Comparative genomics analysis of the MYB gene family in barley: preliminary insights into evolution and biological function in Blue Qingke

Introduction

Qingke (Hordeum vulgare L. var. nudum Hook. f), a variety of barley in the gramineous family that is highly genetically similar to barley (Hordeum vulgare L.), is also called naked barley or hulless barley in the Tibetan Plateau of China, as the lemma and palea are separated from the caryopsis when the grain is ripe (Guo et al., 2020; Yao et al., 2021). Qingke has a nutrient composition structure of “three high and two low” (high protein, fiber, and vitamins and low fat and sugar) and has an extremely high nutritional and dietary value (Guo et al., 2020). The grain color of highland barley is related to the accumulation of different pigments in the aleurone layer, pericarp, and lemma (Jende-Strid, 1993; Strygina, Börner & Khlestkina, 2017), and there are mainly four colors: blue (anthocyanin deposited in the aleurone layer), purple (anthocyanin deposited in the aleurone layer and pericarp), black (pigment deposited in the glume or pericarp), and white (without pigment) (Jia et al., 2016; Suriano et al., 2018; Zeven, 1991). Studies have shown that blue and purple are caused by flavonoid phenolic compounds, and black is caused by plant melanin, oxidation, and polymerization of phenolic compounds (Shoeva et al., 2016). The phenylpropanoid pathway produces lignin, flavonoids, and other metabolites and is regulated by MYB transcription factors (Ma & Constabel, 2019). The blue aleurone trait in barley has only recently evolved, with most wild barley varieties displaying blue aleurone layers, while local and cultivated varieties have blue, purple, and white aleurone layers in various colors (Jia et al., 2020). The blue grain traits of barley are mainly derived from three incomplete dominant Ba genes: Ba1, Ba2, and BaThb (Shen et al., 2013; Singh et al., 2007; Yu et al., 2017; Zheng et al., 2006). In barley, the combination of dominant alleles controlling three transcription factors, MYB, bHLH, and WD40 (MBW complex), regulates anthocyanin synthesis in the aleurone layer (Strygina, Börner & Khlestkina, 2017). Delphinium 3-glucoside is the primary anthocyanin in blue grain barley (Mullick et al., 1958). Previous studies have suggested that anthocyanin accumulation in blue grain barley is positively correlated with the expression levels of the Glutathione S-transferase (HvGST) gene and MbHF35 cluster genes (HvMYB4H, HvMYC4H, and HvF3′5′H). The quantitative trait locus (QTL) associated with the HvGST gene has been preliminarily located at 2.80 Mb (486.18–488.98 Mb) of chromosome 7HL (Jia et al., 2020; Xu et al., 2023).

The MYB family is large and diverse and one of the most widely distributed transcription factor families in plants, being present in all eukaryotes (Ambawat et al., 2013; Dubos et al., 2010). MYB transcription factors have a highly conserved N-terminal MYB DNA-binding domain (DBD), consisting of 1–4 incomplete amino acid repeats (R) and containing about 52 amino acid residues, each encoding three alpha-helices (H1, H2, and H3), of which H2 and H3 form a helix-turn-helix (HTH) structure (Ng, Abeysinghe & Kamali, 2018). MYB transcription factors can bind to target DNA through the HTH structure to regulate the expression of target genes (Rosinski & Atchley, 1998). Of the four MYB proteins, the R2R3-MYB protein is the most abundant type and is specific to plants, responding to both biotic and abiotic stress, as well as primary and secondary metabolism. The R2R3-MYB gene may mainly regulate specific processes in plant evolution during formation (Kranz, Scholz & Weisshaar, 2000; Wilkins et al., 2009). The R2 domain contains a conserved DNA-binding site, and the R3 domain contains a conserved domain that can bind to the bHLH protein (Yin et al., 2021). In plants, the first tryptophan in R3 is replaced by phenylalanine or isoleucine (Ambawat et al., 2013). Studies have found that MYB genes are targets of microRNAs (miRNAs) (Allen et al., 2007). MYB transcription factors are involved in plant development and stress response by binding to MYB cis-elements in target gene promoters, which can regulate the growth of plant roots, stems, leaves, and flowers, and also participate in the biosynthesis of plant secondary metabolites, including anthocyanins, flavonols, and lignin (Lama et al., 2020; Li, Ng & Fan, 2015; Wang, Niu & Zheng, 2021). Numerous studies have shown that MYB transcription factors are widely distributed in plants, are involved in the abscisic acid (ABA) response, and interact with other transcription factors (Ng, Abeysinghe & Kamali, 2018). The first MYB gene identified was v-MYB of avian myeloblastosis virus (AMV) (Klempnauer, Gonda & Bishop, 1982). The first MYB protein discovered was factor C1 in Zea mays (Kranz, Scholz & Weisshaar, 2000). The animal c-MYB gene contains three HTH-encoding repeats (R1R2R3 class genes); thus, it has been suggested that the R2R3-MYB gene is the plant equivalent of c-MYB (Kranz, Scholz & Weisshaar, 2000). Subsequently, MYB transcription factors of numerous diverse species have been identified, most of which activate MYBs and bind to cis-elements in gene promoters to regulate transcription mechanisms and activate gene expression (Ma & Constabel, 2019).

