Transcriptome-wide identification and characterization of WD40 genes, as well as their tissue-specific expression profiles and responses to heat stress in Dimocarpus longan Lour

The WD40 transcription factor (TF) family is widespread in plants and plays important roles in plant growth and development, transcriptional regulation, and tolerance to abiotic stresses. WD40 TFs have been identified and characterized in a diverse series of plant species. However, little information is available on WD40 genes from D. longan. In this study, a total of 45 DlWD40 genes were identified from D. longan RNASeq data, and further analysed by bioinformatics tools. Also, the expression patterns of DlWD40 genes in roots and leaves, as well as responses to heat stress, were evaluated using quantitative real-time PCR (qRT-PCR). We found that the 45 DlWD40 proteins, together with 80 WD40 proteins from Arabidopsis and Zea mays, could be categorized into six groups. Of these, the DlWD40-4 protein was highly homologous to Arabidopsis WDR5a, a protein participating in tolerance to abiotic stresses. Moreover, a total of 25 cis-acting elements, such as abiotic stress and flavonoid biosynthesis elements, were found in the promoters of DlWD40 genes. The DlWD40-33 gene is targeted by miR3627, which has been proposed to be involved in flavonoid biosynthesis. Using qRT-PCR, ten of the 45 DlWD40 genes were demonstrated to have diverse expression patterns between roots and leaves, and these ten DlWD40 genes could also respond to varying durations of a 38 °C heat stress in roots and leaves. The results reported here will provide a basis for the further functional verification of DlWD40 genes in D. longan.


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where the growth environments of D. longan are very friendly (Hu et al., 2018b). The pulps, leaves, flowers, and roots of D. longan contain a variety of bioactive ingredients, such as flavonoids, tannins, and polysaccharides, thereby serving as traditional Chinese medicine to treat wounds, acariasis, and influenza (Ho et al., 2007;Xue et al., 2015). However, compared with the wide consumption of D. longan juicy fruit, the applications of D. longan roots and leaves as Chinese medicines have been constrained due to the little accumulation of active secondary metabolites. Moreover, the adverse environmental condition is another important factor negatively affecting the growth and quality of D. longan. Our previous studies suggested that the 4 bZIP genes that were differently expressed between D. longan roots and leaves might be involved in the secondary metabolite accumulations (Zheng et al., 2020). In D. longan, the up-regulated expressions of WRKY1, WRKY8, and WRKY50 correspondingly contributed to the tolerance to drought, heat, and cold stresses, respectively (Jue et al., 2018). Therefore, it is urgent to investigate the key TF which not only participate in flavonoid biosynthesis but also enhance abiotic resistances.
WD40, a member of the broadly spread TF family in eukaryote species, is composed of WD40 repeats, which are characterized by a sequence of 40-60 amino acids ( van Nocker and Ludwig 2003;Hu et al., 2018a).
Each WD40 repeat typically starts with a Glycine-Histidine (GH) pair at the N-terminus and ends with a tryptophan-aspartic acid (WD) pair at the C-terminus ( van Nocker and Ludwig 2003;Higa and Zhang 2007). Each WD40 repeat is composed of four-stranded antiparallel ß-sheets, and can interact with other WD40 repeats to fold into a β -propeller, an important structure that enables WD40 to perform many functions, such as mediating the protein-protein interactions (Nash et al., 2001;Fonseca and Rubio 2019;Ma et al., 2019). In addition, WD40 proteins are also involved in a variety of biological processes and tolerance to abiotic stresses (Li et al., 2014;Hu et al., 2018a). WD40 proteins can regulate flavonoid accumulation in various tissues by mediating the expression of flavonoid biosynthesis genes, including CHS, CHI, F3H, and DFR (Wu et al., 2015). For example, MdTTG1, a member of the WD40 family from apple, can interact with both bHLH and MYB to initiate the expression of DFR, a gene whose high expression promotes the biosynthesis of anthocyanin . The PhAN11 protein, the first identified WD40 family protein from Petunia hybrida Vilm, was demonstrated to increase the anthocyanin accumulation via activating the expression of the DFR gene (de Vetten et al., 1997). Arabidopsis TTG1 (homologs to PhAN11) could form four distinct protein complexes (TT2-TT8-TTG1, MYB5-TT8-TTG1, TT2-GL3-TTG1, and TT2-EGL3-TTG1) with MYB or bHLH proteins; these four complexes could up-regulate the expression of the DFR gene to increase anthocyanin accumulation (Xu et al., 2014).
