1. Introduction
Due to their “fixed” growth characteristics, plants are usually susceptible to unfavorable environmental factors during growth. After a long period of domestication, agricultural plants have developed a unique regulatory mechanism to adapt to these stresses. Upon sensing stress signals, transcription factors further regulate the expression of functional genes that enable plants to generate specific metabolic and physiological responses to mitigate the effects of stress [1,2]. MYB is a very important class of genes in plants. The presence of the MYB structural domain at its N-terminus is a hallmark feature of the MYB family. Its C-terminus is highly differentiated, which also creates the diversity of MYB proteins [3,4]. The structural domains of MYB genes usually contain varying numbers of R-repeat sequences, and based on their number, the MYB family genes are categorized as 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB. Each R-repeat sequence contains approximately 50–55 amino acids, forms three α-helices, has an HTH structure between the last two α-helices, and is involved in the NDA-binding process [5,6]. The 3R-MYB gene is reportedly generated with the occurrence of R1 duplication within the R2R3-MYB gene. However, it has also been reported that the R2R3-MYB gene is generated due to loss of R1 within the 3R-MYB gene [7].
MYB family genes are functionally diverse and play important roles in plant development [8,9,10,11]. For example, AtMYB96 enhances plant drought tolerance by controlling epidermal wax biosynthesis through the ABA signaling pathway [12]. Overexpression of the OsMYB48-1 gene in rice significantly enhances the tolerance of rice to mannitol and PEG [13]. The FtMYB9 gene further reduces salt-stress damage by regulating the synthesis of proline [14]. MdSIMYB1 enhances plant tolerance to salt, drought, and low temperature by upregulating the expression of stress-response genes (NtDREB1A, NtERD10B, and NtERD10C) in transgenic tobacco [15]. ZmMYB-IF35 expression alleviates oxidative damage in Zea mays under low-temperature stress [16]. OsMYB30 enhances the protective effect of cell membranes by regulating amylolysis and maltose accumulation, resulting in enhanced cold tolerance in rice. In addition, OsMYB30 plays an important role in the resistance of rice brown planthopper mediated by the phenol propane pathway [17,18]. MdMYB30 binds to the promoter of the wax synthesis-related gene MdKCS1 and activates its transcription to enhance the biosynthesis of epidermal waxes to regulate plant resistance to pathogens [19].
Sugarcane (Saccharum officinarum L.) is one of the most important cash crops and provides 80% of the world’s sugar and 90% of China’s sugar. Additionally, sugarcane is a promising renewable-energy crop with significant development potential [20]. However, in the region of Yunnan Province, China, adversities such as drought and low temperature often hinder the growth and development of sugarcane, and the precise and effective enhancement of sugarcane resistance has become an important direction for sugarcane breeding at this stage. E. fulvus, which is a closely related wild species of sugarcane, is characterized by high brix and easy flowering and exhibits a high degree of stress tolerance, which are traits that are valuable for sugarcane breeding [21]. Therefore, the excellent resistance genes in E. fulvus should be fully explored and utilized for improving the resistance of sugarcane in a targeted manner.
Although E. fulvus is very important, few studies have investigated MYB family genes in E. fulvus. The number of members, gene structure, evolutionary features, gene expression properties, and functions of the MYB family of genes in E. fulvus are unknown. Thus, we identified MYB family genes in the whole genome of E. fulvus and systematically analyzed the gene structure, conserved structural domains, chromosomal localization, promoter cis-acting elements, gene duplication, and protein-interaction relationships. In addition, we investigated the effects of low temperature, drought stress, and ABA and MeJA treatments on EfMYB gene expression. These results can enable us to obtain a better systematic understanding of the EfMYB family of genes and lay the foundation for our subsequent studies on EfMYB gene function, regulatory mechanisms, and transgenic breeding of sugarcane.
2. Materials and Methods
2.1. Identification of EfMYB Genes
The E. fulvus genome information and related files were downloaded from the E. fulvus Genome Database (
2.2. Phylogenetic Tree Analysis
Multiple sequence comparison of the EfR2R3-MYB proteins and 126 Arabidopsis thaliana AtR2R3-MYB proteins was carried out using MEGA 7.0 software with the neighbor-joining method (NJ method) and a bootstrap value of 1000 to construct the phylogenetic tree [27,28].
2.3. Protein-Properties, Conserved-Motifs, and Gene-Structure Analysis
The protein properties and subcellular localization of EfMYB family members were predicted using the online tools ExPASy (
2.4. Chromosomal-Location, Gene-Duplication, and Synteny Analysis of EfMYB Genes
Chromosomal localization data of the EfMYB genes were extracted using Docker and visualized using MapChart [33]. The MCScan tool was used to analyze the tandem-duplication (TD) and segmental-duplication (SD) events of EfMYB genes and for the synteny analysis of MYB genes between E. fulvus and A. thaliana, Zea mays, Oryza sativa, Saccharum spontaneum, and Sorghum bicolor [34]. Finally, we analyzed nonsynonymous (ka) and synonymous (ks) substitutions in the EfMYB genes that underwent replication events using the KaKs_Calculator 2.0 tool [35].
2.5. Identification of Cis-Acting Elements in EfMYB Genes
The promoter sequence 2000 bp upstream of the EfMYB gene sequence was extracted from the genome file using the Docker tool. The cis-acting elements were then predicted using the PlantCARE online tool (
2.6. Protein-Interaction Network and RNA-Seq Data Analysis of EfMYB Genes
EfMYB proteins were uploaded to OrthoVenn2 to identify EfMYB proteins homologous to A. thaliana MYB proteins (
2.7. Material Processing and Gene Expression
First, the E. fulvus clones were planted in pots, and the following stress treatments were executed after the plants had grown five leaves. The low-temperature-stress treatments were conducted as follows. The plant material was transferred to a cold incubator at 4 °C and sampled after 0 h (CK), 24 h, and 72 h of stress treatment. Drought-stress treatments were conducted by stopping the supply of water to the plants, and samples were taken after 0 d (CK), 3 d, 6 d, and 9 d of the stress treatment. The soil water content at each drought-stress time point was 70 ± 5%, 50 ± 5%, 30 ± 5%, and 15 ± 5%. For exogenous hormone treatments, E. fulvus asexual tissue culture seedlings were sprayed with abscisic acid (ABA) and methyl jasmonate (MeJA) solutions at a concentration of 100 µM. Samples were collected after 0 h (CK), 6 h, and 12 h of treatment. After performing the above treatments, the samples collected were all of the first fully expanded leaves of the plant material, counted from top to bottom. RNA was extracted using TRIzol reagent from Tiangen Biochemical Technology (Beijing, China), and the quality and concentration of RNA were then detected using 1% agarose gel electrophoresis and UV spectrophotometry. Finally, the better-quality RNA was reverse transcribed into cDNA. qRT-PCR analysis was performed with an Applied Biosystems ABI 7500 instrument using 2 × RealStar Fast SYBR qPCR Mix (GenStar) (Table S8). We used the 25S rRNA gene as an internal reference to calculate the expression levels of the EfMYB genes with the 2−ΔΔct method [39].
3. Results
3.1. Identification and Protein Characterization of EfMYB Genes
A total of 133 EfMYB genes were identified in the E. fulvus genome, including 48 Ef1R-MYB, 84 EfR2R3-MYB, and 1 Ef3R-MYB genes (Table S1). The EfMYBs were named EfMYB1~EfMYB133 based on their arrangement order on chromosomes. The predicted protein physicochemical properties are shown in Table S2. The molecular weight (MW) was 12,252 Da (EfMYB45)~231,536.9 Da (EfMYB106), the isoelectric point (pI) was 4.38 (EfMYB82)~11.49 (EfMYB67), the amino acid lengths ranged from 110 to 2143 aa, and the average amino acid lengths for MW and pI were 448.37 aa, 48,681.63 Da and 8.01, respectively. The predicted subcellular localization of the EfMYBs showed that a few members were located in chloroplasts (7.5%), while the majority were located in the nucleus (92.5%) (Table S2), indicating that these EfMYB transcription factors may play regulatory roles in the chloroplast and nucleus, respectively.
