, 6, 49]. At least one SlbHLH protein was observed in each Arabidopsis subfamily [6, 48], indicating that the differences of bHLH TFs may be earlier than dicots and monocots, while some new members and subfamilies were generated as evolution proceeded. In addition, the results of classification of subfamilies exhibited differences and similarities compared with that of other plants, manifesting more diversity in the function and structure of bHLH proteins in different plants [78]. Previously, phylogenetic studies classified rice [49] and radish [79] bHLHs into 22 subfamilies, and 21, 15, 20, 27, and 27 subfamilies were divided in the bHLHs of Arabidopsis [47], tobacco [60], passion fruit [71], barley [80] and Mango [70], respectively. These results indicate that bHLH proteins show diversity in diverse plants, which results in the difference and complexity of the bHLH classifications. Moreover, the conserved domain and motif detections also corroborated these classifications, the close SlbHLHs proteins of the same subgroups generally harbored the common compositions of domains and motifs.

Gene duplication events such as tandem, genome or segmental duplication are one of the significant driving forces in the expansion and evolution of numerous gene families of plant species, which can drive the generation of species and novel functional genes, hence plants can survive in adverse environment in the time of evolution [56, 60, 81]. The results of collinearity analysis exhibited that many SlbHLHs were identified as the segmental and tandem duplications, and the contributions of duplications to the increased SlbHLHs are similar. It is worth mentioning that several duplicated SlbHLHs participate in the tolerance to various abiotic stress. Previous studies in Arabidopsis and rice [5, 49], tobacco [60], pepper [61], Chinese cabbage [66], cauliflower [69] and apple [82] indicated that the tandem, genome or segmental duplication events may explain the expansion and evolution of tomato bHLH TFs family. Analogously, many SlbHLH gene members were identified as the segmental and tandem duplications via collinearity analysis, suggesting that some SlbHLHs may be generated by gene duplication events in tomato, further explaining the mechanism that results in bHLH gene expansion. Moreover, the SlbHLH genes displaying segmental duplication and tandem repeat are members from the same subfamily, the expansions of SlbHLH proteins from the specific subfamilies may be of benefit to the adaptation of tomato to changing environmental conditions during growth and development [80, 83]. In summary, our results further demonstrate that segmental and tandem duplications may contribute to the SlbHLH gene expansion that had reported in many other plant species, such as Chinese cabbage [66], cauliflower [69], Raphanus sativus [79], and apple [82], and so on.

In addition, the synteny analysis evaluating the relationships of SlbHLHs and their counterparts of ten plant species were investigated, including model plants Arabidopsis thaliana and Oryza sativa, representative Brassica, Solanaceae, Cucurbitaceae and Gramineae plant species. Among them, the orthologous gene number of tomato and Solanum tuberosum was the largest and located in similar locations on chromosomes, which supported the evolutionary relationships between them, following by cucumber, pepper, citrullus lanatus, watermelon and Arabidopsis. These orthologous gene pairs may derive from the common ancestor of tomato and corresponding plants. Furthermore, the more intricate relations such as single Solanum tuberosum-to-multiple tomato genes were found, suggesting that these homologous bHLH genes may have significant roles in the evolutions of tomato SlbHLH genes. But only one or two homologous genes were found between tomato and three gramineae plants (Zea mays, Triticum aestivum and Oryza sativa), this phenomenon probably because of the fact that enormous chromosomal fusions or rearrangements happened in their genomes, and the identifications of synteny relationships were severely obscured by selective gene loss [84]. These findings may be related to the evolutionary relationships between tomato and these ten kinds of plants. And large-scale gene and fragment duplication might precede the divergences of a number of plants and harbored significant funtions in the expansions of bHLH gene family.

3.3 Transcriptome analysis of DEGs responding to salt stress

In plants species, numerous members of transcription factor families, such as bHLH [67, 71], NAC [85, 86], GRAS [87, 88], MADS [89, 90], bZIP [4, 91–93], MYB [94, 95], WRKY [96–98], Dof [99, 100] et al., have been reported to take part in response to biotic and/or abiotic stress. Previous study had reported that numerous members of different gene families have been indentified in response to abiotic stress via the RNA-seq [101]. Also, our RNA-seq showed that 5548 DEGs with 2996 DEGs up-regulated and 2552 DEGs down-regulated were indentified as the significance cut-off in DESeq2. Among these 5548 DEGs, 2089 DEGs blongs to members of 398 transcription factors families, such as the AP2/ERF, B3, C3H, bZIP, DEAD, GRAS, bHLH, Homeobox, MYB, NAC, WRKY, MADS-box, and Dof family, and so on. And other 3459 DEGs also blong to different gene families, such as cytochrome P450, E3 ubiquitin-protein ligase, zinc finger protein, pectinesterase (PE), polygalacturonase (PG), expansin et. al. All these DEGs are related to enzyme activity, DNA binding, cell wall components, cell wall modification and metabolic, signal transduction of plant hormone, stress response, transmembrane transport and carbohydrate metabolic process, and so on. These data indicate that plenty of genes play significant roles in response to salt stress via regulating or impacting the transcripts of genes related to cell component modification, amino acid metabolism, enzyme activity, signal transduction, transcription and transmembrane transport et.al. In brief, our data provide a research basis for screening available gene resources in tomato that respond strongly to salt stress.

3.4 Expression profiling and functional prediction of SlbHLHs in tomato

To date, bHLH transcription factors have received wide attentions because of their extensive involvements in regulating diverse biological processes during plant development, especially in the regulation of the adaptation to numerous abiotic stress, including resistance to salt, drought, low/high temperature stresses, oxidative stress, iron deficiency, heavy metal stress, and osmotic stress [9, 102–106]. In addition, they also participate in the crosstalk of different hormones signaling, such as brassinosteroid (BR), salicylic acid (SA), abscisic acid (ABA), ethylene (ET), and jasmonic acid (JA) [74], and they are crucial for the growth and survival of plant species in environment. The increasing number of investigations related to the significant roles of bHLH proteins in controlling plant responses to diverse adverse environmental stimulus manifest that bHLHs are promising candidates for increasing the tolerance of crops to numerous environmental stresses through molecular breeding.

Drought, high salinity and cold stress are the major environmental factors which can cause biochemical and physiological effects on plants. These effects are mainly demonstrated in reduced enzyme activities, disordered hormone metabolism, decreased photosynthesis efficiency, and plant vigor and germination rates, leading to irreversible damage to plant yield, crop quality and growth [9, 107, 108]. Many studies have reported that numerous bHLH TFs play significant roles in regulating numerous abiotic stresses tolerance in various plant species [36, 102, 109, 110]. For instance, AtbHLH068 play a significant role responding to the drought stress by the ABA-dependent pathway via regulating the components of ABA metabolism and/or signaling [38]. AtbHLH122 positively regulate the response to NaCl, osmotic and drought stress in Arabidopsis [111]. Arabidopsis AtbHLH112 is involved in regulating genes related to abiotic stress tolerance (salt, drought and ABA) via binding to their GCG-box and E-box motifs [112]. Overexpression of a bHLH transcription gene OsWIH2 in rice led to significantly higher tolerance to drought [113]. The bHLH transcription factor ZmPTF1 regulates the tolerance to drought stress through promoting ABA biosynthesis and root development in maize [77]. The AtbHLH148 (AIF2/RITF1) participates in the ROS detoxification generated by salt stress via interacting with RSA1 (SHORT ROOT IN SALT MEDIUM 1) to regulate salt stress related genes [114]. AtbHLH106 confers salt, drought, cold, and ABA resistance on Arabidopsis by regulating multiple genes via binding to their G-box motif [115]. Overexpression of rice bHLH gene OsbHLH38 which participates in ABA-dependent salt tolerance enhanced the tolerance to salt stress [116]. The rice bHLH TF OsbHLH062 can interacts with OsJAZ9 to endow plants salt tolerance via modulating the Jasmonic acid (JA) signaling pathway [117]. Ectopic expression of tomato bHLH gene SlICE1a in tobacco results in enhanced cold and salt stress tolerance of transgenic plants [118]. Overexpression the bHLH gene AtICE1(AtbHLH116) increased the cold tolerance through ABA independent pathway in transgenic Arabidopsis [119]. Silencing of CabHLH035 in pepper decreased the resistance of transgenic pepper to cold stress, while overexpression of CabHLH035 resulted in increased the tolerance of transgenic Arabidopsis to cold stress [120].