Currently, studies on the MYB gene family have focused on Arabidopsis (Dubos et al., 2010), rice (Katiyar et al., 2012), and maize (Du et al., 2012), while fewer studies have been conducted on the Qingke MYB family. Previous researchers have identified a MYB family gene, HvMYB4H, located at the Blx1 locus on chromosome 4H, which controls the blue aleurone trait in barley (Jia et al., 2020). However, the anthocyanin-related genes that control Qingke’s blue aleurone trait have not been fully elucidated. Moreover, MYB transcription factor is the key factor controlling the anthocyanin phenylpropanoid (Zeng et al., 2020). Therefore, based on published reports of MYB genes in Arabidopsis and rice (Katiyar et al., 2012), firstly, 92 Qingke HvMYB genes were obtained by comparative genomics analysis. Chromosome localization, gene and protein structure, protein physicochemical properties, conserved domains, protein conserved motifs, promoter cis-acting elements, phylogenetic relationships, collinearity with Arabidopsis, rice, and maize were analyzed using bioinformatics methods. Notably, only R2R3-MYB transcription factors have been reported in barley (Tombuloglu et al., 2013), and that the method was one-sided as it was screening R2R3 MYB TFs from barley whole RNA-seq data. Therefore, the identification in this study is crucial to ensure the accuracy and comprehensiveness of the barley MYB gene family. The interaction between HvMYB and HvMYC proteins on cell nucleus and cell membrane was verified by the BiFC method, which laid a foundation for the research and application of HvMYB transcription factors in the grain color of cereal crops.

Materials and methods

Results

Identification and location of HvMYB genes on chromosomes

A total of 92 Qingke HvMYB genes were identified by comparative genomics analysis and named HvMYB1–HvMYB92 based on their distribution on chromosomes. Ninety-two HvMYB genes were selectively distributed on seven Qingke chromosomes (Fig. 1). Twenty HvMYB genes were distributed on chromosome 3, which had the highest gene density (21.73%), and seven HvMYB genes were distributed on chromosome 2, which had the lowest gene density (7.60%). A total of 18, nine, 15, 10, and 13 HvMYBs were distributed on chromosomes 1, 4, 5, 6, and 7, respectively. The distribution of MYB genes on chromosomes showed that most of the MYB genes were located at the ends of the chromosomes.

Construction of the MYB protein phylogenetic tree and distribution of conserved domains

According to the characteristics of MYB family structural domains and transcription factors, the 92 HvMYB genes were classified into five categories, which was similar to the previous classification based on the three repeat sequences (R1, R2, and R3) of c-Myb. To further analyze the phylogenetic relationships of the HvMYB family in Qingke, we constructed a phylogenetic tree using 197 AtMYB proteins from Arabidopsis, 155 OsMYB proteins from rice, and 92 HvMYB proteins from Qingke, for a total of 444 MYB proteins (Fig. 2). Ninety-two HvMYB proteins were divided into five groups (A–E). One HvMYB was placed in Group A. Fifty-five HvMYBs were placed in Group B. Eight HvMYBs were placed in Group C. Thirteen HvMYBs were placed in Group D. Fifteen HvMYBs were placed in Group E.