3.2. Phylogenetic, Conserved-motif, and Gene-Structure Analyses of the EfMYBs
As previously reported [40], compared with other types of EfMYB genes, EfR2R3-MYB genes accounted for the largest percentage (63.16%) of the EfMYB family. To analyze the characteristics of EfR2R3-MYB proteins, we built a phylogenetic tree of 84 EfR2R3-MYB proteins with 126 AtR2R3-MYB proteins (Figure 1). EfR2R3-MYB was divided into 20 subgroups (E1~E20) based on multiple sequence alignment. Among them, E1, E2 (S21), E3, E4 (S23), E5 (S18), E6 (S20), E7, E8, E9 (S14), E10 (S1), E11 (S5), E12 (S7), E13 (S13), E14 (S16), E15 (S17), E16 (S2), E17, E18 (S4), E19 (S11), and E20 (S9) all contained E. fulvus and A. thaliana R2R3-MYB genes, whereas S22, S25, S15, S19, S6, S3, S12, S24, and S10 contained only the AtR2R3-MYB genes. Overall, subgroups E13 (S13) and E15 (S17) had the highest number of EfR2R3-MYB genes (nine and seven, respectively); subgroups E3 and E17 had the fewest number of EfR2R3-MYB genes (only one in both groups); and most of the other subgroups contained three to four genes.
It is generally believed that motifs may contain specific binding sites and be involved in specific biological functions. We predicted 20 motifs (Motif 1~Motif 20) for EfMYB family genes (Table S4). The results showed that almost all the EfR2R3-MYB genes contained Motif 3, Motif 2, and Motif 1, and more than half of the EfR2R3-MYB genes contained Motif 4. In addition, only Motif 15 appeared in EfMYB16 and EfMYB17, and only Motif 14 appeared in EfMYB14 and EfMYB15. Motif 16 and Motif 13 were unique to the E14 (S16) subgroup and E1 subgroup, respectively (Figure 2a). In summary, most EfR2R3-MYB genes in the same subgroup usually have similar motif types, and motifs between different subgroups may lead to differences in EfMYB gene function. Compared to EfR2R3-MYB, Ef1R-MYB contained a markedly smaller number of motifs (Figure 3a). However, EfMYB94 did not generate motif under the parameters we set, which may be due to sequence differences between EfMYB94 and other proteins. Interestingly, there were multiple repetitions of Motif 3, Motif 2, and Motif 16 in Ef3R-MYB (EfMYB26). Motifs can bind to transcription factors, regulate gene expression, or participate in protein-protein interactions. This suggests that the repeated occurrence of these motifs in EfMYB26 may be recognized by specific transcription factors for binding to regulatory regions of genes and thereby exert certain unique biological effects [41]. We further analysed the core motifs of the EfR2R3-MYB structural domain and found that many amino acid residues were conserved in the R2 and R3 repeat sequences. The R2 and R3 repeat motifs contained 3 and 2 conserved tryptophan residues (W), respectively (Supplementary Figure S1).
Diversity in gene structure may be a basis for the evolution of gene families. To understand the structural characteristics of EfMYB family genes, we analyzed the exon and intron distributions of EfR2R3-MYB, Ef1R-MYB, and Ef3R-MYB. The results showed that the number of exons in the EfR2R3-MYB genes ranged 1~23, with 36.9% of the members containing two introns and 23.8% containing one intron. Notably, EfMYB20, EfMYB57, EfMYB112, EfMYB71, and EfMYB59 contained no introns, while EfMYB12 contained 22 introns (Figure 2b). The numbers of introns in the Ef1R-MYB genes were mainly one, two or four. However, EfMYB42 had the highest number of introns (19), and three genes (EfMYB45, EfMYB94, EfMYB39) had no introns (Figure 3b). Many EfMYB genes demonstrated a domain with a cross-intron structure (Figure 2b and Figure 3b). These results indicated that the structures of the EfMYB genes were diverse.
3.3. Chromosomal-Distribution and Gene-Duplication Analysis of EfMYB Genes
The results showed that three genes (EfMYB131, EfMYB132, and EfMYB133) were not localized on any chromosome, and the remaining 130 EfMYB genes were unevenly distributed on Chr1-Chr10. The highest number of genes was distributed on Chr4 (30), followed by Chr1 and Chr7 (19 and 16). The lowest number of genes (6) was found on Chr2, Chr9, and Chr10 (Figure 4).
Tandem duplications and segmental duplications are events that occur frequently in plant genomes, and they contribute to the expansion of gene families, which gives rise to new members and functions that drive plant evolution [42]. To investigate the expansion of EfMYB family genes, we analyzed tandem duplications and segmental duplications of EfMYB genes using the MCScanX tool (Table S3). As shown in Figure 4, four tandem duplicate gene pairs were found on Chr1 (EfMYB14-EfMYB15), Chr4 (EfMYB62-EfMYB63), Chr5 (EfMYB73-EfMYB74), and Chr10 (EfMYB128-EfMYB129). In addition, we identified 21 EfMYB segmental-duplication gene pairs (Figure 5). The EfMYB gene segmental-duplication events occurred mainly in Chr3 (6 genes), Chr4 (5 genes), Chr5 (5 genes), Chr6 (6 genes), and Chr7 (5 genes), indicating that segmental duplication and tandem duplication events are important factors driving the expansion of EfMYB genes in E. fulvus.
We further calculated the Ka/Ks values of these duplicated gene pairs to more clearly analyze the evolutionary process of the EfMYB genes (Table S3). The results showed that the Ka/Ks values ranged from 0.07 to 1027. A total of 58.33% of the gene pairs had values < 1, indicating that these genes may have evolved under purifying selection pressure, whereas another 41.67% of the genes may have evolved under positive selection pressure after replication (Ka/Ks > 1). In addition, we found that the timing of gene duplication events was 17.83 Mya~204.86 Mya.
3.4. Synteny Analysis
We performed MYB gene synteny analysis of five representative plants (S. bicolor, Z. mays, A. thaliana, O. sativa, and S. spontaneum) with E. fulvus to better understand the evolutionary features of MYB genes (Table S5; Figure 6). The highest number of MYB homologous genes with sucrose was found in S. bicolor (84 pairs), followed by O. sativa (74 pairs). However, the lowest numbers of MYB homologues were found in E. fulvus and A. thaliana (3 pairs). Notably, 13 EfMYB genes (EfMYB13, EfMYB19, EfMYB20, EfMYB36, EfMYB38, EfMYB28, EfMYB43, EfMYB61, EfMYB56, EfMYB77, EfMYB88, EfMYB91, and EfMYB109) have syntenic gene pairs with rice, sorghum, Saccharum spontaneum and maize MYB genes. This finding shows that MYB family genes are highly conserved in Gramineae.