In addition, the Arabidopsis AtbHLH009/PIF4 play significant roles in many other biological processes, including high temperature-mediated adaptations of plant architecture, phytochrome B signaling, and anthocyanin accumulation [32, 40, 121]. The bHLH transcription factor DcTT8 regulates the biosynthesis of anthocyanin in Dendrobium candidum [122]. Two bHLH transcription factors AtbHLH104 and AtbHLH115 play crucial roles in iron (Fe) homeostasis maintenance in Arabidopsis [123, 124]. Overexpression of FtbHLH3, a bHLH gene of tartary buckwheat, enhances the tolerance to drought/oxidative stress in transgenic Arabidopsis [125]. Three bHLH TFs MYC2 (AtbHLH002), MYC3 (AtbHLH005), and MYC4 (AtbHLH004) are needed for jasmonate-mediated suppression of Arabidopsis flowering [126]. The phytochrome-interacting bHLH TF PIL5 (AtbHLH015) directly regulates GAI and RGA expression to control gibberellin responsiveness in Arabidopsis seeds [127]. The bHLH transcription factor HFR1/AtbHLH026 is involved in phytochrome A and cryptochrome signalling [128]. In Arabidopsis, PRE3 (AtbHLH135), a non-DNA‐binding bHLH TF, participates in the control of light signaling pathway [129]. Three bHLH TFs PIF3 (AtbHLH008), PIF4 (AtbHLH009), and PIF5 (AtbHLH065) play signifcant roles in dark-induced and age-triggered leaf senescence [130].

In our study, the data of RNA-seq analyses and qRT-PCR displayed that most of the examined SlbHLHs exhibited obvious different expression patterns under different kinds of abiotic stresses and hormone treatments, suggesting the diverse and crucial functions of tomato bHLH TFs in resistance to diversified adverse environmental conditions. For instance, the transcripts of multiple SlbHLHs, particularly SlbHLH031, SlbHLH083, SlbHLH147, SlbHLH191, were dramatically induced by various abiotic stresses. These increased transcript accumulation of these bHLH genes may help plants to decrease the damage generated by adverse abiotic stresses. Previous studies reported that the bHLH members of close subfamily might have similar functions in resistance to different abiotic stresses [74, 130]. Moreover, the potential roles of SlbHLHs in stress resistance were further certified by cis-element and phylogenetic analysis. Recent investigations displayed that bHLH members from multiple subgroups, including AtbHLH-068/-148 of subgroup orphan, AtbHLH122 of subgroup IX, AtbHLH112 of subgroup X, AtbHLH116 of subgroup IIIB, AtbHLH-009/-015/-026 of subgroup VII were all in connection with resistance to a variety of abiotic stresses as described above [74, 102, 103]. Therefore, the transcripts of SlbHLH018 in subgroup orphan, SlbHLH053 in subgroup IX, SlbHLH053 in subgroup X, SlbHLH-031/-147/-191 in subgroup IIIB, SlbHLH123 in subgroup VII were significantly increased by various stresses, indicating that they might also participate in stress response pathways.

Phytohormones, especially SA, JA, ACC and ABA, play vital functions in assisting plant species to resist various adverse environmental stresses [58]. For instance, AtbHLH068 regulates the response of transgenic Arabidopsis to drought stress through the ABA-dependent pathway [38]. OsbHLH062 could endow plant species with tolerance to salt stress by modulating the JA signaling pathway [117]. Furthermore, the cis-elements containing in the promoter regions of functional genes play crucial roles in regulating gene transcription. In this study, our data diplayed that numerous cis-elements related to different stresses and hormones including the DRE, P-box, CGTCA-motif, TCA-element, ABRE, MBS, LTR, GARE-motif and ERE were observed in most SlbHLHs promoters. These results were similar to the previous studies of pepper[61], Nicotiana tabacum [60], Passiflora edulis [71], Raphanus sativus [79]. In this study, the transcripts of most detected SlbHLHs (SlbHLH-010/-017/-018/-031/-053/-078/-089/-160/-191) were dramatically induced or decreased by one or a few phytohormones, such as ABA, ACC, IAA, MeJA and SA. However, the detailed biological functions of most tomato SlbHLHs require to be further investigated. Therefore, investigating the participation of SlbHLHs in the regulation of environmental stresses could provide valuable insights to study their potential functions in stress tolerance.

In this sutdy, numerous genes and transcription factors from different families were identified to be the potential and significant candidates that in response to salt stress through RNA-seq analysis. Finally, a total of 195 bHLH TFs were identified and characterized from tomato genome. Among these bHLH genes, new members which had not been identfied from previous studies of SlbHLH gene family were found in tomato, such as SlbHLH033 (PRE2), SlbHLH072 (PRE5), SlbHLH113 (PRE3) et al. The phylogenetic relationship, syntenic analysis, molecular characterization, gene structure, chromosome localization, and conserved domain and motifs of these 195 bHLH TFs were investigated systematically in this study. The relationships between SlbHLHs and abiotic stress were studied. Segmental and tandem duplications are conducive to the SlbHLHs expansions in tomato, and collinearity analysis of orthologous genes in eleven representative plant species further provided significant clues to the phylogenetic relationships of SlbHLHs. The SlbHLH genes in relation to abiotic stress were identified via RNA-seq analysis and qRT-PCR data. The transcriptional levels of multiple SlbHLH genes, particularly the SlbHLH031, SlbHLH083, SlbHLH123, SlbHLH147 and SlbHLH191, were dramatically increased under numerous abiotic stresses and hormone treatments, providing the promising candidates for the genetic engineering of tomato with enhanced stress resistance. Further verification of the regulatory mechanisms and specific roles of the excellent candidate SlbHLH genes in response to stress tolerance is necessary. In summary, all results obtained in this study will provide more valuable information to further understand the significance and regulatory mechanism of the bHLH transcription factor family, and expect the promising prospects of multiple SlbHLH members in regulating tomato tolerance to abiotic stresses.

5.1 Plant material and growth conditions

In this study, the tomato AC⁺⁺ (Solanum lycopersicum Mill. cv. Ailsa Craig) was employed as the wild-type (WT). All the WT tomato seedlings were cultivated under the standard cultivation environment (16/8 h day/night cycle at 25/18°C, 250 µmol m^–2 s^–1, 80% humidity) until they were used.

5.2 Identification of tomato bHLH genes

The GFF annotations and genome sequences of Solanum lycopersicum and other plant species were obtained from Ensembl Plants database (http://plants.ensembl.org/index.html). All the amino acid sequences of Arabidopsis AtbHLHs were obtained from TAIR database (https://www.arabidopsis.org/) based on the previous studies [47, 49]. After that, these Arabidopsis bHLH proteins were used as inquires to blast the tomato protein sequence database using the BLASTp program with E-value (1e-5) to obtain the candidate protein sequences. Subsequently, the Batch CD-Search database (https://www.ncbi.nlm.nih.gov/cdd/Structure/cdd/wrpsb.cgi), PROSITE database (https://prosite.expasy.org/) and the Pfam database (http://pfam.xfam.org/) were employed to confirm these collected candidate bHLH members whether contain a bHLH domain. Ultimately, the screened non-redundant sequences which were regarded as the putative bHLH proteins were used for further study. The nucleotide and protein sequences of each SlbHLH were listed in Supplementary file 1.