In addition to their involvement in the biosynthesis of secondary metabolites, WD40 proteins have also been shown to participate in the tolerance to abiotic stresses, such as salt stress, drought stress, and heat stress.
For example, the TTG1 gene from the Recretohalophyte limonium bicolor could help transgenic Arabidopsis to resist salt tolerance through both reducing epidermal ion accumulation and increasing osmotic pressure (Yuan et al., 2019). The TaWD40 transcription factor identified from wheat could respond to abiotic stresses. Heterologous over-expression of the TaWD40 significantly enhanced the tolerance to salt stress and osmotic stress in Arabidopsis (Kong et al., 2015). The tolerance to drought stress was strengthened in Medicago sativa L. by enhancing the expression of WD40-1, the mechanism of which was due to the increasing content of anthocyanin (Feyissa et al., 2019). The TAWD protein, a member of the WD40 TF family from Arabidopsis, was involved in improving Arabidopsis resistance to high-temperature stress through activating the expression of heat-shock response (HSR) genes (Xia 2019).
Although the WD40 TF gene family has been intensively investigated in a variety of species, including potato, rice, and Arabidopsis ( van Nocker and Ludwig 2003;Ouyang et al., 2012;Tao et al., 2019), little is known about WD40 TF genes for D. longan. In this study, we identified 45 DlWD40 genes from D. longan RNA-Seq data and used bioinformatics tools to analyse their physiochemical properties, conserved motifs, evolutionary relationships, GO annotations, gene structures, promoter function prediction, protein-protein interactions, and miRNA targets. Additionally, qRT-PCR was used to detect their expression patterns in roots and leaves, as well as in response to heat stress, in order to identify key WD40 TF genes that both function in 3 flavonoid accumulation and in tolerance to heat stress. The results of this study will provide a genetic resource for not only improving D. longan tolerance to abiotic stresses but also increasing flavonoid accumulation in D. longan leaves and roots through genetic engineering.

Materials and Methods
Materials and growth conditions D. longan plants were planted in a greenhouse maintained at 25 °C and relative humidity of 50%. After 2 months of growth, leaves (picked from the upper peripheral branches) and roots gathered from ten plants.
For the heat stress, the 2-month-old D. longan plants were exposed to a 38 °C heat stress for 1, 4, 8, or 24 h. Then, the roots and leaves from D. longan were collected after heat stress at the given time points. All samples were immediately immersed in liquid nitrogen and then stored in a − 80 °C freezer for quantitative real-time PCR (qRT-PCR) analysis.
Identification and bioinformatics analysis of DlWD40 proteins Based on RNA-Seq data of D. longan (NCBI accession number: SRP155595), a total of 45 putative DlWD40 genes were identified. Using the NCBI CD-Search and the SMART tools, the WD40 domain and the quantities of WD40 repeat for 45 DlWD40 proteins were identified. The molecular weights, protein sequence lengths, aliphatic indices, instability indices, grand average of hydropathicity scores, isoelectric points, subcellular protein localization, motif predictions, miRNA targets, and functional regulatory networks of the D. longan DlWD40 proteins were comprehensively investigated using bioinformatics software ( Table 1). The D. longan miRNAs used for the analysis of miRNA targets were downloaded from the results of Lin et al. (2013). Genes from the D. longan genome that were highly homologous to the 45 DlWD40 genes identified from RNA-Seq data were used as representatives to study the DlWD40 gene structures and cis-acting elements. The visualization analysis of gene structures and conserved motifs were achieved using TBtools . Forty-five DlWD40 proteins, together with 80 WD40 proteins from Arabidopsis thaliana and Zea mays, were used to construct a phylogenetic tree in MEGA 7.0 using the neighbour-joining method with 1000 bootstrap iterations. The functional classification of DlWD40 proteins into biological process, molecular function, and cellular component were generated by Blast2GO v5.0. RNA isolation and quantitative real-time PCR analysis RNA was extracted from the root and leaf tissues of D. longan using the cetyltrimethylammonium bromide (CTAB) method (Jaakola et al., 2001). TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen Biotech, Beijing, China) were used to obtain the first-strand cDNAs following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) reactions were performed to evaluate 4 the expression levels of DlWD40 genes using the TransStart® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. The reaction system includes 10 µL of 2× TransStart® Top Green qPCR SuperMix, l µL of cDNA template, 7 µL of ddH2O, 1 µL of forwarding primer, and 1 µL of reverse primer ( Table 2). The D. longan tubulin gene was chosen as a reference gene. The qRT-PCR reaction parameters were set as follows: 95 °C for 1 min, followed by 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. All assays were performed in triplicate. The 2 −ΔΔCT method was used to calculate the relative gene expression (Schmittgen and Livak 2008).