3.5. Cis-Acting Regulatory Elements in the Promoters of EfMYBs
The study of promoter cis-acting elements is crucial for understanding the transcriptional regulatory properties of EfMYB genes and inferring their potential functions. Therefore, we predicted numerous elements using PlantCARE that may regulate EfMYB genes (Table S6). The cis-acting elements in the EfMYB promoter can be mainly classified into several categories related to plant secondary metabolism, environmental-stress response, light response, hormonal response and plant development. A total of 48 low-temperature-related cis-acting elements (LTR) were found in the promoter of EfR2R3-MYB, suggesting that they may play a role in E. fulvus under cold stress. Some MYB-binding sites related to the response to drought and the regulation of flavonoid biosynthesis were found in the promoters of EfMYB46, EfMYB105, EfMYB87, and EfMYB25. In addition, some regulatory elements involved in seed-specific regulation and meristem expression were also discovered. A total of 64 auxin-related elements, 58 gibberellin-response-related elements, and 41 salicylic-acid-related elements were identified. Notably, we found that regulatory elements associated with MeJA and ABA were most common in EfR2R3-MYB, with 374 and 292, respectively (Figure 7). Similar phenomena were also found in the Ef1R-MYB genes (Figure 8). The EfMYB genes are perhaps involved in the regulation of various types of plant responses through the hormone signaling pathway.
3.6. Protein-Interaction-Network Analysis of the EfMYB Gene
To explore the interaction relationship of EfMYB proteins, Arabidopsis proteins homologous to EfMYB proteins were mapped in OrthoVenn2, and protein-interaction networks were further constructed (Table S7). The protein-interaction network consists of a total of 24 EfMYB family proteins and 14 other family proteins. Among them, EfMYB9, EfMYB72, and EfMYB106 interacted with many EfMYB family proteins, and EfMYB14, EfMYB51, and EfMYB2 interacted with AT1G06720, AT1G63810, RPL4, PRPL11, SAR3, NUP98A, NUP155, NUP160, HOS15, BSH, and ARP4 (Figure 9).
3.7. Effects of Low-Temperature and Drought Stress on EfMYB Gene Expression
We used TBtools to map the heatmap of EfMYB gene expression in E. fulvus leaves under low-temperature stress (0 h, 24 h, and 72 h) (Figure 10). A total of 20 EfR2R3-MYB genes and 11 Ef1R-MYB genes exhibited downregulation of expression after low-temperature stress. After 24 h of low-temperature stress, 26 EfR2R3-MYB genes and 20 Ef1R-MYB genes showed upregulated expression. After 72 h of low-temperature stress, 32 EfR2R3-MYB genes and 12 Ef1R-MYB genes showed upregulated expression. Among them, nine EfMYB genes (EfMYB1, EfMYB30, EfMYB39, EfMYB70, EfMYB81, EfMYB84, EfMYB101, EfMYB124, and EfMYB130) were strongly induced by low-temperature stress. The results imply that all of these genes, which showed significantly upregulated or downregulated expression under cold stress, may play important roles in the low-temperature response of E. fulvus.
We used qRT-PCR to further verify whether these nine EfMYB genes are cold-responsive genes (Figure 11). The results showed that the expression of three genes (EfMYB30, EfMYB70, EfMYB84) could be significantly induced by low temperature, and the expression trend was consistent with the FPKM values. In addition, four other genes (EfMYB1, EfMYB39, EfMYB81, EfMYB124) whose expression could also be induced by low temperature were also identified, but their expression trend was not completely consistent with the FPKM values. It is hypothesized that these seven EfMYB genes play important roles in early or late events of the cold stress response in E. fulvus. In contrast, the expression of EfMYB101 and EfMYB130 was not induced by low temperature.
We then analyzed the effects of drought stress on the expression of these nine EfMYB genes (Figure 12). Drought stress significantly induced the expression of EfMYB30, EfMYB70, EfMYB81, and EfMYB101. After 9 d of stress, the expression levels of EfMYB39, EfMYB124, and EfMYB84 were also somewhat increased compared with those in the CK group. However, the expression levels of EfMYB1 and EfMYB130 were downregulated after drought stress.
3.8. Effects of ABA and MeJA Treatments on EfMYB Gene Expression
Related studies have reported that MYB genes regulate environmental-stress responses in plants through hormone signaling pathways [13]. Our previous analysis showed that MeJA- and ABA-related regulatory elements are most common in the promoters of EfMYB genes. Therefore, we further investigated the effects of ABA and MeJA treatments on EfMYB gene expression. We found that five genes, EfMYB1, EfMYB30, EfMYB39, EfMYB84, EfMYB101, and EfMYB130, were significantly upregulated with ABA treatment for 12 h. ABA treatment had no significant effect on the expression of EfMYB70 and EfMYB81, but significantly downregulated the expression of EfMYB124 (Figure 13). The expression of all nine EfMYB genes was significantly induced with MeJA treatment. Except EfMYB124, which reached its maximal expression after 6 h of MeJA treatment, the expression of the remaining eight genes was highest after 12 h of treatment (Figure 14).
4. Discussion
The MYB family genes are functionally diverse, and the members of the family are widely distributed across various plant species. Since the discovery of the first plant MYB gene (c1) [43], with the continuous development of sequencing technology, MYB genes have been identified in an increasing number of species, including A. thaliana (198), rice (155), tomato (127), and potato (158) [44,45,46,47]. Researchers recently completed whole-genome sequencing of E. fulvus, which provides favorable support for us to carry out EfMYB gene identification, structure, and evolutionary characterization [48]. Based on this recent work, we identified a total of 133 EfMYB genes from the whole genome of E. fulvus (genome size: 902 Mb). The distribution of MYB genes in plants is not directly related to the size of the plant’s own genome. For example, A. thaliana (genome size: 125 Mb) has a relatively large number of MYB genes (198), but sorghum (genome size: 818 Mb) has only 145 MYB genes [49]. Consistent with other reports [50], in plants, the number of EfR2R3-MYB subgroup genes was also the highest among all EfMYB genes. However, we did not find Ef4R-MYB genes present in the E. fulvus genome, and such genes may have been lost during gene evolution.
By constructing phylogenetic trees of EfR2R3-MYB and AtR2R3-MYB, we found that S22, S25, S15, S19, S6, S3, S12, S24, and S10 contained only AtR2R3-MYB; E1, E2 (S21), E3, E4 (S23), E5 (S18), E6 (S20), E7, E8, E9 (S14), E10 (S1), E11 (S5), E12 (S7), E13 (S13), E14 (S16), E15 (S17), E16 (S2), E18 (S4), E19 (S11), and E20 (S9) all contained EfR2R3-MYB and AtR2R3-MYB. This finding suggests that these R2R3-MYB genes may have a common ancestor but underwent species-specific differentiation during the evolutionary process [51]. Gene structure is strongly linked to gene evolution and function, so we analyzed 20 motifs of the EfMYB gene. We found that the types and numbers of conserved motifs of EfR2R3-MYB genes located in the same phylogenetic tree branch are similar, which also supports our grouping. The motif composition of the Ef1R-MYB subgroup of genes shows diversity. In addition, we found that the number of motifs in Ef3R-MYB (EfMYB26) was the highest (16). Interestingly, the motifs of EfMYB26 were essentially duplications of Motif 3, Motif 2, and Motif 16, and these duplicated motifs may be directly related to the function of this gene. In addition, we found that the number of introns in the EfMYB gene was mainly 1~2, which was similar to that reported in other plants [52,53]. However, there are also some genes that have a more prominent number of introns, such as EfMYB12 (EfR2R3-MYB), EfMYB42 (Ef1R-MYB), and EfMYB26 (Ef3R-MYB), which contain 22, 19, and 14 introns, respectively, suggesting that EfMYB genes may have undergone intron loss and gain during the evolutionary process [54].