Analysis of conserved HvMYB protein motifs and domains

To further study the homologous motif characteristics of 92 HvMYB protein members, the MEME tool was applied to analyze the motif distribution region and the frequency of the most common amino acids at each location. A total of 10 motifs (motifs 1–10) were identified in the 92 HvMYB proteins (Fig. 3; Table S5). The motifs of MYB proteins in the same group were similar in classification order, but the number of MYB protein motifs differed among diverse groups. Motif 6 was annotated as Myb_DNA-binding, which is a basic feature of MYB transcription factors. Motif 3 was found in all HvMYB proteins, except HvMYB12, HvMYB27, HvMYB76, HvMYB3, HvMYB92, and HvMYB84. Motifs 1, 2, 3, and 5 were present on the N-terminal of most HvMYB proteins. Based on the structural domain composition, the 92 HvMYB proteins were classified into 17 subfamilies, of which the most numerous was the PLN03091 superfamily domain of subfamily 2, which contained a total of 53 genes (Fig. 4; Table S4).

Analysis of covariance between Qingke and Arabidopsis, rice, and maize MYB families

We performed a collinearity analysis of the genomes of Hordeum vulgare (Morex_V3), Arabidopsis, Oryza sativa, and Zea mays to explore the evolutionary history of MYB genes (Fig. 5; Table S6). Only 21 copies of the Qingke MYB gene were found in the Arabidopsis genome, while 85 and 121 MYB gene pairs were found in the rice and maize genomes, respectively. The number of collinear gene pairs between Qingke and monocotyledonous plants (rice and maize) was much larger than that between Qingke and Arabidopsis, indicating that the collinearity of MYB family members between Arabidopsis and Qingke was low.

HvMYB gene structural diversity and main promoter cis-acting elements regulation analysis

To explore the cis-acting elements in all HvMYB gene promoters, 2 kb upstream coding sequences (CDSs) of each gene were extracted and searched in the PlantCARE database. The cis-acting elements of the 92 HvMYB genes promoter regions were analyzed (Fig. 6; Table S7), the results showed that the promoter regions of 92 genes contained 42 cis-acting elements associated with stress and hormones (Table S8). Among them are abscisic acid responsive, auxin, and gibberellin, which are related to plant physiological regulation, light regulation, and response to biological stress. Abiotic stress and immune response elements, such as MYB-binding sites, low temperature, methyl jasmonate, salicylic acid, defense, and stress response, were also included. Most HvMYB genes contained ABA-responsive, light-responsive, and MeJA-responsive elements. HvMYB genes could be associated with plant physiology, biotic and abiotic stress, and immune responses. HvMYB genes have been speculated to be inducible promoters. The presence of various cis-acting elements in gene promoters indicates that these genes have diverse functions.

Screening of the HvMYB genes related to anthocyanin synthesis

To further understand the expression levels of 92 HvMYB genes in different Qingke varieties, RNA-seq was performed on ‘White 91-97-3’ and ‘Blue Qingke’ at the milk, soft dough, and mature stages. White 91-97-3 and Blue Qingke contained 10,940 differential genes. The 92 HvMYB genes intersected with the differential genes of the two varieties, which contained 41 differential genes (Fig. 7). The differential genes of both Blue Qingke and White Qingke have similar expression patterns, that is, the expression level was higher in the mature stage, while the expression level was lower in the milk stage. It is inferred that genes regulating Qingke blue grain traits mainly play a significant role in the middle stage of grain development (Fig. 8; Table S9). In view of the location interval of blue grain Qingke in this experiment, HvMYB53 was identified as the key gene controlling blue grain Qingke, and HvMYC interacting with HvMYB53 was screened in RNA-seq, these two genes were selected as key genes for subsequent analysis. Subsequently, we performed qRT-PCR analysis of these two genes, the primer sequence was shown in Table 1. RNA-seq and qRT-PCR (Table S10) analysis showed that the two genes had similar expression patterns: that is, the two genes were highly expressed in the soft dough stage (Figs. 9A and 9B).