Results
Identification and structural analysis of DlWD40 TFs from D. longan Based on Nr annotation, we obtained 45 putative DlWD40 genes from RNA-Seq data of D. longan leaves and roots (accession number: SRP155595). Then CD search and SMART tools were used to further confirm their identities. For convenience, these genes were numbered from DlWD40-1 to  according to the order in which they were identified from RNA-Seq data. In total, 45 DlWD40 proteins could be divided into two groups ( Figure 1). The first group, including 34 DlWD40 proteins, was characterized as having only the WD40 domain in each DlWD40. The second group (11 DlWD40 proteins) not only contained the WD40 domain but also possessed other identified domains, such as Cytadhesin-P30, Med15, Bromo-WDR9-I-like, Coatomer-WDAD, Ge1-WD40, DUF4678, NLE, CAF1C-H4-bd, Katanin-con80, ATG16, Ufd2P-core, and ANAPC4. The physicochemical properties of DlWD40 proteins, including the length of open reading frames (ORF), molecular weights (MW), isoelectric points (pI), aliphatic indices, instability indices (II), grand average of hydropathicity (GRAVY) scores, and subcellular localizations were evaluated using ExPASY. The results shown in Table 3 indicate that the length of DlWD40 proteins varied from 124 bp to 1286 bp, and the molecular weight ranges from 13,370.22 to 141,013.77 Da. Among 45 DlWD40 proteins, 17 DlWD40s were acidic proteins, and 28 DlWD40s were basic proteins. The subcellular localization analysis showed that nearly half of the DlWD40 proteins were predicted to localize in the nuclear compartment, and the remaining were predicted to localize in the chloroplast, cytoplasm, plasma, mitochondrial, or peroxisome.

Conserved motifs and gene structures
The conserved motifs of 45 DlWD40 proteins were investigated by MEME. To analyse the gene structures, genes from the D. longan genome that were highly homologous to 45 DlWD40 genes from D. longan RNA-Seq data were chosen as representatives (Table 4). TBtools was used to display the evolutionary relationship, conserved motifs, and gene structures of the 45 DlWD40 proteins (Figure 2). A total of 15 motifs were identified from DlWD40 proteins. Of these, the motif2 was present in all the 45 DlWD40 proteins. The motif3 and motif4 were also widely spread, conserved by 41 and 43 DlWD40 proteins, respectively. The gene structure analysis showed that the quantities of introns contained by DlWD40 genes varied significantly. Particularly, there were no introns found in DlWD40-1, DlWD40-10,  In addition, in group A or group B, formed based on the results of the phylogenetic tree, had similar motif compositions and quantities of introns/exons. 6

Phylogenetic analysis
An unrooted phylogenetic tree was constructed using MEGA 7.0 to analyze the evolutionary relationships among 125 WD40 proteins (45 DlWD40 proteins from D. longan, 40 WD40 proteins from Arabidopsis thaliana, and 40 WD40 proteins from Zea mays) (Figure 3). Apart from six WD40 proteins not classified into any group, a total of 119 WD40 proteins were categorized into six groups following the strategy that was adopted in rice and Arabidopsis thaliana (Ouyang et al., 2012). Group 1 was the largest, containing 49 WD40 proteins, whereas group 6 was the smallest, with only 5 WD40 proteins in this group. Importantly, the DlWD40-4 proteins were highly homologous to the Arabidopsis thaliana WDR5a protein, a protein involved in tolerance to abiotic stresses . Although the TTG1 protein from Arabidopsis thaliana was another important WD40 protein functioning in anthocyanin accumulation, we, unfortunately, did not find any DlWD40 proteins that were highly homologous to the TTG1 protein. The functions of DlWD40 proteins were annotated using Blast2GO v5.2.5. All 45 DlWD40 proteins were classified into three major categories: biological process, cellular component, and molecular function ( Figure 4). In the biological process, 45 DlWD40 proteins could be further divided into 13 functional subcategories, of which the cellular process (21 DlWD40 proteins), metabolic process (19 DlWD40 proteins), and cellular component organization or biogenesis (13 DlWD40 proteins) were the first three largest functional subcategories. In molecular function, all 45 DlWD40 proteins were functionally related to binding; thirteen DlWD40 proteins were predicted to be associated with catalytic activity. Three terms were assigned to cellular components, which were intracellular (20 DlWD40 proteins), cellular anatomical entity (21 DlWD40 proteins) and protein-containing complex (16 DlWD40 proteins). Analysis of promoter and miRNA targets To further predict the putative functions of DlWD40 genes, PlantCARE was used to analyse the cisacting elements in the promoter region of the 45 DlWD40 genes. The results are shown in Figure 5: 25 main types of cis-regulatory elements were detected, including hormone response elements (abscisic acid, gibberellin, methyl jasmonate, and salicylic acid), abiotic stress response elements (anaerobic, low-temperature, light, drought, and wound), MYB response elements, and other response elements related to plant growth and development. The compositions and quantities of cis-elements in the promoter of each DlWD40 gene are systematically displayed in Figure 5. The light response elements (G-box, I-box, AE-box, and Box-4) that were conserved by all the 45 DlWD40 proteins were found to present in a large number.