The MYB structural domain is the core region of MYB transcription factors, which can specifically bind to the promoters of target genes and thus regulate target-gene expression. We further analyzed the core motifs of the EfR2R3-MYB structural domain and found that many amino acid residues are conserved in the R2 and R3 repeat sequences. The R2 and R3 repeat motifs contained 3 and 2 conserved tryptophan residues (W), respectively (Supplementary Figure S1). The first W in the R3 repeat motif was replaced by F/I/L (phenylalanine/isoleucine/leucine), and these phenomena were also observed in Arabidopsis, Morella rubra, and Liriodendron [55,56,57]. In addition to the highly conserved W, Glu-11, Gly-23, Leu-36, and Arg-44 in the R2 repeat motif and Gly-25, Arg-38, and Asn-45 in the R3 repeat motif are also conserved in the EfR2R3-MYB protein. These conserved amino acid residues may together with tryptophan residues maintain the HTH structure of the MYB domain.
Gene duplication events, including whole-genome duplication (WGD), SD, and TD, are important factors driving the expansion of gene families. The occurrence of these duplication events may result in the derivation of new gene functions [58]. In this study, we found 21 segmental-duplication and 4 tandem-duplication EfMYB gene pairs in the whole E. fulvus genome, and these quantities are relatively fewer than the MYB duplication gene pairs in S. spontaneum, Tripterygium wilfordii, and ginger, which also explains the relatively small number of EfMYB genes [59,60]. It has been reported that there are usually three types of new genes derived from gene replication events, namely, nonfunctionalization, new functionalization, and subfunctionalization genes [61]. The type of MYB duplicated genes in E. fulvus needs to be further explored. Synteny analysis can be used to understand the evolutionary relationships of homologous gene pairs and to estimate the time of divergence between species [62]. In this study, E. fulvus and S. bicolor MYB homologous gene pairs were densely distributed across the chromosomes, followed by those with O. sativa MYB homologous gene pairs. Notably, 13 EfMYB genes have syntenic gene pairs with O. sativa, S. bicolor, S. spontaneum, and Z. mays MYB genes, suggesting that these genes may have undergone similar environmental selection in these species.
Several studies have shown that MYB proteins function by interacting with other family proteins. In barley, the MYB family protein (HvANT1) can further regulate the transcriptional activation of the anthocyanin synthesis-related gene HvDFR after binding to the bHLH family protein (HvANT2) and WD40 family protein (HvWD40-140) to form MBW complexes [63]. In protein-interaction-network analysis, we found that EfMYB proteins are tightly linked to other family proteins (ARP4, H0S15, ELF3, PRPL11, and NUP98A). Studies have shown that the NUP98A and ARP4 proteins affect flowering and leaf size in plants [64,65]. The H0S15 gene regulates the expression of the cold-tolerance-related gene COR, thereby affecting cold tolerance in A. thaliana [66]. Overexpression of the ELF3 gene makes transgenic A. thaliana more tolerant to salt stress [67]. PRPL11 has also been reported in studies related to plant salt-tolerance responses. [68]. These results suggest that MYB proteins may interact with these proteins and then regulate various types of responses. In addition, several studies have shown the existence of transcriptional cross-autoregulation within transcription-factor families [69]. In our study, numerous MYB proteins interacted with each other in the interaction network, suggesting that potential transcription-factor self-regulation may also exist within the EfMYB family.
MYB family genes are important in the plant adversity-stress response. Drought stress can induce expression of the PtoMYB142 gene. Its overexpression leads to an increase in wax accumulation in poplar leaves and significantly enhances drought tolerance [70]. Drought stress also rapidly induces AtMYB12 gene expression, and overexpressing transgenic plants accumulate more flavonoids than the wild type, which mitigates damage from drought and oxidative stress [71]. In this study, we found that EfMYB30, EfMYB70, EfMYB81, and EfMYB101 were upregulated under drought stress, indicating that they may play important roles in E. fulvus response to drought stress. Transcriptional regulation of the plant cold-stress response involves a complex network of multiple genes. The ICE-CBF/DREB-COR cascade pathway is considered the most typical pathway regulating the cold stress response [72]. The expression of COR genes has been shown to play a crucial role in the plant cold-stress response. According to reports, CBF genes regulate only 10%-20% of genes, while various transcription factors, such as MYB73, ZAT10, CZF2, and WRKY33, can also induce the expression of COR genes under cold stress [73]. Studies have also indicated that the R2R2-MYB transcription factor gene AtMYB14 can regulate the cold-stress response by modulating CBF gene expression [74]. AtMYB15 can interact with ICE1 to activate CBF gene expression [75]. Additionally, Li et al. found that cold stress can rapidly induce the expression of the maize gene ZmMYB31, and many cold-responsive genes (AtCBF1, AtCBF2, AtCBF3, AtCOR1, AtCOR2, AtGSTU5) were upregulated in transgenic lines overexpressing ZmMYB31 [76]. Genes such as SlMYB102, RmMYB108, and GmMYBJ1 have also been demonstrated to play positive regulatory roles in the plant cold-stress response [77,78,79]. In a previous study, we found numerous MYB binding sites in the promoter region of the E. fulvus EfCBF/DREB genes, indicating that the EfMYB transcription factor may play an important role in regulating CBF gene expression [80]. In this study, we found that EfMYB39, EfMYB84, and EfMYB124 could be induced by low temperature very significantly using qRT-PCR analysis, suggesting that these genes may play important roles in E. fulvus response to cold stress. In addition, further research is needed to determine whether EfMYB transcription factors regulate EfCBF gene expression under cold stress.
Some studies have shown that MYB genes further regulate plant growth, development, and stress responses through hormone signaling pathways. For example, PtrMYB94 responds to drought stress and ABA induction, and its overexpression upregulates some ABA and drought-responsive genes (ABA1, DREB2B) in plants and thus enhances their drought resistance [81]. SiMYB56 enhances the drought tolerance of transgenic rice plants by regulating lignin biosynthesis and ABA signaling pathways [82]. SiMYB75 positively regulates the responses to drought, salt, and osmotic stress through an ABA-mediated pathway [83]. TcMYB8 and SbMYB12 can respond to MeJA treatment [84,85], and GlMYB4 and GlMYB88 play important roles in the synthesis of flavonoids mediated using MeJA signaling [86]. In this study, we found that regulatory elements associated with MeJA and ABA were most common in EfMYB; thus, we also analyzed the expression characteristics of the nine EfMYB genes after ABA and MeJA treatments. EfMYB1, EfMYB30, EfMYB39, EfMYB84, and EfMYB130 responded positively to ABA treatment. Among them, EfMYB30 also had a positive response to drought stress, indicating that the gene may be involved in the regulation of drought stress in E. fulvus through ABA signaling. In addition, we found that these nine EfMYB genes respond to MeJA treatment to varying degrees. In this study, the gene structure and evolutionary characteristics of EfMYB family genes were comprehensively studied, and EfMYB genes with potential functions in drought and cold resistance were identified. We will study the function and regulatory pathway of these candidate genes in future research.
5. Conclusions
In this study, a total of 133 EfMYB genes were identified in the whole E. fulvus genome, and phylogenetic, gene structure, evolutionary, protein-interaction, and expression-characterization analyses were performed to illustrate the structural features, possible evolutionary mechanisms, and potential functions of the gene family members. The results of this study provide a good basis for further study of the biological function and regulatory mechanism of the EfMYB gene and the genetic improvement of transgenic sugarcane.
Conceptualization, methodology, software, X.R.; writing—original draft preparation, X.R. and Z.Q.; writing—review and editing, Z.Q., L.H. and F.L.; formal analysis, Z.Q. and L.X.; investigation, H.W.; Data curation, Q.L. and Q.Z.; visualization, X.R. and Z.Q.; and supervision, project administration, funding acquisition, L.H. and F.L. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data are contained within the article or
We thank the laboratory staff for their help with this study and the reviewers for their constructive suggestions.