5.3 Phylogenetic tree construction and sequence alignment of tomato bHLH proteins

For phylogenetic analysis, a total of 169 Arabidopsis AtbHLHs which were identified by many studies were used in this study [5, 47, 131]. But in Bailey’s and Toledo-Ortiz’s reports, we found that AtbHLH133 (At2g20095) and AtbHLH152 (At1g22380) were not bHLH proteins, so these two proteins were not used in this study. Then a new bHLH protein (AT2G20100.3) was renamed AtbHLH133, but the serial number AtbHLH152 was not used in this study. In addition, other 8 AtbHLHs were named AtbHLH163-170 following Carretero-Paulet’s study [5]. After that, these 169 AtbHLH protein sequences in Arabidopsis were used to perform the construction of unrooted phylogenetic tree with tomato SlbHLH proteins. First, a total of 365 bHLH protein sequences were used for the sequence alignment employing the ClustalW software with default parameters. Then the MEGA-X software was employed to perform the construction of the phylogenetic tree using the obtained data by the Maximum Likelihood method [132]. The following parameters were employed: the best WGA + G + F evolutionary model calculated via MEGA-X with 1000 bootstrap value and partial deletions. The construction of phylogenetic tree of 195 SlbHLH proteins also were performed using the same method.

5.4 Protein property, conserved domain, motif and protein interaction analysis of SlbHLHs

The online ExPASy database (http://expasy.org/) was employed for the evaluation of the Mw (molecular weight) and the theoretical pI (isoelectric point) of 195 SlbHLH proteins. The online NetPhos 3.1 database (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1) and the Plant-mPLoc database (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) was employed to predict the phosphorylation sites and the subcellular locations of each SlbHLH protein, respectively. The Batch CD-Search database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and the MEME 5.5.0 (https://meme-suite.org/meme/tools/meme) were applied to investigate the conserved domain and the motif of each SlbHLH protein, respectively. Subsequently, the STRING 12.0 website (https://cn.string-db.org/) was employed to construct the potential protein interacting network.

5.5 Gene structure of SlbHLH genes and cis‑element analyses of their promotors

The intron-exon structure of each SlbHLH gene was illustrated using the genomic sequence and the GFF annotation data of 195 SlbHLHs, and the TBtools software v1.106 was used to present the results [133]. The 2.0 kb sequence of each SlbHLH gene promoter were eatrated from tomato genome by Tbtools software to investage the potential cis-elements that associated with hormone-, growth and devleopment and stress in the promoter region of 195 SlbHLHs using the plantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Finally, statistical analysis was performed to investigate the cis-elements of each SlbHLH promoter. The TBtools software and NovoMagic tool (https://magic.novogene.com/customer/main#/homeNew) were used to visualize all obtained results.

5.6 Chromosomal distribution and collinearity analysis of tomato SlbHLHs

The chromosomal distribution of 195 SlbHLH genges was analysed on account of the GFF annotation information of tomato genomes provided by Sol Genomics Network (https://solgenomics.net/). For the collinearity analysis between SlbHLHs and associated genes in other plants, the genome and the GFF annotation data of tomato and ten representative plants including Oryza sativa, Arabidopsis thaliana, Capsicum annuum, Solanum tuberosum, Brassica oleracea, Brassica rapa, Cucumis sativus, Citrullus lanatus, Zea mays and Triticum aestivum were downloaded from TAIR, Sol Genomics Network, phytozome (https://phytozome-next.jgi.doe.gov/) and EnsemblPlants (http://plants.ensembl.org/index.html) and. The Multiple Collinearity Scan toolkit (MCScanX) with the default parameter was performed to evaluate the collinearity relationships and gene duplications [134]. And finally, the TBtools softwares and circos were employed to finish the visualization of results, the value 30 was set to the minimum block size [133, 135].

5.7 Transcriptome-wide analysis of salt-responsive SlbHLH genes

The 8-week-old uniform WT tomato seedlings were selected for the salt stress treatment. Salt stress were carried out by submerging the roots of WT tomato seedlings in 300 mM NaCl for 24h, and the untreated WT tomato seedlings were used as the control. Then the roots of untreated and salt-treated tomato plants were harvested respectively from more than six different seedlings. Two biological replicates of untreated and salt-treated samples were used to perform the RNA-seq analyses. Then 3 µg total RNA from each sample was obtained and employed as the input material for the construction of sequencing libraries. Then 4 samples were sent to the Novogene Bioinformatics Institute (Beijing, China) for assembly and transcriptome sequencing.

All samples were sequenced and transcriptome analysis was performed by the Illumina HiSeq 2500 platform. Clean reads were filtered by removing reads including poly-N, adapters, low-quality reads and duplication sequences from the raw data. The transcriptome assembly was carried out by stringTie (v. 1.3.3b) [136]. The tomato reference genomes database (https://solgenomics.net/organism/Solanum_lycopersicum/genome) was employed as the annotation of gene function. Read counts was used to estimate gene expression, and genes with |log2 (fold change)| >1 and padj/q-value < 0.05 (adjusted P-value) determined by DESeq2 were considered as differentially expressed genes (DEGs) [137]. The clusterProfiler R package [138] was used to implement the GO enrichment analysis of DEGs and to detected the statistical enrichment of DEGs by KEGG pathway enrichment analysis (https://www.genome.jp/kegg) [139–141]. The pathway of DEGs with padj (adjust P-value) < 0.05 was identified as the dramatically enriched GO terms and KEGG pathway enrichment. Ultimately, the SlbHLH genes and other gene families which response to salt stress were screened from the obtained RNA-seq data. Read counts were used to detect the gene expression and DEGs were screened by Log2 (fold change) and false discovery rate (FDR) as previously described [142]. The RNA-seq data are available in the National Center for Biotechnology Information (NCBI) database under the accession numbers (SRR8184599; SRR8184598) of our privous study [142] and the accession numbers (SAMN38113668, SAMN38113669) of submission.

5.8 Abiotic stress and hormone treatments

Tomato seedlings of AC⁺⁺ were planted in a green house, then potted 8-week-old uniform WT tomato seedlings were selected for the performance of all treatments. The drought and salt stresses were performed by respectively submerging tomato roots in 20% (m/v) PEG6000 and 300 mM NaCl solutions, then roots and leaves at the same position were harvested. Heat and cold stresses were performed by respectively culturing WT seedlings at 4°C and 42°C, and then leaves located at the same position were harvested. For the hormone treatments, all needed WT seedlings were sprayed, respectively, with 2 mM SA or 0.1 mM ACC, ABA, IAA, MeJA solutions, and leaves located at the same position were harvested. After that, all samples were harvested at 0, 1, 6, 12, 24, 48 h post different treatments. Three independent biological replicates were used for each treatment. All samples were harvested and liquid nitrogen was used to immediately frozen all samples. Finally, All harvested samples were stored at -80°C until being used to performed RNA extraction.

5.9 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis of salt‑responsive SlbHLH genes

Total RNA was extracted from each sample using the plant RNA Extraction Kits (omega, USA, www.omegabiotek.com) with gDNA Eraser following the manufacturers’ instruction. Reverse transcription and the first-strand cDNA synthesis was performed using the same methods described before [143]. Then the synthesized cDNAs were diluted to 15 ng/µL using the RNase/DNase-free water. Then the CFX96™ Real-Time System (Bio-Rad, USA) was used to implement the qRT-PCR following the same the same method as described before [143]. The stomato EF1α gene was used as the internal standard [144]. All needed primers had been listed in Table S7. Part of salt‑responsive SlbHLH genes which were identified as the differentially expressed genes were tested using three independent biological replicates. Meanwhile, three technical replicates were also needed in each biological replicate.

Statistical analyses

Data were presented as mean ± SE standard deviation. It was adopted that a cut-off value of two-fold for the expression of each differential gene was considered as the biological significance [145]. The graphs were generated using the originPro software (v8.0, SAS Institute)

Institutional Review Board Statement

Not applicable

Informed Consent

Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (no. ZR2021MC079, no. ZR2020QB154). Science and technology Huimin demonstration special of Qingdao (solanaceous vegetables new varieties breeding and demonstration promotion, 23-2-8-xdny-15-nsh). Chongqing Postdoctoral Science Foundation (no. CSTB2022NSCQ-BHX0037).