miRNAs are a class of single-stranded non-coding RNAs (18-25 nt in length) that negatively regulate gene expression by cleaving targeted mRNAs and repressing translation. In this study, we used psRNATarget to explore the D. longan miRNAs that could target on DlWD40 genes. The results shown in Figure 6 indicate that the regulatory networks of miRNA-gene were complicated. It can be seen that a DlWD40 gene could be targeted by more than one miRNA, suggesting that the expressions of DlWD40 genes might be repressed by various miRNAs. It is noteworthy that the expressions of DlWD40-5, DlWD40-6, and DlWD40-25 genes were predicted to be negatively regulated by five diverse miRNAs via either cleaving targeted mRNAs or repressing translation. It is noteworthy that miRNA1150, miRNA1078, miRNA2595, miRNA2673a, miRNA2930, and miRNA5170 could regulate different DlWD40 genes in the same way, while miRNA1150 operated by a different mechanism.  The miRNA regulating more than one gene were marked by purple boxes. The top E-value was no more than 3.5 (lower E-values correspond to higher credibility of miRNA regulatory genes) 12

Protein-protein interactions
The STRING tool was used in this study to investigate the protein-protein interactions among DlWD40 proteins. STRING was unable to directly study the DlWD40 protein interactions because the D. longan genomic data were not available in the STRING database. To solve this problem, the Arabidopsis WD40 proteins that were highly homologous to DlWD40 proteins were chosen as representative sequences for the analysis of protein interactions. To a certain extent, the Arabidopsis WD40 protein interactions could be used to reveal the interactions of the DlWD40 proteins in D. longan, since orthologous proteins usually have similar biological functions (Li et al., 2005).
As shown in Figure 7, 17 DlWD40 proteins (DlWD40-2,  linked with other proteins by purple lines, indicating that the protein-protein interactions were validated experimentally. Among these, DlWD-44 (homologous to AT5G14530), DlWD40-28 (homologous to WDR5b), DlWD40-11 (homologous to AT3G21060), DlWD40-5 (homologous to FY), DlWD40-40 (homologous to APC4), and DlWD40-4 (homologous to WDR5a) could each interact with more than three proteins. Unfortunately, the DlWD39 protein was not found to be homologous to any Arabidopsis WD40 proteins. Also, 13 DlWD40 proteins (DlWD40-1, DlWD40-7, DlWD40-9, DlWD40-10,  were not found to have protein-protein interactions. Expression levels of DlWD40 genes in root and leaf tissues from D. longan The expression levels of DlWD40 genes in D. longan roots and leaves were retrieved from D. longan RNA-Seq data and shown as a heat map in Figure 8. Ten DlWD40 genes were identified to exhibit varied expression levels in roots and leaves . Next, the expression patterns of these ten DlWD40 genes were further verified by qRT-PCR, the results of which were generally consistent with that acquired from RNA-Seq data (Figure 9). DlWD40-13, DlWD40-22, DlWD40-32, DlWD40-36, and DlWD40-38 were highly expressed in roots, whereas DlWD40-4, DlWD40-20, DlWD40-21, DlWD40-25, and DlWD40-31 were highly expressed in leaves. It is noteworthy that the expression levels of the DlWD40-20 between roots and leaves were significantly different. The expression level of the DlWD40-20 in leaves was nearly 15-fold higher than that in roots. Expression levels of DlWD40 genes in root and leaf tissues under heat treatments qRT-PCR was used to further study whether or not these ten tissue-specific expression DlWD40 genes could respond under 38 °C heat treatments for 1, 4, 8, or 24 h. We found that the expression levels of all ten DlWD40 genes could be influenced by high-temperature stresses (Figure 10). In general, the expression levels of ten DlWD40 genes first increased and then decreased during the heat stresses from 0 to 24 h, although some DlWD40 genes rebounded at 24 h. In roots, the expression levels of the DlWD40-4, DlWD40-20, DlWD40-25, and DlWD40-31 genes under heat stresses were more than 3-fold higher than the expression levels of 0 h. Of these, the expression level of the DlWD40-25 gene in leaves was also very high after 24 h heat stress, approximately 3.5-fold that at the 0 h time point. Asterisks