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. Phylogenetic tree of R2R3-MYB proteins between E. fulvus and A. thaliana. EfR2R3-MYB is marked in color. AtR2R3-MYB is marked in black. The violet marking for the AtR2R3-MYB genes indicates that they belong to the unclassified-gene category.
Enlarge this image.Figure 2. Conserved motifs and gene structure of EfR2R3-MYB genes. (a) The motif compositions of the EfR2R3-MYB. Colored boxes represent different motifs. (b) Structural characterization of the EfR2R3-MYB. The green, yellow, and pink boxes represent the UTR, CDS, and MYB domains, respectively.
Enlarge this image.Figure 3. Conserved motifs and gene structure of Ef1R-MYB and Ef3R-MYB genes. (a) The motif compositions of the Ef1R-MYB and Ef3R-MYB. Colored boxes represent different motifs. (b) Structural characterization of the Ef1R-MYB and Ef3R-MYB. The green, yellow, and pink boxes represent the CDS, MYB domains, and UTR, respectively.
Enlarge this image.Figure 4. Chromosomal-localization and tandem-duplication analysis of the EfMYB gene. The red genes on the chromosome scaffold represent EfMYB tandem-duplicated gene pairs.
Enlarge this image.Figure 5. Segmental-duplication analysis of the EfMYB gene within the genome. The gray line represents the collinear region in the E. fulvus genome, and the orange line represents duplicated EfMYB gene pairs.
Enlarge this image.Figure 6. Synteny analysis of EfMYB genes with MYB genes from other species. The grey background represents collinear regions within the genomes of sorghum and the exemplified species, whereas the colored lines represent MYB gene pairs with collinearity.
Enlarge this image.Figure 7. Cis-regulatory-element prediction of EfR2R3-MYB genes. Each cis-regulatory element is distinguished using a box of a different color.
Enlarge this image.Figure 8. Cis-regulatory-element prediction of Ef1R-MYB and Ef3R-MYB genes. Each cis-regulatory element is distinguished using a box of a different color.
Enlarge this image.Figure 9. Analysis of the EfMYB protein-interaction network. Red color represents EfMYB family proteins, and other family proteins are indicated in green. The thickness of the connecting line represents the strength of the interaction between proteins.
Enlarge this image.Figure 10. Heatmap of EfMYB gene expression in leaves under low-temperature stress. (a) Heatmap of EfR2R3-MYB gene expression. (b) Heatmap of Ef1R-MYB gene expression. A heatmap was generated using FPKM values. The blue, white, and red boxes represent low, moderate, and high gene expression, respectively.
Enlarge this image.Figure 11. Expression profile of EfMYB genes under low-temperature stress. All graphs were generated using the means of three independent replicate experiments, and significant (p < 0.05) and highly significant (p < 0.01) increases or decreases relative to the CK group are indicated using * and **, respectively. The line graph represents the FPKM values.
Enlarge this image.Figure 12. Expression profile of EfMYB genes under drought stress. All graphs were generated using the means of three independent replicate experiments, and significant (p < 0.05) and highly significant (p < 0.01) increases or decreases relative to the CK group are indicated with * and **, respectively.
Enlarge this image.Figure 13. Expression profile of EfMYB genes under ABA treatment. All graphs were generated using the means of three independent replicate experiments, and significant (p < 0.05) and highly significant (p < 0.01) increases or decreases relative to the CK group are indicated with * and **, respectively.
Enlarge this image.Figure 14. Expression profile of the EfMYB genes under MeJA treatment. All graphs were generated using the means of three independent replicate experiments, and significant (p < 0.05) and highly significant (p < 0.01) increases or decreases relative to the CK group are indicated with * and **, respectively.
Supplementary Materials
The following supporting information can be downloaded at
References
1. Golldack, D.; Lüking, I.; Yang, O. Plant tolerance to drought and salinity: Stress regulating transcription factors and their functional significance in the cellular transcriptional network. Plant Cell Rep.; 2008; 30, pp. 1383-1391. [DOI: https://dx.doi.org/10.1007/s00299-011-1068-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21476089]
2. Zhu, J.K. Abiotic Stress Signaling and Responses in Plants. Cell; 2016; 167, pp. 313-324. [DOI: https://dx.doi.org/10.1016/j.cell.2016.08.029] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27716505]
3. Kranz, H.; Scholz, K.; Weisshaar, B. c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages. Plant J.; 2000; 21, pp. 231-235. [DOI: https://dx.doi.org/10.1046/j.1365-313x.2000.00666.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10743663]
4. Jin, H.; Martin, C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol. Biol.; 1999; 41, pp. 577-585. [DOI: https://dx.doi.org/10.1023/A:1006319732410]
5. Lipsick, J.S. One billion years of Myb. Oncogene; 1996; 13, pp. 223-235. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8710361]
6. Dubos, C.; Stracke, R.; Grotewold, E.; Weisshaar, B.; Martin, C.; Lepiniec, L. MYB transcription factors in Arabidopsis. Trends Plant Sci.; 2010; 15, pp. 573-581. [DOI: https://dx.doi.org/10.1016/j.tplants.2010.06.005]
7. Rosinski, J.A.; Atchley, W.R. Molecular evolution of the Myb family of transcription factors: Evidence for polyphyletic origin. J. Mol. Evol.; 1998; 46, pp. 74-83. [DOI: https://dx.doi.org/10.1007/PL00006285]
8. Zhang, S.; Zhao, Q.; Zeng, D.; Xu, J.; Zhou, H.; Wang, F.; Ma, N.; Li, Y. RhMYB108, an R2R3-MYB transcription factor, is involved in ethylene-and JA-induced petal senescence in rose plants. Hortic. Res.; 2019; 6, 131. [DOI: https://dx.doi.org/10.1038/s41438-019-0221-8]
9. Zhu, X.; Liang, W.; Cui, X.; Chen, M.; Yin, C.; Luo, Z.; Zhu, J.; Lucas, W.J.; Wang, Z.; Zhang, D. Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther, a MYB domain protein. Plant J.; 2015; 82, pp. 570-581. [DOI: https://dx.doi.org/10.1111/tpj.12820]
10. Cao, H.; Chen, J.; Yue, M.; Xu, C.; Jian, W.; Liu, Y.; Song, B.; Gao, Y.; Cheng, Y.; Li, Z. Tomato transcriptional repressor MYB70 directly regulates ethylene-dependent fruit ripening. Plant J.; 2020; 104, pp. 1568-1581. [DOI: https://dx.doi.org/10.1111/tpj.15021]