Author Contribution

Conceptualization: J.L.Z., Z.H.Z., Y.P.; Experimental design: J.L.Z., Z.H.Z., Y.P.; Writing-original draft preparation: J.L.Z., Z.H.Z.; Writing–review and editing: J.L., M.K.Z., Y.P.; Data Collection: J.L.Z., X.Y.L., D.D.; Data analysis: J.L.Z, M.K.Z, J.L.; Software: J.L.Z., M.K.Z.; Validation: X.Y.L., M.K.Z.; Supervision: Z.H.Z., J.L., Y.P.; Data curation: J.L.Z., M.K.Z., D.D.; Project administration: Z.H.Z., Y.Y.S.; Funding acquisition: J.L.Z., Z.H.Z, D.D., Y.Y.S.; All authors have read and agreed to the published version of the manuscript.

Data Availability

Most data generated or analysed during this study are included in this published article and its supplementary information files. Sequence data that support the findings of this study have been deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/sra) with the accession numbers (SRR26696993, SRR26696994, SRR26696995, SRR26696996).

Waseem, M.; Nkurikiyimfura, O.; Niyitanga, S.; Jakada, B. H.; Shaheen, I.; Aslam, M. M., GRAS transcription factors emerging regulator in plants growth, development, and multiple stresses. Molecular Biology Reports 2022, 49, (10), 9673–9685.
Erpen, L.; Devi, H. S.; Grosser, J. W.; Dutt, M., Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants. Plant Cell, Tissue and Organ Culture (PCTOC) 2018, 132, (1), 1–25.
Wang, Y.; Zhang, J.; Hu, Z.; Guo, X.; Tian, S.; Chen, G., Genome-Wide Analysis of the MADS-Box Transcription Factor Family in Solanum lycopersicum. International Journal of Molecular Sciences 2019, 20, (12), 2961–2983.
Liu, S.; Zhang, C.; Zhu, Q.; Guo, F.; Chai, R.; Wang, M.; Deng, X.; Dong, T.; Meng, X.; Zhu, M., Genome- and transcriptome-wide systematic characterization of bZIP transcription factor family identifies promising members involved in abiotic stress response in sweetpotato. Scientia Horticulturae 2022, 303, 111185.
Carretero-Paulet, L.; Galstyan, A.; Roig-Villanova, I.; Martínez-García, J. F.; Bilbao-Castro, J. R.; Robertson, D. L. J. P. p., Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae^1[W]. 2010, 153, (3), 1398–1412.
Pires, N.; Dolan, L., Origin and Diversification of Basic-Helix-Loop-Helix Proteins in Plants. Molecular Biology and Evolution 2010, 27, (4), 862–874.
Atchley, W. R.; Terhalle, W.; Dress, A., Positional Dependence, Cliques, and Predictive Motifs in the bHLH Protein Domain. Journal of Molecular Evolution 1999, 48, (5), 501–516.
Massari, M. E.; Murre, C., Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms. Molecular and Cellular Biology 2000, 20, (2), 429–440.
Sun, X.; Wang, Y.; Sui, N., Transcriptional regulation of bHLH during plant response to stress. Biochemical and biophysical research communications 2018, 503, (2), 397–401.
Murre, C.; McCaw, P. S.; Baltimore, D., A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 1989, 56, (5), 777–783.
Ferré-D'Amaré, A. R.; Pognonec, P.; Roeder, R. G.; Burley, S. K., Structure and function of the b/HLH/Z domain of USF. The EMBO journal 1994, 13, (1), 180–9.
Jones, S., An overview of the basic helix-loop-helix proteins. Genome Biology 2004, 5, (6), 226.
Grotewold, E.; Sainz, M. B.; Tagliani, L.; Hernandez, J. M.; Bowen, B.; Chandler, V. L., Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proceedings of the National Academy of Sciences 2000, 97, (25), 13579–13584.
Murre, C., Helix-loop-helix proteins and the advent of cellular diversity: 30 years of discovery. Gene Dev 2019, 33, (1–2), 6–25.
Dennis, D. J.; Han, S.; Schuurmans, C., bHLH transcription factors in neural development, disease, and reprogramming. Brain research 2019, 1705, 48–65.
Zhu, Z.; Chen, G.; Guo, X.; Yin, W.; Yu, X.; Hu, J.; Hu, Z., Overexpression of SlPRE2, an atypical bHLH transcription factor, affects plant morphology and fruit pigment accumulation in tomato. Sci Rep 2017, 7, (1), 5786.
Zhu, Z.; Liang, H.; Chen, G.; Li, F.; Wang, Y.; Liao, C.; Hu, Z., The bHLH transcription factor SlPRE2 regulates tomato fruit development and modulates plant response to gibberellin. Plant cell reports 2019, 38, (9), 1053–1064.
Menand, B.; Yi, K.; Jouannic, S.; Hoffmann, L.; Ryan, E.; Linstead, P.; Schaefer, D. G.; Dolan, L., An Ancient Mechanism Controls the Development of Cells with a Rooting Function in Land Plants. Science 2007, 316, (5830), 1477–1480.
Pillitteri, L. J.; Sloan, D. B.; Bogenschutz, N. L.; Torii, K. U., Termination of asymmetric cell division and differentiation of stomata. Nature 2007, 445, (7127), 501–505.
Morohashi, K.; Zhao, M.; Yang, M.; Read, B.; Lloyd, A.; Lamb, R.; Grotewold, E., Participation of the Arabidopsis bHLH Factor GL3 in Trichome Initiation Regulatory Events. Plant Physiology 2007, 145, (3), 736–746.
Gremski, K.; Ditta, G.; Yanofsky, M. F., The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana. Development 2007, 134, (20), 3593–3601.
Rajani, S.; Sundaresan, V., The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence. Current Biology 2001, 11, (24), 1914–1922.
Groszmann, M.; Paicu, T.; Alvarez, J. P.; Swain, S. M.; Smyth, D. R., SPATULA and ALCATRAZ, are partially redundant, functionally diverging bHLH genes required for Arabidopsis gynoecium and fruit development. The Plant Journal 2011, 68, (5), 816–829.
Xu, J.; Ding, Z.; Vizcay-Barrena, G.; Shi, J.; Liang, W.; Yuan, Z.; Werck-Reichhart, D.; Schreiber, L.; Wilson, Z. A.; Zhang, D., ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis. The Plant Cell 2014, 26, (4), 1544–1556.
Szécsi, J.; Joly, C.; Bordji, K.; Varaud, E.; Cock, J. M.; Dumas, C.; Bendahmane, M., BIGPETALp, a bHLH transcription factor is involved in the control of Arabidopsis petal size. The EMBO journal 2006, 25, (16), 3912–3920.
Heisler, M. G. B.; Atkinson, A.; Bylstra, Y. H.; Walsh, R.; Smyth, D. R., SPATULA, a gene that controls development of carpel margin tissues in Arabidopsis, encodes a bHLH protein. Development 2001, 128, (7), 1089–1098.
Wu, H.; Fan, L.; Guo, M.; Liu, L.; Liu, L.; Hou, L.; Zheng, L.; Qanmber, G.; Lu, L.; Zhang, J.; Li, F.; Yang, Z., GhPRE1A promotes cotton fibre elongation by activating the DNA-binding bHLH factor GhPAS1. Plant Biotechnology Journal 2023, 21, (5), 896–898.
Xie, X.-B.; Li, S.; Zhang, R.-F.; Zhao, J.; Chen, Y.-C.; Zhao, Q.; Yao, Y.-X.; You, C.-X.; Zhang, X.-S.; Hao, Y.-J., The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant, Cell & Environment 2012, 35, (11), 1884–1897.
Baudry, A.; Heim, M. A.; Dubreucq, B.; Caboche, M.; Weisshaar, B.; Lepiniec, L., TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. The Plant Journal 2004, 39, (3), 366–380.
Smolen, G. A.; Pawlowski, L.; Wilensky, S. E.; Bender, J., Dominant Alleles of the Basic Helix-Loop-Helix Transcription Factor ATR2 Activate Stress-Responsive Genes in Arabidopsis. Genetics 2002, 161, (3), 1235–1246.
Ni, M.; Tepperman, J. M.; Quail, P. H., PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein. Cell 1998, 95, (5), 657–67.
Huq, E.; Quail, P. H., PIF4, a phytochrome-interacting bHLH factor, functions as a negative regulator of phytochrome B signaling in Arabidopsis. The EMBO journal 2002, 21, (10), 2441–2450.
Fairchild, C. D.; Schumaker, M. A.; Quail, P. H., HFR1 encodes an atypical bHLH protein that acts in phytochrome A signal transduction. Genes Dev 2000, 14, (18), 2377–91.
Friedrichsen, D. M.; Nemhauser, J.; Muramitsu, T.; Maloof, J. N.; Alonso, J.; Ecker, J. R.; Furuya, M.; Chory, J., Three Redundant Brassinosteroid Early Response Genes Encode Putative bHLH Transcription Factors Required for Normal Growth. Genetics 2002, 162, (3), 1445–1456.
Arnaud, N.; Girin, T.; Sorefan, K.; Fuentes, S.; Wood, T. A.; Lawrenson, T.; Sablowski, R.; Østergaard, L., Gibberellins control fruit patterning in Arabidopsis thaliana. Genes & Devlopment 2010, 24, (19), 2127–32.