indicate that the value is significantly different from that of the 0 h treatment (* p < 0.05, ** p < 0.01) Discussion D. longan, a sort of very delicious fruit, can serve as traditional Chinese herbal medicine because of the pharmacological activities of its chemical constituents (Xue et al., 2015). D. Longan has been extensively used to promote blood metabolism and relieve insomnia (Huang et al., 2019). By contrast, the D. longan roots and leaves are also used as traditional Chinese herbal medicine, but are not widely applied due to the little accumulation of flavonoids and other important secondary metabolites. Furthermore, the circumstance (e.g., high temperature) where the D. longan usually grows was another important factor adversely affecting their qualities and yields (Jue et al., 2018). Studies have shown that the biosynthesis of flavonoids, a complex biological process, is regulated by the WD40 TF family in plants (Liu et al., 2013). WD40 TFs can interact with bHLH and MYB TFs to form complexes that could regulate the transcriptional levels of related genes, thereby affecting the anthocyanin synthesis in plants (Qin et al., 2020). In addition, the WD40 TFs have also been shown to be involved in tolerance to abiotic stresses, such as heat, cold, and salt. For example, under the condition of dehydration, the expression level of SmWD40-170 gene decreased to enhance the drought tolerance of Salvia miltiorrhiza by promoting stomatal closure . Thus, studying the multifunctional DlWD40 TF family from D. longan might be of great importance for both increasing the flavonoid accumulation and enhancing their tolerance to abiotic stresses.
In this study, a total of 45 DlWD40 genes were found from RNA-Seq data based on Nr annotation, and their identities were further confirmed using SMART and NCBI CD search tools. An unrooted phylogenetic tree was constructed to analyse the evolutionary relationships of DlWD40 proteins from D. longan. Based on the classification strategy followed in the rice and Arabidopsis (Ouyang et al., 2012), 45 DlWD40 proteins, together with WD40 proteins from Arabidopsis thaliana (monocot) and Zea mays (dicot), were divided into six groups. Among these, two WD40 proteins from Arabidopsis, TTG1 and WDR5a, attracted our attention due to their involvement in flavonoid biosynthesis. The Arabidopsis TTG1 can promote anthocyanin synthesis by positively regulating the expression of DFR and other genes. Petunia AN11 and Fragaria ananassa TTG1, homologous to Arabidopsis TTG1, have also been reported to be involved in the biosynthesis of anthocyanin (Xu et al., 2014). Petunia AN11 can increase anthocyanin accumulation by enhancing the expression of the DFR gene (de Vetten et al., 1997). By contrast, the way in which F. ananassa TTG1 increases anthocyanin accumulation is different. F. ananassa TTG1 increases anthocyanin accumulation by regulating the BAN gene (Schaart et al., 2013). However, none of the DlWD40 genes identified from the D. longan RNA-Seq data were homologous to the Arabidopsis TTG1. Fortunately, we found a gene (accession NO. Dlo_032775.1) highly homologous to Arabidopsis TTG1 in the D. longan genome. The reasons why the homologous gene was found in the D. longan genome rather than transcriptome is likely because it is expressed neither in roots nor in leaves under normal growth environment. However, we could not exclude that its expression might be induced by other factors. For example, the expression of the Arabidopsis TTG1 gene could be induced by abscisic acid (ABA), thereby increasing anthocyanin accumulation . The Arabidopsis WDR5a gene has been shown to be involved in tolerance to drought stress. Under drought stress, the WDR5a gene is overexpressed, promoting the accumulation of nitric oxide in roots . Studies have indicated that the massive accumulation of flavonoids in maize and Arabidopsis was a consequence of the increased nitric oxide (Mongin et al., 2012;Tossi et al., 2011). According to the results shown in the phylogenetic tree, the DlWD40-4 gene was highly homologous to the Arabidopsis WDR5a gene. Consequently, it is inferred that the DlWD40-4 gene might function as a WDR5a gene, participating in tolerance to drought stress and flavonoid accumulation.