11. Sun, B.; Zhu, Z.; Chen, C.; Chen, G.; Cao, B.; Chen, C.; Lei, J. Jasmonate-Inducible R2R3-MYB Transcription Factor Regulates Capsaicinoid Biosynthesis and Stamen Development in Capsicum. J. Agric. Food Chem.; 2019; 67, pp. 10891-10903. [DOI: https://dx.doi.org/10.1021/acs.jafc.9b04978] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31505929]
12. Seo, P.J.; Lee, S.B.; Suh, M.C.; Park, M.J.; Go, Y.S.; Park, C.M. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis. Plant Cell.; 2011; 23, pp. 1138-1152. [DOI: https://dx.doi.org/10.1105/tpc.111.083485] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21398568]
13. Xiong, H.; Li, J.; Liu, P.; Duan, J.; Zhao, Y.; Guo, X.; Li, Y.; Zhang, H.; Ali, J.; Li, Z. Overexpression of OsMYB48-1, a novel MYB-related transcription factor, enhances drought and salinity tolerance in rice. PLoS ONE; 2014; 9, e92913. [DOI: https://dx.doi.org/10.1371/journal.pone.0092913] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24667379]
14. Gao, F.; Zhou, J.; Deng, R.Y.; Zhao, H.X.; Li, C.L.; Chen, H.; Suzuki, T.; Park, S.U.; Wu, Q. Overexpression of a tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis. J. Plant Physiol.; 2017; 214, pp. 81-90. [DOI: https://dx.doi.org/10.1016/j.jplph.2017.04.007] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28460279]
15. Wang, R.K.; Cao, Z.H.; Hao, Y.J. Overexpression of a R2R3 MYB gene MdSIMYB1 increases tolerance to multiple stresses in transgenic tobacco and apples. Physiol. Plant.; 2014; 150, pp. 76-87. [DOI: https://dx.doi.org/10.1111/ppl.12069] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23647377]
16. Meng, C.; Sui, N. Overexpression of maize MYB-IF35 increases chilling tolerance in Arabidopsis. Plant Physiol. Biochem.; 2019; 135, pp. 167-173. [DOI: https://dx.doi.org/10.1016/j.plaphy.2018.11.038]
17. Lv, Y.; Yang, M.; Hu, D.; Yang, Z.; Ma, S.; Li, X.; Xiong, L. The OsMYB30 Transcription Factor Suppresses Cold Tolerance by Interacting with a JAZ Protein and Suppressing β-Amylase Expression. Plant Physiol.; 2017; 173, pp. 1475-1491. [DOI: https://dx.doi.org/10.1104/pp.16.01725]
18. He, J.; Liu, Y.; Yuan, D.; Duan, M.; Liu, Y.; Shen, Z.; Yang, C.; Qiu, Z.; Liu, D.; Wen, P. et al. An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia-lyase pathway in rice. Proc. Natl. Acad. Sci. USA; 2020; 117, pp. 271-277. [DOI: https://dx.doi.org/10.1073/pnas.1902771116]
19. Zhang, Y.L.; Zhang, C.L.; Wang, G.L.; Wang, Y.X.; Qi, C.H.; Zhao, Q.; You, C.X.; Li, Y.Y.; Hao, Y.J. The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis. BMC Plant Biol.; 2019; 19, 362. [DOI: https://dx.doi.org/10.1186/s12870-019-1918-4]
20. Hoang, N.V.; Furtado, A.; Botha, F.C.; Simmons, B.A.; Henry, R.J. Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels. Front. Bioeng. Biotechnol.; 2015; 3, 182. [DOI: https://dx.doi.org/10.3389/fbioe.2015.00182]
21. Xian, H.W.; Qing, H.Y.; Fu, S.L.; Li, L.H.; Shun, C.H. Characterization of the Chromosomal Transmission of Intergeneric Hybrids of Saccharum spp. and Erianthus fulvus by Genomic in situ Hybridization. Crop Sci.; 2010; 50, pp. 1642-1648.
22. Qian, Z.; Li, X.; He, L.; Gu, S.; Shen, Q.; Rao, X.; Zhang, R.; Di, Y.; Xie, L.; Wang, X. et al. EfGD: The Erianthus fulvus genome database. Database; 2022; 31, baac076. [DOI: https://dx.doi.org/10.1093/database/baac076] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36043401]
23. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J. et al. Pfam: The protein families database in 2021. Nucleic Acids Res.; 2021; 49, pp. D412-D419. [DOI: https://dx.doi.org/10.1093/nar/gkaa913] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33125078]
24. Lu, S.; Wang, J.; Chitsaz, F.; Derbyshire, M.K.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Marchler, G.H.; Song, J.S. et al. CDD/SPARCLE: The conserved domain database in 2020. Nucleic Acids Res.; 2020; 48, pp. D265-D268. [DOI: https://dx.doi.org/10.1093/nar/gkz991]
25. Letunic, I.; Bork, P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res.; 2018; 46, pp. D493-D496. [DOI: https://dx.doi.org/10.1093/nar/gkx922] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29040681]
26. Potter, S.C.; Luciani, A.; Eddy, S.R.; Park, Y.; Lopez, R.; Finn, R.D. HMMER web server: 2018 update. Nucleic Acids Res.; 2018; 46, pp. W200-W204. [DOI: https://dx.doi.org/10.1093/nar/gky448]
27. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol.; 2016; 33, pp. 1870-1874. [DOI: https://dx.doi.org/10.1093/molbev/msw054]
28. Du, J.; Zhang, Q.; Hou, S.; Chen, J.; Meng, J.; Wang, C.; Liang, D.; Wu, R.; Guo, Y. Genome-Wide Identification and Analysis of the R2R3-MYB Gene Family in Theobroma cacao. Genes; 2022; 13, 1572. [DOI: https://dx.doi.org/10.3390/genes13091572]
29. Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.C.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol.; 1999; 112, pp. 531-552.
30. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res.; 2015; 43, pp. W39-W49. [DOI: https://dx.doi.org/10.1093/nar/gkv416]
31. Crooks, G.E.; Hon, G.; Chandonia, J.M.; Brenner, S.E. WebLogo: A sequence logo generator. Genome Res.; 2004; 14, pp. 1188-1190. [DOI: https://dx.doi.org/10.1101/gr.849004]
32. 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. Mol. Plant.; 2020; 13, pp. 1194-1202. [DOI: https://dx.doi.org/10.1016/j.molp.2020.06.009] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32585190]
33. Voorrips, R.E. MapChart: Software for the graphical presentation of linkage maps and QTLs. J. Hered.; 2002; 93, pp. 77-78. [DOI: https://dx.doi.org/10.1093/jhered/93.1.77] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12011185]
34. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.H.; Jin, H.; Marler, B.; Guo, H. et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res.; 2012; 40, e49. [DOI: https://dx.doi.org/10.1093/nar/gkr1293] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22217600]
35. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform.; 2010; 8, pp. 77-80. [DOI: https://dx.doi.org/10.1016/S1672-0229(10)60008-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20451164]
36. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res.; 2002; 30, pp. 325-327. [DOI: https://dx.doi.org/10.1093/nar/30.1.325] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11752327]
37. Xu, L.; Dong, Z.; Fang, L.; Luo, Y.; Wei, Z.; Guo, H.; Zhang, G.; Gu, Y.Q.; Coleman-Derr, D.; Xia, Q. et al. OrthoVenn2: A web server for whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Res.; 2019; 47, pp. W52-W58. [DOI: https://dx.doi.org/10.1093/nar/gkz333]
38. Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N.S.; Wang, J.T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res.; 2003; 13, pp. 2498-2504. [DOI: https://dx.doi.org/10.1101/gr.1239303]
39. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods; 2001; 25, pp. 402-408. [DOI: https://dx.doi.org/10.1006/meth.2001.1262]
40. Xia, H.; Liu, X.; Lin, Z.; Zhang, X.; Wu, M.; Wang, T.; Deng, H.; Wang, J.; Lin, L.; Deng, Q. et al. Genome-Wide Identification of MYB Transcription Factors and Screening of Members Involved in Stress Response in Actinidia. Int. J. Mol. Sci.; 2022; 23, 2323. [DOI: https://dx.doi.org/10.3390/ijms23042323]
41. Xu, B.; Chen, B.; Qi, X.; Liu, S.; Zhao, Y.; Tang, C.; Meng, X. Genome-wide Identification and Expression Analysis of RcMYB Genes in Rhodiola crenulata. Front. Genet.; 2022; 13, 831611. [DOI: https://dx.doi.org/10.3389/fgene.2022.831611]
42. Wei, H.; Chen, S.; Niyitanga, S.; Liu, T.; Qi, J.; Zhang, L. Genome-wide identification and expression analysis response to GA3 stresses of WRKY gene family in seed hemp (Cannabis sativa L). Gene; 2022; 822, 146290. [DOI: https://dx.doi.org/10.1016/j.gene.2022.146290]
43. Paz-Ares, J.; Ghosal, D.; Wienand, U.; Peterson, P.A.; Saedler, H. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J.; 1987; 6, pp. 3553-3558. [DOI: https://dx.doi.org/10.1002/j.1460-2075.1987.tb02684.x]
44. Yanhui, C.; Xiaoyuan, Y.; Kun, H.; Meihua, L.; Jigang, L.; Zhaofeng, G.; Zhiqiang, L.; Yunfei, Z.; Xiaoxiao, W.; Xiaoming, Q. et al. The MYB transcription factor superfamily of Arabidopsis: Expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol. Biol.; 2006; 60, pp. 107-124. [DOI: https://dx.doi.org/10.1007/s11103-005-2910-y]
45. Katiyar, A.; Smita, S.; Lenka, S.K.; Rajwanshi, R.; Chinnusamy, V.; Bansal, K.C. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis. BMC Genom.; 2012; 13, 544. [DOI: https://dx.doi.org/10.1186/1471-2164-13-544]
46. Li, Z.; Peng, R.; Tian, Y.; Han, H.; Xu, J.; Yao, Q. Genome-Wide Identification and Analysis of the MYB Transcription Factor Superfamily in Solanum lycopersicum. Plant Cell Physiol.; 2016; 57, pp. 1657-1677. [DOI: https://dx.doi.org/10.1093/pcp/pcw091]
47. Sun, W.; Ma, Z.; Chen, H.; Liu, M. MYB Gene Family in Potato (Solanum tuberosum L.): Genome-Wide Identification of Hormone-Responsive Reveals Their Potential Functions in Growth and Development. Int. J. Mol. Sci.; 2019; 20, 4847. [DOI: https://dx.doi.org/10.3390/ijms20194847]
48. Kui, L.; Majeed, A.; Wang, X.; Yang, Z.; Chen, J.; He, L.; Di, Y.; Li, X.; Qian, Z.; Jiao, Y. et al. A chromosome-level genome assembly for Erianthus fulvus provides insights into its biofuel potential and facilitates breeding for improvement of sugarcane. Plant Commun.; 2023; 21, 100562. [DOI: https://dx.doi.org/10.1016/j.xplc.2023.100562] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36814384]
49. Baillo, E.H.; Kimotho, R.N.; Zhang, Z.; Xu, P. Transcription Factors Associated with Abiotic and Biotic Stress Tolerance and Their Potential for Crops Improvement. Genes; 2019; 10, 771. [DOI: https://dx.doi.org/10.3390/genes10100771] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31575043]
50. Xia, M.; Tu, L.; Liu, Y.; Jiang, Z.; Wu, X.; Gao, W.; Huang, L. Genome-wide analysis of MYB family genes in Tripterygium wilfordii and their potential roles in terpenoid biosynthesis. Plant Direct.; 2022; 6, e424. [DOI: https://dx.doi.org/10.1002/pld3.424] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35898558]
51. Yang, J.; Zhang, B.; Gu, G.; Yuan, J.; Shen, S.; Jin, L.; Lin, Z.; Lin, J.; Xie, X. Genome-wide identification and expression analysis of the R2R3-MYB gene family in tobacco (Nicotiana tabacum L.). BMC Genom.; 2022; 23, 432. [DOI: https://dx.doi.org/10.1186/s12864-022-08658-7] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35681121]
52. Du, H.; Feng, B.R.; Yang, S.S.; Huang, Y.B.; Tang, Y.X. The R2R3-MYB transcription factor gene family in maize. PLoS ONE; 2012; 7, e37463. [DOI: https://dx.doi.org/10.1371/journal.pone.0037463] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22719841]
53. Ke, Y.J.; Zheng, Q.D.; Yao, Y.H.; Ou, Y.; Chen, J.Y.; Wang, M.J.; Lai, H.P.; Yan, L.; Liu, Z.J.; Ai, Y. Genome-Wide Identification of the MYB Gene Family in Cymbidium ensifolium and Its Expression Analysis in Different Flower Colors. Int. J. Mol. Sci.; 2021; 22, 13245. [DOI: https://dx.doi.org/10.3390/ijms222413245] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34948043]
54. Fedorova, L.; Fedorov, A. Introns in gene evolution. Genetica; 2003; 118, pp. 123-131. [DOI: https://dx.doi.org/10.1023/A:1024145407467] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12868603]
55. Stracke, R.; Werber, M.; Weisshaar, B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol.; 2001; 4, pp. 447-456. [DOI: https://dx.doi.org/10.1016/S1369-5266(00)00199-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11597504]
56. Cao, Y.; Jia, H.; Xing, M.; Jin, R.; Grierson, D.; Gao, Z.; Sun, C.; Chen, K.; Xu, C.; Li, X. Genome-Wide Analysis of MYB Gene Family in Chinese Bayberry (Morella rubra) and Identification of Members Regulating Flavonoid Biosynthesis. Front. Plant Sci.; 2021; 12, 691384. [DOI: https://dx.doi.org/10.3389/fpls.2021.691384] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34249063]
57. Yang, L.; Liu, H.; Hao, Z.; Zong, Y.; Xia, H.; Shen, Y.; Li, H. Genome-Wide Identification and Expression Analysis of R2R3-MYB Family Genes Associated with Petal Pigment Synthesis in Liriodendron. Int. J. Mol. Sci.; 2021; 22, 11291. [DOI: https://dx.doi.org/10.3390/ijms222011291]
58. Clark, J.W.; Donoghue, P.C.J. Whole-Genome Duplication and Plant Macroevolution. Trends Plant Sci.; 2018; 23, pp. 933-945. [DOI: https://dx.doi.org/10.1016/j.tplants.2018.07.006]
59. Yuan, Y.; Yang, X.; Feng, M.; Ding, H.; Khan, M.T.; Zhang, J.; Zhang, M. Genome-wide analysis of R2R3-MYB transcription factors family in the autopolyploid Saccharum spontaneum: An exploration of dominance expression and stress response. BMC Genom.; 2021; 22, 622. [DOI: https://dx.doi.org/10.1186/s12864-021-07689-w]
60. Yao, X.; Meng, F.; Wu, L.; Guo, X.; Sun, Z.; Jiang, W.; Zhang, J.; Wu, J.; Wang, S.; Wang, Z. et al. Genome-wide identification of R2R3-MYB family genes and gene response to stress in ginger. Plant Genome; 2022; 9, e20258. [DOI: https://dx.doi.org/10.1002/tpg2.20258]
61. Jiang, C.; Gu, J.; Chopra, S.; Gu, X.; Peterson, T. Ordered origin of the typical two- and three-repeat Myb genes. Gene; 2004; 326, pp. 13-22. [DOI: https://dx.doi.org/10.1016/j.gene.2003.09.049]