Verma, D.; Jalmi, S. K.; Bhagat, P. K.; Verma, N.; Sinha, A. K. J. T. F. j., A bHLH transcription factor, MYC2, imparts salt intolerance by regulating proline biosynthesis in Arabidopsis. The FEBS journal 2020, 287, (12), 2560–2576.
Zhang, H.; Guo, J.; Chen, X.; Zhou, Y.; Pei, Y.; Chen, L.; Lu, M.; Gong, H.; Chen, R. J. H. R., Pepper bHLH transcription factor CabHLH035 contributes to salt tolerance by modulating ion homeostasis and proline biosynthesis. 2022, 9.
Le Hir, R.; Castelain, M.; Chakraborti, D.; Moritz, T.; Dinant, S.; Bellini, C., AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana. Physiologia Plantarum 2017, 160, (3), 312–327.
Feng, X.-M.; Zhao, Q.; Zhao, L.-L.; Qiao, Y.; Xie, X.-B.; Li, H.-F.; Yao, Y.-X.; You, C.-X.; Hao, Y.-J., The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple. BMC Plant Biology 2012, 12, (1), 22.
Koini, M. A.; Alvey, L.; Allen, T.; Tilley, C. A.; Harberd, N. P.; Whitelam, G. C.; Franklin, K. A., High Temperature-Mediated Adaptations in Plant Architecture Require the bHLH Transcription Factor PIF4. Current Biology 2009, 19, (5), 408–413.
Lau, O. S.; Song, Z.; Zhou, Z.; Davies, K. A.; Chang, J.; Yang, X.; Wang, S.; Lucyshyn, D.; Tay, I. H. Z.; Wigge, P. A.; Bergmann, D. C., Direct Control of SPEECHLESS by PIF4 in the High-Temperature Response of Stomatal Development. Current biology: CB 2018, 28, (8), 1273–1280.e3.
Seo, J.-S.; Joo, J.; Kim, M.-J.; Kim, Y.-K.; Nahm, B. H.; Song, S. I.; Cheong, J.-J.; Lee, J. S.; Kim, J.-K.; Choi, Y. D., OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. The Plant Journal 2011, 65, (6), 907–921.
Jiang, Y.; Yang, B.; Deyholos, M. K., Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress. Molecular genetics and genomics: MGG 2009, 282, (5), 503–16.
Abe, H.; Urao, T.; Ito, T.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K., Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling. Plant Cell 2003, 15, (1), 63–78.
Yadav, V.; Mallappa, C.; Gangappa, S. N.; Bhatia, S.; Chattopadhyay, S., A Basic Helix-Loop-Helix Transcription Factor in Arabidopsis, MYC2, Acts as a Repressor of Blue Light–Mediated Photomorphogenic Growth. The Plant Cell 2005, 17, (7), 1953–1966.
Buck, M. J.; Atchley, W. R., Phylogenetic analysis of plant basic helix-loop-helix proteins. J Mol Evol 2003, 56, (6), 742–50.
Toledo-Ortiz, G.; Huq, E.; Quail, P. H., The Arabidopsis Basic/Helix-Loop-Helix Transcription Factor Family^[W]. The Plant Cell 2003, 15, (8), 1749–1770.
Heim, M. A.; Jakoby, M.; Werber, M.; Martin, C.; Weisshaar, B.; Bailey, P. C., The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 2003, 20, (5), 735–47.
Li, X.; Duan, X.; Jiang, H.; Sun, Y.; Tang, Y.; Yuan, Z.; Guo, J.; Liang, W.; Chen, L.; Yin, J.; Ma, H.; Wang, J.; Zhang, D., Genome-Wide Analysis of Basic/Helix-Loop-Helix Transcription Factor Family in Rice and Arabidopsis. Plant Physiology 2006, 141, (4), 1167–1184.
Wang, J.; Hu, Z.; Zhao, T.; Yang, Y.; Chen, T.; Yang, M.; Yu, W.; Zhang, B., Genome-wide analysis of bHLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato (Solanum lycopersicum). BMC genomics 2015, 16, (1), 39.
Sun, H.; Fan, H. J.; Ling, H. Q., Genome-wide identification and characterization of the bHLH gene family in tomato. BMC genomics 2015, 16, (1), 9.
Zhu, Z.; Luo, M.; Li, J.; Cui, B.; Liu, Z.; Fu, D.; Zhou, H.; Zhou, A., Comparative transcriptome analysis reveals the function of SlPRE2 in multiple phytohormones biosynthesis, signal transduction and stomatal development in tomato. Plant Cell Reports 2023, 42, (5), 921–937.
Guo, P.; Yang, Q.; Wang, Y.; Yang, Z.; Xie, Q.; Chen, G.; Chen, X.; Hu, Z., Overexpression of SlPRE3 alters the plant morphologies in Solanum lycopersicum. Plant Cell Reports 2023.
Li, J.; Gong, J.; Zhang, L.; Shen, H.; Chen, G.; Xie, Q.; Hu, Z. J. J. o. P. P., Overexpression of SlPRE5, an atypical bHLH transcription factor, affects plant morphology and chlorophyll accumulation in tomato. 2022, 273, 153698.
Xu, J.; Xu, H.; Zhao, H.; Liu, H.; Xu, L.; Liang, Z., Genome-wide investigation of bHLH genes and expression analysis under salt and hormonal treatments in Andrographis paniculata. Industrial Crops and Products 2022, 183, 114928.
Cannon, S. B.; Mitra, A.; Baumgarten, A.; Young, N. D.; May, G., The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC plant biology 2004, 4, (1), 1–21.
Zhao, J.; Zhai, Z.; Li, Y.; Geng, S.; Song, G.; Guan, J.; Jia, M.; Wang, F.; Sun, G.; Feng, N.; Kong, X.; Chen, L.; Mao, L.; Li, A., Genome-Wide Identification and Expression Profiling of the TCP Family Genes in Spike and Grain Development of Wheat (Triticum aestivum L.). Frontiers in Plant Science 2018, 9.
Verma, V.; Ravindran, P.; Kumar, P. P., Plant hormone-mediated regulation of stress responses. BMC plant biology 2016, 16, 1–10.
Bari, R.; Jones, J. D., Role of plant hormones in plant defence responses. Plant molecular biology 2009, 69, 473–488.
Bano, N.; Patel, P.; Chakrabarty, D.; Bag, S. K. J. P.; Plants, M. B. o., Genome-wide identification, phylogeny, and expression analysis of the bHLH gene family in tobacco (Nicotiana tabacum). 2021, 27, (8), 1747–1764.
Zhang, Z.; Chen, J.; Liang, C.; Liu, F.; Hou, X.; Zou, X., Genome-Wide Identification and Characterization of the bHLH Transcription Factor Family in Pepper (Capsicum annuum L.). Frontiers in Genetics 2020, 11.
Wang, R.; Zhao, P.; Kong, N.; Lu, R.; Pei, Y.; Huang, C.; Ma, H.; Chen, Q., Genome-wide identification and characterization of the potato bHLH transcription factor family. Genes 2018, 9, (1), 54.
Fan, Y.; Lai, D.; Yang, H.; Xue, G.; He, A.; Chen, L.; Feng, L.; Ruan, J.; Xiang, D.; Yan, J. J. B. G., Genome-wide identification and expression analysis of the bHLH transcription factor family and its response to abiotic stress in foxtail millet (Setaria italica L.). 2021, 22, (1), 1–18.
Wang, L.; Xiang, L.; Hong, J.; Xie, Z.; Li, B., Genome-wide analysis of bHLH transcription factor family reveals their involvement in biotic and abiotic stress responses in wheat (Triticum aestivum L.). 3 Biotech 2019, 9, (6), 236–248.
Zhang, T.; Lv, W.; Zhang, H.; Ma, L.; Li, P.; Ge, L.; Li, G., Genome-wide analysis of the basic Helix-Loop-Helix (bHLH) transcription factor family in maize. Bmc Plant Biology 2018, 18, (1), 235.
Song, X.-M.; Huang, Z.-N.; Duan, W.-K.; Ren, J.; Liu, T.-K.; Li, Y.; Hou, X.-L., Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis). Molecular Genetics and Genomics 2014, 289, (1), 77–91.
Li, G.; Jin, L.; Sheng, S., Genome-Wide Identification of bHLH Transcription Factor in Medicago sativa in Response to Cold Stress. Genes 2022, 13, (12), 2371.
Chang, S.; Li, Q.; Huang, B.; Chen, W.; Tan, H., Genome-wide identification and characterisation of bHLH transcription factors in Artemisia annua. BMC Plant Biology 2023, 23, (1), 63.
Jiang, H.; Liu, L.; Shan, X.; Wen, Z.; Zhang, X.; Yao, X.; Niu, G.; Shan, C.; Sun, D. J. P.; Plants, M. B. o., Genome-wide identification and expression analysis of the bHLH gene family in cauliflower (Brassica oleracea L.). 2022, 28, (9), 1737–1751.
Salih, H.; Tan, L.; Htet, N. N. W., Genome-Wide Identification, Characterization of bHLH Transcription Factors in Mango. Tropical Plant Biology 2021, 14, (1), 72–81.
Liang, J.; Fang, Y.; An, C.; Yao, Y.; Wang, X.; Zhang, W.; Liu, R.; Wang, L.; Aslam, M.; Cheng, Y. J. I. J. o. B. M., Genome-wide identification and expression analysis of the bHLH gene family in passion fruit (Passiflora edulis) and its response to abiotic stress. 2022.
Xu, G.; Guo, C.; Shan, H.; Kong, H., Divergence of duplicate genes in exon–intron structure. Proceedings of the National Academy of Sciences 2012, 109, (4), 1187–1192.