Diverse cis-elements were found in the promoter regions of 45 DlWD40 genes using PlantCARE. Some of the cis-elements were associated with stress responses, suggesting that the DlWD40 TF family from D. longan might be involved in tolerance to inhospitable growth conditions. Also, the MeJA-responsiveness element was found in many of the DlWD40 genes, and its functions have been explored in diverse plant species.
For example, MeJA could increase anthocyanin accumulation in apple through enhancing the expression levels of related genes; all these related genes were proved to contain MeJA response elements (Yan et al., 2020). Therefore, the DlWD40 genes that contain the MeJA-responsiveness element in promoters could be expected to associate with anthocyanin biosynthesis under MeJA induction. In addition, the cis-element of MYB binding site involved in flavonoid was found in the promoters of both DlWD40-24 and DlWD40-38, which suggest that the expression of these two genes might be triggered by MYB TFs and then participate in the flavonoid biosynthesis in D. longan. miRNAs were also believed to be pivotal factors influencing plant growth and metabolism through posttranscriptional regulation (Wang et al., 2017). A total of 25 DlWD40 genes were predicted to be targeted by 41 miRNAs using the psRNATarget. Previous studies have shown that the miR3627 is involved in anthocyanin biosynthesis in Vitis vinifera L. (Sunitha et al., 2019) Accordingly, we speculate that the DlWD40-33 gene is targeted by miR3627 and might be related to anthocyanin biosynthesis.
The expression patterns of 45 DlWD40 genes were evaluated by RNA-Seq and qRT-PCR, ten members of which were shown to have different expression levels between root and leaf tissues of D. longan. Gene expressions were believed to have close relationships with secondary metabolite accumulations. For example, the high expressions of AcMYB123 or AcbHLH42 transcription factor genes in Actinidia inner pericarp were proved to contribute to anthocyanin synthesis . As a result, the DlWD40-20 gene that was highly expressed in leaves was regarded as a candidate gene for further investigation of its involvement in secondary metabolite biosynthesis. One of our future work will focus on the function of DlWD40-20 gene participating in the accumulation of secondary metabolites by using transgenic technology. Also, these ten tissue-specific DlWD40 genes were shown to respond to a 38 °C heat stress, whereas they showed diverse expression levels in both roots and leaves. The TAWD gene, a member of the WD40 gene family from Arabidopsis, was found to help resist high temperatures (Hu et al., 2018). Therefore, we speculate that DlWD40 genes with strong thermal responses, including DlWD40-4, DlWD40-20, DlWD40-25, and DlWD40-31, deserve further investigation for their putative functions in resisting heat stress.

Conclusions
In this study, bioinformatics tools were used to analyse 45 DlWD40 genes identified from D. longan, including physical and chemical properties, conserved domains, cis-acting elements, protein-protein interactions, evolutionary relationships, GO annotations, and miRNA targets. Moreover, the expression pattern of DlWD40 genes in roots and leaves, as well as the responses to heat stress (38 °C), were analysed by qRT-PCR. Ten of the 45 DlWD40 genes  were differentially expressed in roots and leaves. Of these, the DlWD40-20 gene in leaves was nearly 15-fold higher than that in roots. All of these ten DlWD40 genes could respond to heat stress (38 °C). The DlWD40-4, DlWD40-20, DlWD40-25, and DlWD40-31 genes responded most strongly to heat stress. The data reported here expand our understanding of WD40 TFs and provide gene resources for increasing the content of flavonoid through genetic engineering. Also, our findings will provide a basis for improving abiotic stress resistance in plants using transgenic technologies.

Authors' Contributions
ZW and ZZW conceived and designed the study, as well as wrote the manuscript. ZZW performed the experiments. YXF reviewed and edited the manuscript. XTT, CN and LWL analyzed the data. All authors read and approved the final manuscript.