Subcellular localization and BiFC analysis of HvMYB and HvMYC

We found that HvMYB and HvMYC proteins interact, based on the results of protein-protein interaction (PPI) of RNA-seq in our laboratory (Table S11). To understand the protein characteristics of HvMYB and HvMYC, the subcellular localization of this proteins were detected. Nicotiana benthamiana leaves expressing the green fluorescent protein (GFP) were analyzed using a confocal microscope. HvMYB-GFP was expressed in the nucleus and cell membrane, HvMYC-GFP was expressed in the nucleus, cell membrane, and cytoplasm. Therefore, HvMYB was localized in the nucleus and cell membrane, HvMYC was localized in the nucleus, cell membrane, and cytoplasm (Fig. 10). HvMYB and HvMYC fluoresce yellow in the nucleus and cell membrane, indicating an interaction (Fig. 11).

Discussion

The MYB gene family is associated with a variety of plant-specific cellular functions (Shen et al., 2013). The main feature of the MYB protein is the localization of three regular tryptophan (W) residues with each DBD, and the abundance of the MYB gene in a species may be related to genome duplication (segmentation/tandem) rather than genome size (Zheng et al., 2006). MYB genes are characterized as transcription factors when they contain at least two MYB repeats (R) and explicitly recognize DNA motifs to regulate gene transcription (Yu et al., 2017). MYB transcription factors play an extensive role in plant growth and development, hormone signaling, and coping with stress responses. The gene has been studied more in Arabidopsis (Kirik et al., 1998), rice (Katiyar et al., 2012; Mullick et al., 1958), and maize (Du et al., 2012) and less in Qingke. Transcription factors are usually co-expressed with their downstream structural genes, and similar expression patterns are often an effective way to identify transcription factors that regulate secondary metabolism (Yu et al., 2021). Therefore, studying the Qingke MYB gene family and comparing it with its homologous plant genes will help explain the evolution of this gene family during the natural formation of Qingke.

The 92 HvMYB genes in this study were named HvMYB1–HvMYB92 based on their location on the chromosome, which distinguishes them from previous studies that named them based on gene annotation and structural domain type. Analysis of motifs and intron–exon patterns of full-length protein sequences helps identify various domains and further confirms the results of phylogenetic analysis (Giacomelli et al., 2010; Wang et al., 2011). The MYB family members in the same group have a similar structure, with all MYB proteins except HvMYB12, HvMYB27, HvMYB76, HvMYB3, HvMYB92, and HvMYB84 containing motif 3 (pink) and most MYB proteins containing motifs 1 (dark blue) and 2 (yellow). Motifs 1, 2, 3, and 5 appeared sequentially at the N-terminus of HvMYB proteins, which is consistent with the findings of Yu et al. (2021). In addition, motif 5 was distributed before motif 3, and motif 7 occurred before motif 1 in most HvMYB proteins, suggesting that HvMYB proteins are highly conserved and that motif differences among HvMYB proteins may be related to the biological functions of their specific genes. Exon–intron structure diversity and cleavage pattern significantly affected the number and expansion of gene families (Wei et al., 2016). According to rice studies, intron loss occurs faster than intron addition after fragment replication (Lin et al., 2006). Our study found that the number of introns was proportional to the length of the gene. In Qingke, HvMYB75, HvMYB59, HvMYB72, HvMYB91, HvMYB15, HvMYB16, HvMYB18, HvMYB18, HvMYB62, HvMYB63, and HvMYB8 had no introns, indicating that these MYB branches evolved later than the other groups.

The highly conserved tryptophan (W) residues distributed in the third helix are significant for the DNA-binding activity of MYB proteins, which indicates functional conservation between different plant species, but sequence conservation between plants does not necessarily imply functional conservation (Du et al., 2012). In this study, 92 HvMYB genes were classified into five categories, the resulting functional conservation between the Qingke HvMYB genes and their homologs suggests the origin and evolutionary diversity of plant MYB genes; species-specific groups/subgroups may evolve or be lost during evolution, leading to functional divergence (Du et al., 2012). Transcription factors specifically bind to cis-acting elements to regulate the expression of related target genes, and multiple cis-acting sites determine the diversity of the regulatory functions of transcription factors (Lei et al., 2021; Ning et al., 2017; Zhao et al., 2021). Further analysis of the cis-acting elements of diverse genes showed that HvMYB genes are presumably associated with plant physiology, biotic and abiotic stress, and immune responses, and it has been hypothesized that HvMYB genes are inducible promoters and that the presence of various cis-acting elements in the promoters of the genes may imply that these genes have diverse functions.