Feller, A.; Machemer, K.; Braun, E. L.; Grotewold, E., Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal 2011, 66, (1), 94–116.
Hao, Y.; Zong, X.; Ren, P.; Qian, Y.; Fu, A. J. I. J. o. M. S., Basic Helix-Loop-Helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis. 2021, 22, (13), 7152.
Lu, R.; Zhang, J.; Wu, Y.-W.; Wang, Y.; Zhang, J.; Zheng, Y.; Li, Y.; Li, X.-B. J. P. P., bHLH transcription factors LP1 and LP2 regulate longitudinal cell elongation. 2021, 187, (4), 2577–2591.
Liang, Y.; Ma, F.; Li, B.; Guo, C.; Hu, T.; Zhang, M.; Liang, Y.; Zhu, J.; Zhan, X., A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato. 2022.
Li, Z.; Liu, C.; Zhang, Y.; Wang, B.; Ran, Q.; Zhang, J., The bHLH family member ZmPTF1 regulates drought tolerance in maize by promoting root development and abscisic acid synthesis. Journal of Experimental Botany 2019, 70, (19), 5471–5486.
Ledent., V.; Vervoort., M., The Basic Helix-Loop-Helix Protein Family: Comparative Genomics and Phylogenetic Analysis. Genome Research 2001, 11, (5), 754–770.
Wang, R.; Li, Y.; Gao, M.; Han, M.; Liu, H., Genome-wide identification and characterization of the bHLH gene family and analysis of their potential relevance to chlorophyll metabolism in Raphanus sativus L. BMC genomics 2022, 23, (1), 548.
Quan, X.; Liang, X.; Xie, C.; Yin, N.; Zhang, N.; Li, H.; He, W., Genome-wide identification and characterization of barley bHLH transcription factors and their expression in response to low nitrogen stress. 2020.
Lynch, M.; Conery, J. S., The Evolutionary Fate and Consequences of Duplicate Genes. Science 2000, 290, (5494), 1151–1155.
Yang, J.; Gao, M.; Huang, L.; Wang, Y.; van Nocker, S.; Wan, R.; Guo, C.; Wang, X.; Gao, H., Identification and expression analysis of the apple (Malus × domestica) basic helix-loop-helix transcription factor family. Scientific Reports 2017, 7, (1), 28.
Fan, Y.; Yang, H.; Lai, D.; He, A.; Cheng, J. J. B. G., Genome-wide identification and expression analysis of the bHLH transcription factor family and its response to abiotic stress in sorghum [Sorghum Bicolor (L.) Moench]. 2021, 22, (1).
Paterson, A. H.; Wang, X.; Tang, H.; Lee, T. H., Synteny and Genomic Rearrangements. In Plant Genome Diversity Volume 1: Plant Genomes, their Residents, and their Evolutionary Dynamics, Wendel, J. F.; Greilhuber, J.; Dolezel, J.; Leitch, I. J., Eds. Springer Vienna: Vienna, 2012; pp 195–207.
Guo, F.; Liu, S.; Zhang, C.; Dong, T.; Meng, X.; Zhu, M., Genome-wide systematic survey and analysis of NAC transcription factor family and their response to abiotic stress in sweetpotato. Scientia Horticulturae 2022, 299, 111048.
Shiriga, K.; Sharma, R.; Kumar, K.; Yadav, S. K.; Hossain, F.; Thirunavukkarasu, N., Genome-wide identification and expression pattern of drought-responsive members of the NAC family in maize. Meta Gene 2014, 2, 407–417.
Zhang, C.; Liu, S.; Liu, D.; Guo, F.; Yang, Y.; Dong, T.; Zhang, Y.; Ma, C.; Tang, Z.; Li, F.; Meng, X.; Zhu, M., Genome-wide survey and expression analysis of GRAS transcription factor family in sweetpotato provides insights into their potential roles in stress response. BMC Plant Biology 2022, 22, (1), 232.
Wang, T.-T.; Yu, T.-F.; Fu, J.-D.; Su, H.-G.; Chen, J.; Zhou, Y.-B.; Chen, M.; Guo, J.; Ma, Y.-Z.; Wei, W.-L.; Xu, Z.-S., Genome-Wide Analysis of the GRAS Gene Family and Functional Identification of GmGRAS37 in Drought and Salt Tolerance. Frontiers in Plant Science 2020, 11, (2024), 1–19.
Yang, Z.; Nie, G.; Feng, G.; Xu, X.; Li, D.; Wang, X.; Huang, L.; Zhang, X., Genome-wide identification of MADS-box gene family in orchardgrass and the positive role of DgMADS114 and DgMADS115 under different abiotic stress. International Journal of Biological Macromolecules 2022, 223, 129–142.
Castelán-Muñoz, N.; Herrera, J.; Cajero-Sánchez, W.; Arrizubieta, M.; Trejo, C.; García-Ponce, B.; Sánchez, M. d. l. P.; Álvarez-Buylla, E. R.; Garay-Arroyo, A., MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants. Frontiers in Plant Science 2019, 10, (853).
Banerjee, A.; Roychoudhury, A., Abscisic-acid-dependent basic leucine zipper (bZIP) transcription factors in plant abiotic stress. Protoplasma 2017, 254, (1), 3–16.
Liu, H.; Tang, X.; Zhang, N.; Li, S.; Si, H., Role of bZIP Transcription Factors in Plant Salt Stress. International Journal of Molecular Sciences 2023, 24, (9), 7893.
Gai, W.-X.; Ma, X.; Qiao, Y.-M.; Shi, B.-H.; ul Haq, S.; Li, Q.-H.; Wei, A.-M.; Liu, K.-K.; Gong, Z.-H., Characterization of the bZIP Transcription Factor Family in Pepper (Capsicum annuum L.): CabZIP25 Positively Modulates the Salt Tolerance. Frontiers in Plant Science 2020, 11, (139), 1–18.
Roy, S., Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome. Plant Signaling & Behavior 2016, 11, (1), e1117723.
Li, J.; Zhou, H.; Xiong, C.; Peng, Z.; Du, W.; Li, H.; Wang, L.; Ruan, C., Genome-wide analysis R2R3-MYB transcription factors in Xanthoceras sorbifolium Bunge and functional analysis of XsMYB30 in drought and salt stresses tolerance. Industrial Crops and Products 2022, 178, 114597.
Liu, S.; Zhang, C.; Guo, F.; Sun, Q.; Yu, J.; Dong, T.; Wang, X.; Song, W.; Li, Z.; Meng, X.; Zhu, M., A systematical genome-wide analysis and screening of WRKY transcription factor family engaged in abiotic stress response in sweetpotato. BMC Plant Biology 2022, 22, (1), 616.
Jiang, J.; Ma, S.; Ye, N.; Jiang, M.; Cao, J.; Zhang, J., WRKY transcription factors in plant responses to stresses. Journal of integrative plant biology 2017, 59, (2), 86–101.
Li, W.; Pang, S.; Lu, Z.; Jin, B., Function and Mechanism of WRKY Transcription Factors in Abiotic Stress Responses of Plants. In 2020; Vol. 9.
Liu, J.; Meng, Q.; Xiang, H.; Shi, F.; Ma, L.; Li, Y.; Liu, C.; Liu, Y.; Su, B., Genome-wide analysis of Dof transcription factors and their response to cold stress in rice (Oryza sativa L.). BMC genomics 2021, 22, (1), 800.
Zhang, C.; Dong, T.; Yu, J.; Hong, H.; Liu, S.; Guo, F.; Ma, H.; Zhang, J.; Zhu, M.; Meng, X., Genome-wide survey and expression analysis of Dof transcription factor family in sweetpotato shed light on their promising functions in stress tolerance. Frontiers in Plant Science 2023, 14.
Meng, X.; Liu, S.; Dong, T.; Xu, T.; Ma, D.; Pan, S.; Li, Z.; Zhu, M., Comparative transcriptome and proteome analysis of salt-tolerant and salt-sensitive sweet potato and overexpression of IbNAC7 confers salt tolerance in Arabidopsis. Frontiers in Plant Science 2020, 11, 1342.
Guo, J.; Sun, B.; He, H.; Zhang, Y.; Tian, H.; Wang, B., Current Understanding of bHLH Transcription Factors in Plant Abiotic Stress Tolerance. International Journal of Molecular Sciences 2021, 22, (9), 4921.
Qian, Y.; Zhang, T.; Yu, Y.; Gou, L.; Yang, J.; Xu, J.; Pi, E., Regulatory Mechanisms of bHLH Transcription Factors in Plant Adaptive Responses to Various Abiotic Stresses. Frontiers in Plant Science 2021, 12, 677611.
Wang, K.; Liu, H.; Mei, Q.; Yang, J.; Ma, F.; Mao, K., Characteristics of bHLH transcription factors and their roles in the abiotic stress responses of horticultural crops. Scientia Horticulturae 2023, 310, 111710.
Zhao, Q.; Ren, Y.-R.; Wang, Q.-J.; Yao, Y.-X.; You, C.-X.; Hao, Y.-J., Overexpression of MdbHLH104 gene enhances the tolerance to iron deficiency in apple. Plant Biotechnology Journal 2016, 14, (7), 1633–1645.
Babitha, K. C.; Vemanna, R. S.; Nataraja, K. N.; Udayakumar, M., Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress. PLOS ONE 2015, 10, (9), e0137098.
Song, J.; Fan, H.; Zhao, Y.; Jia, Y.; Du, X.; Wang, B., Effect of salinity on germination, seedling emergence, seedling growth and ion accumulation of a euhalophyte Suaeda salsa in an intertidal zone and on saline inland. Aquatic Botany 2008, 88, (4), 331–337.
Shabala, S., Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops. Annals of Botany 2013, 112, (7), 1209–1221.
Ren, Y.-R.; Yang, Y.-Y.; Zhao, Q.; Zhang, T.-E.; Wang, C.-K.; Hao, Y.-J.; You, C.-X. J. E.; Botany, E., MdCIB1, an apple bHLH transcription factor, plays a positive regulator in response to drought stress. 2021, 188, 104523.
Qiu, J.-R.; Huang, Z.; Xiang, X.-Y.; Xu, W.-X.; Wang, J.-T.; Chen, J.; Song, L.; Xiao, Y.; Li, X.; Ma, J. J. B. P. B., MfbHLH38, a Myrothamnus flabellifolia bHLH transcription factor, confers tolerance to drought and salinity stresses in Arabidopsis. 2020, 20, (1), 1–14.
Liu, W.; Tai, H.; Li, S.; Gao, W.; Zhao, M.; Xie, C.; Li, W.-X., bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism. New Phytologist 2014, 201, (4), 1192–1204.
Liu, Y.; Ji, X.; Nie, X.; Qu, M.; Zheng, L.; Tan, Z.; Zhao, H.; Huo, L.; Liu, S.; Zhang, B.; Wang, Y., Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytologist 2015, 207, (3), 692–709.
Gu, X.; Gao, S.; Li, J.; Song, P.; Zhang, Q.; Guo, J.; Wang, X.; Han, X.; Wang, X.; Zhu, Y.; Zhu, Z., The bHLH transcription factor regulated gene OsWIH2 is a positive regulator of drought tolerance in rice. Plant Physiology and Biochemistry 2021, 169, 269–279.
Guan, Q.; Wu, J.; Yue, X.; Zhang, Y.; Zhu, J., A Nuclear Calcium-Sensing Pathway Is Critical for Gene Regulation and Salt Stress Tolerance in Arabidopsis. PLOS Genetics 2013, 9, (8), e1003755.
Ahmad, A.; Niwa, Y.; Goto, S.; Ogawa, T.; Shimizu, M.; Suzuki, A.; Kobayashi, K.; Kobayashi, H., bHLH106 Integrates Functions of Multiple Genes through Their G-Box to Confer Salt Tolerance on Arabidopsis. PLOS ONE 2015, 10, (5), e0126872.
Du, F.; Wang, Y.; Wang, J.; Li, Y.; Zhang, Y.; Zhao, X.; Xu, J.; Li, Z.; Zhao, T.; Wang, W.; Fu, B., The basic helix-loop-helix transcription factor gene, OsbHLH38, plays a key role in controlling rice salt tolerance. Journal of Integrative Plant Biology 2023, 65, (8), 1859–1873.
Wu, H.; Ye, H.; Yao, R.; Zhang, T.; Xiong, L., OsJAZ9 acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice. Plant Science 2015, 232, 1–12.
Feng, H.-L.; Ma, N.-N.; Meng, X.; Zhang, S.; Wang, J.-R.; Chai, S.; Meng, Q.-W., A novel tomato MYC-type ICE1-like transcription factor, SlICE1a, confers cold, osmotic and salt tolerance in transgenic tobacco. Plant Physiology and Biochemistry 2013, 73, 309–320.
Viswanathan, C.; Masaru, O.; Siddhartha, K.; Byeong-Ha, L.; Xuhui, H.; Manu, A.; Jian-Kang, Z., ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 2003, 17, (8), 1043–1054.
Zhang, H.; Guo, J.; Chen, X.; Zhou, Y.; Pei, Y.; Chen, L.; Haq, S. u.; Zhang, M.; Gong, H.; Chen, R., Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper. Horticultural Plant Journal 2023.
Qin, J.; Zhao, C.; Wang, S.; Gao, N.; Wang, X.; Na, X.; Wang, X.; Bi, Y., PIF4-PAP1 interaction affects MYB-bHLH-WD40 complex formation and anthocyanin accumulation in Arabidopsis. Journal of Plant Physiology 2022, 268, 153558.
Jia, N.; Wang, J.-J.; Liu, J.; Jiang, J.; Sun, J.; Yan, P.; Sun, Y.; Wan, P.; Ye, W.; Fan, B., DcTT8, a bHLH transcription factor, regulates anthocyanin biosynthesis in Dendrobium candidum. Plant Physiology and Biochemistry 2021, 162, 603–612.
Liang, G.; Zhang, H.; Li, X.; Ai, Q.; Yu, D., bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana. Journal of Experimental Botany 2017, 68, (7), 1743–1755.
Zhang, J.; Liu, B.; Li, M.; Feng, D.; Jin, H.; Wang, P.; Liu, J.; Xiong, F.; Wang, J.; Wang, H.-B., The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis. The Plant Cell 2015, 27, (3), 787–805.
Yao, P.-F.; Li, C.-L.; Zhao, X.-R.; Li, M.-F.; Zhao, H.-X.; Guo, J.-Y.; Cai, Y.; Chen, H.; Wu, Q., Overexpression of a Tartary Buckwheat Gene, FtbHLH3, Enhances Drought/Oxidative Stress Tolerance in Transgenic Arabidopsis. Frontiers in Plant Science 2017, 8.
Wang, H.; Li, Y.; Pan, J.; Lou, D.; Hu, Y.; Yu, D., The bHLH transcription factors MYC2, MYC3, and MYC4 are required for Jasmonate-mediated inhibition of flowering in Arabidopsis. Molecular Plant 2017, 10, (11), 1461–1464.
Oh, E.; Yamaguchi, S.; Hu, J.; Yusuke, J.; Jung, B.; Paik, I.; Lee, H.-S.; Sun, T.-p.; Kamiya, Y.; Choi, G., PIL5, a Phytochrome-Interacting bHLH Protein, Regulates Gibberellin Responsiveness by Binding Directly to the GAI and RGA Promoters in Arabidopsis Seeds. The Plant Cell 2007, 19, (4), 1192–1208.
Duek, P. D.; Fankhauser, C., HFR1, a putative bHLH transcription factor, mediates both phytochrome A and cryptochrome signalling. The Plant Journal 2003, 34, (6), 827–836.
Castelain, M.; Le Hir, R.; Bellini, C., The non-DNA-binding bHLH transcription factor PRE3/bHLH135/ATBS1/TMO7 is involved in the regulation of light signaling pathway in Arabidopsis. Physiologia Plantarum 2012, 145, (3), 450–460.
Song, Y.; Yang, C.; Gao, S.; Zhang, W.; Li, L.; Kuai, B., Age-triggered and dark-induced leaf senescence require the bHLH transcription factors PIF3, 4, and 5. Molecular plant 2014, 7, (12), 1776–1787.
Bailey, P. C.; Martin, C.; Toledo-Ortiz, G.; Quail, P. H.; Huq, E.; Heim, M. A.; Jakoby, M.; Werber, M.; Weisshaar, B., Update on the Basic Helix-Loop-Helix Transcription Factor Gene Family in Arabidopsis thaliana. The Plant Cell 2003, 15, (11), 2497–2502.
Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K., MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution 2018, 35, (6), 1547–1549.
Chen, C.; Chen, H.; Zhang, Y.; Thomas, H. R.; Frank, M. H.; He, Y.; Xia, R., TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Molecular plant 2020, 13, (8), 1194–1202.
Wang, Y.; Tang, H.; DeBarry, J. D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-h.; Jin, H.; Marler, B.; Guo, H.; Kissinger, J. C.; Paterson, A. H., MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research 2012, 40, (7), e49-e49.
Krzywinski, M.; Schein, J.; Birol, I.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S. J.; Marra, M. A., Circos: An information aesthetic for comparative genomics. Genome research 2009, 19, (9), 1639–1645.
Pertea, M.; Pertea, G. M.; Antonescu, C. M.; Chang, T. C.; Mendell, J. T.; Salzberg, S. L., StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature biotechnology 2015, 33, (3), 290–295.
Love, M. I.; Huber, W.; Anders, S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology 2014, 15, (12), 550–570.
Yu, G.; Wang, L.-G.; Han, Y.; He, Q.-Y., clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters. Omics: A Journal of Integrative Biology 2012, 16, (5), 284–287.
Kanehisa, M.; Goto, S., KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research 2000, 28, (1), 27–30.
Kanehisa, M., Toward understanding the origin and evolution of cellular organisms. Protein Science 2019, 28, (11), 1947–1951.
Kanehisa, M.; Furumichi, M.; Sato, Y.; Kawashima, M.; Ishiguro-Watanabe, M., KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Research 2022, 51, (D1), D587-D592.
Meng, X.; Cai, J.; Deng, L.; Li, G.; Sun, J.; Han, Y.; Dong, T.; Liu, Y.; Xu, T.; Liu, S.; Li, Z.; Zhu, M., SlSTE1 promotes abscisic acid-dependent salt stress-responsive pathways via improving ion homeostasis and reactive oxygen species scavenging in tomato. Journal of Integrative Plant Biology 2020, 62, (12), 1942–1966.
Zhang, J.; Hu, Z.; Yao, Q.; Guo, X.; Nguyen, V.; Li, F.; Chen, G., A tomato MADS-box protein, SlCMB1, regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening. Scientific reports 2018, 8, (1), 3413–3427.
Expósito-Rodríguez, M.; Borges, A. A.; Borges-Pérez, A.; Pérez, J. A., Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC plant biology 2008, 8, (1), 131–142.
Mingku, Z.; Guoping, C.; Tingting, D.; Lingling, W.; Jianling, Z.; Zhiping, Z.; Zongli, H., SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato. PLoS One 2015, 10, (8), e0133849.