Loss of a structural domain does not always lead to negative consequences, and although gene function is temporarily lost or altered, it may lead to the re-expansion of the gene family (Liu et al., 2022). The major structural domains of MYB proteins are PLN03091 and PLN03212, which is consistent with the findings of Yu et al. (2021). F-box proteins are involved in various life activities, such as cell cycle regulation, apoptosis, and signal transduction, and they play vital roles in maintaining normal plant growth and development and mediating abiotic stress responses (Xu et al., 2023). The SANT structural domain, which is found mainly in proteins involved in chromatin function and usually recognizes histone tails, is a novel motif found in numerous eukaryotic transcriptional regulatory proteins, and it shares homology with the DNA-binding structural domain of c-MYB, although it is unlikely to bind DNA (Boyer et al., 2002; Grüne et al., 2003). Arabidopsis AtMYB96 is an R2R3-type MYB transcription factor that regulates the drought stress response by integrating ABA and auxin signals, and genes in the same branch as this gene may have the same function (Joon et al., 2009). Some of the structural domains in MYBs contain highly conserved SHAQKY (F/Y) motifs in the third predicted α-helix, and the 1R structural domains of OsMYBs and StMYB1 are highly conserved, suggesting that other amino acid residues upstream of the SHAQK (Y/F) F motif and within the predicted third α-helix may contribute to the recognition of specific DNA sequence elements (Lu et al., 2002). The above structural domains are present in only a few MYB proteins, indicating that they are not the major structural domains of MYB proteins. Gene collinearity analysis among diverse species showed that Qingke and maize had the most collinear gene pairs, followed by rice and Arabidopsis; this difference may be related to Qingke’s genome characteristics and evolution.

Garcia-Gimenez et al. (2022) hypothesized that MYB transcription factors may positively regulate the accumulation of (1,3; 1,4)-β-glucan in barley primary cell walls. Recent studies have also described novel roles for MYBs as repressors, negatively affecting traits such as secondary cell wall biosynthesis and cold tolerance (Jende-Strid, 1993; Mullick et al., 1958). Previous studies have located the HvMYB4H gene in blue barley on chromosome 4H (Jia et al., 2020), indicating that the MYB gene is a significant factor in the blue color of wheat crops. Subcellular localization predicts that most HvMYB genes are located primarily in the nucleus, and it is hypothesized that MYB genes perform important functions primarily in the nucleus. The flavonoid biosynthetic pathway is transcriptionally regulated by the MBW complex, whose activation or repression is mainly determined by MYB transcription factors through binding to structural gene promoters and common bHLH and WD40 factors (Kyte & Doolittle, 1982). In potato, when three MYB genes (StAN1, StMYBA1, and StMYB113) are co-expressed with the bHLH gene, anthocyanin synthesis in tobacco can be regulated (Liu et al., 2016). The ZmPAC1 gene in maize can regulate anthocyanin synthesis when co-expressed with ZmR1 (bHLH) and ZmC1 (MYB) (Carey et al., 2004). These results suggest that the combination of MYB and bHLH to regulate anthocyanin synthesis. BiFC results showed that HvMYB and HvMYC can form protein interactions. We speculate that the joint role of HvMYB and HvMYC in the synthesis of anthocyanins in Qingke seed colour may be stronger than that of a single transcription factor. Expression analysis of the differential genes and two reciprocal genes showed that all the genes had higher expression almost at the soft dough stage and the mature stage, and lower expression at the milk stage. It is hypothesised that the genes regulating Blue Qingke mainly start to function in the middle stage of seed development, which may also be related to their involvement in different biological processes or functions.

Conclusion

In this study, we identified 92 Qingke HvMYB genes using protein-motif interactions. Among them, HvMYC protein interacting with HvMYB53 was found in Blue Qingke and White 91-97-3 RNA-seq data, which was also proved by BiFC method. HvMYB53 and HvMYC genes play a significant role in Qingke grain coloring, and their expression patterns are significant differences among different varieties. This study not only proved the key role of HvMYB53 and HvMYC genes in anthocyanin biosynthesis, but also provided a theoretical basis for further research on the regulatory mechanism and functional evolution of Qingke anthocyanin biosynthesis.

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