No competing interests reported.

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Transcriptome- and genome-wide systematic characterization of bHLH transcription factor family identifies promising members that respond to abiotic stress in tomato

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Abstract

Figures

1. Introduction

2. Results

2.1 Identification and characterization of tomato SlbHLH genes

2.2 Chromosome distribution of tomato SlbHLH genes

2.3 Phylogenetic relationship of SlbHLH proteins in tomato

2.4 Collinearity analysis of tomato SlbHLH genes

2.5 Collinearity analysis of SlbHLHs between tomato and other plants

2.6 Motif composition analysis of SlbHLH proteins in tomato

2.7 Conserved domain and gene structure analyses of SlbHLHs

2.8 Cis-element analysis in the SlbHLHs promoter regions

2.9 Protein interaction network analysis of SlbHLHs in tomato

2.10 RNA-seq of tomato roots in response to salt stress

2.11 Transcriptome‑wide identification of salt-responsive SlbHLHs and their expression patterns under multiple abiotic stresses

2.12 Expression profiles of SlbHLHs under different hormone treatments

3. Discussion

3.1 Characterization of SlbHLHs in stomato

3.2 Phylogenetic relationship and collinearity analysis of SlbHLHs tomato

3.3 Transcriptome analysis of DEGs responding to salt stress

3.4 Expression profiling and functional prediction of SlbHLHs in tomato

4. Conclusion

5. Materials and methods

5.1 Plant material and growth conditions

5.2 Identification of tomato bHLH genes

5.3 Phylogenetic tree construction and sequence alignment of tomato bHLH proteins

5.4 Protein property, conserved domain, motif and protein interaction analysis of SlbHLHs

5.5 Gene structure of SlbHLH genes and cis‑element analyses of their promotors

5.6 Chromosomal distribution and collinearity analysis of tomato SlbHLHs

5.7 Transcriptome-wide analysis of salt-responsive SlbHLH genes

5.8 Abiotic stress and hormone treatments

5.9 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis of salt‑responsive SlbHLH genes

Declarations

Institutional Review Board Statement

Conflicts of Interest:

Funding

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Data Availability

References

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