62. Zafar, M.M.; Rehman, A.; Razzaq, A.; Parvaiz, A.; Mustafa, G.; Sharif, F.; Mo, H.; Youlu, Y.; Shakeel, A.; Ren, M. Genome-wide characterization and expression analysis of Erf. gene family in cotton. BMC Plant Biol.; 2022; 22, 134. [DOI: https://dx.doi.org/10.1186/s12870-022-03521-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35317739]
63. Chen, L.; Cui, Y.; Yao, Y.; An, L.; Bai, Y.; Li, X.; Yao, X.; Wu, K. Genome-wide identification of WD40 transcription factors and their regulation of the MYB-bHLH-WD40 (MBW) complex related to anthocyanin synthesis in Qingke (Hordeum vulgare L. var. nudum Hook. f.). BMC Genom.; 2023; 24, 166. [DOI: https://dx.doi.org/10.1186/s12864-023-09240-5] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37016311]
64. Jiang, S.; Xiao, L.; Huang, P.; Cheng, Z.; Chen, F.; Miao, Y.; Fu, Y.F.; Chen, Q.; Zhang, X.M. Nucleoporin Nup98 participates in flowering regulation in a CONSTANS-independent mode. Plant Cell Rep.; 2019; 38, pp. 1263-1271. [DOI: https://dx.doi.org/10.1007/s00299-019-02442-w] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31236659]
65. Nie, W.F.; Wang, J. Actin-Related Protein 4 Interacts with PIE1 and Regulates Gene Expression in Arabidopsis. Genes; 2021; 12, 520. [DOI: https://dx.doi.org/10.3390/genes12040520]
66. Lim, C.J.; Ali, A.; Park, J.; Shen, M.; Park, K.S.; Baek, D.; Yun, D.J. HOS15-PWR chromatin remodeling complex positively regulates cold stress in Arabidopsis. Plant Signal Behav.; 2021; 16, 1893978. [DOI: https://dx.doi.org/10.1080/15592324.2021.1893978]
67. Sakuraba, Y.; Bülbül, S.; Piao, W.; Choi, G.; Paek, N.C. Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways. Plant J.; 2017; 92, pp. 1106-1120. [DOI: https://dx.doi.org/10.1111/tpj.13747] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29032592]
68. Omidbakhshfard, M.A.; Omranian, N.; Ahmadi, F.S.; Nikoloski, Z.; Mueller-Roeber, B. Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana. Plant Signal Behav.; 2012; 7, pp. 1095-1102. [DOI: https://dx.doi.org/10.4161/psb.21218]
69. Sharma, P.; Lin, T.; Grandellis, C.; Yu, M.; Hannapel, D.J. The BEL1-like family of transcription factors in potato. J. Exp. Bot.; 2014; 65, pp. 709-723. [DOI: https://dx.doi.org/10.1093/jxb/ert432]
70. Song, Q.; Kong, L.; Yang, X.; Jiao, B.; Hu, J.; Zhang, Z.; Xu, C.; Luo, K. PtoMYB142, a poplar R2R3-MYB transcription factor, contributes to drought tolerance by regulating wax biosynthesis. Tree Physiol.; 2022; 42, pp. 2133-2147. [DOI: https://dx.doi.org/10.1093/treephys/tpac060]
71. Wang, F.; Kong, W.; Wong, G.; Fu, L.; Peng, R.; Li, Z.; Yao, Q. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Mol. Genet. Genom.; 2016; 291, pp. 1545-1559. [DOI: https://dx.doi.org/10.1007/s00438-016-1203-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27033553]
72. Zhao, C.; Zhang, Z.; Xie, S.; Si, T.; Li, Y.; Zhu, J.K. Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis. Plant Physiol.; 2016; 171, pp. 2744-2759. [DOI: https://dx.doi.org/10.1104/pp.16.00533] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27252305]
73. Ritonga, F.N.; Ngatia, J.N.; Wang, Y.; Khoso, M.A.; Farooq, U.; Chen, S. AP2/ERF, an important cold stress-related transcription factor family in plants: A review. Physiol. Mol. Biol. Plants; 2021; 27, pp. 1953-1968. [DOI: https://dx.doi.org/10.1007/s12298-021-01061-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34616115]
74. Chen, Y.; Chen, Z.; Kang, J.; Kang, D.; Gu, H.; Qin, G. AtMYB14 Regulates Cold Tolerance in Arabidopsis. Plant Mol. Biol. Rep.; 2013; 31, pp. 87-97. [DOI: https://dx.doi.org/10.1007/s11105-012-0481-z]
75. Agarwal, M.; Hao, Y.; Kapoor, A.; Dong, C.H.; Fujii, H.; Zheng, X.; Zhu, J.K. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J. Biol. Chem.; 2006; 281, pp. 37636-37645. [DOI: https://dx.doi.org/10.1074/jbc.M605895200]
76. Li, M.; Lin, L.; Zhang, Y.; Sui, N. ZmMYB31, a R2R3-MYB transcription factor in maize, positively regulates the expression of CBF genes and enhances resistance to chilling and oxidative stress. Mol. Biol. Rep.; 2019; 46, pp. 3937-3944. [DOI: https://dx.doi.org/10.1007/s11033-019-04840-5]
77. Wang, M.; Hao, J.; Chen, X.; Zhang, X. SlMYB102 expression enhances low-temperature stress resistance in tomato plants. PeerJ; 2020; 8, e10059. [DOI: https://dx.doi.org/10.7717/peerj.10059]
78. Dong, J.; Cao, L.; Zhang, X.; Zhang, W.; Yang, T.; Zhang, J.; Che, D. An R2R3-MYB Transcription Factor RmMYB108 Responds to Chilling Stress of Rosa multiflora and Conferred Cold Tolerance of Arabidopsis. Front. Plant Sci.; 2021; 12, 696919. [DOI: https://dx.doi.org/10.3389/fpls.2021.696919]
79. Su, L.T.; Li, J.W.; Liu, D.Q.; Zhai, Y.; Zhang, H.J.; Li, X.W.; Zhang, Q.L.; Wang, Y.; Wang, Y.Q. A novel MYB transcription factor, GmMYBJ1, from soybean confers drought and cold tolerance in Arabidopsis thaliana. Gene; 2014; 538, pp. 46-55. [DOI: https://dx.doi.org/10.1016/j.gene.2014.01.024]
80. Qian, Z.; Rao, X.; Zhang, R.; Gu, S.; Shen, Q.; Wu, H.; Lv, S.; Xie, L.; Li, X.; Wang, X. et al. Genome-Wide Identification, Evolution, and Expression Analyses of AP2/ERF Family Transcription Factors in Erianthus fulvus. Int. J. Mol. Sci.; 2023; 24, 7102. [DOI: https://dx.doi.org/10.3390/ijms24087102]
81. Fang, Q.; Wang, X.; Wang, H.; Tang, X.; Liu, C.; Yin, H.; Ye, S.; Jiang, Y.; Duan, Y.; Luo, K. The poplar R2R3 MYB transcription factor PtrMYB94 coordinates with abscisic acid signaling to improve drought tolerance in plants. Tree Physiol.; 2020; 40, pp. 46-59. [DOI: https://dx.doi.org/10.1093/treephys/tpz113] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31728530]
82. Xu, W.; Tang, W.; Wang, C.; Ge, L.; Sun, J.; Qi, X.; He, Z.; Zhou, Y.; Chen, J.; Xu, Z. et al. SiMYB56 Confers Drought Stress Tolerance in Transgenic Rice by Regulating Lignin Biosynthesis and ABA Signaling Pathway. Front. Plant Sci.; 2020; 11, 785. [DOI: https://dx.doi.org/10.3389/fpls.2020.00785] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32625221]
83. Dossa, K.; Mmadi, M.A.; Zhou, R.; Liu, A.; Yang, Y.; Diouf, D.; You, J.; Zhang, X. Ectopic expression of the sesame MYB transcription factor SiMYB305 promotes root growth and modulates ABA-mediated tolerance to drought and salt stresses in Arabidopsis. AoB Plants; 2019; 12, plz081. [DOI: https://dx.doi.org/10.1093/aobpla/plz081] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32099638]
84. Zhou, L.; Li, J.; Zeng, T.; Xu, Z.; Luo, J.; Zheng, R.; Wang, Y.; Wang, C. TcMYB8, a R3-MYB Transcription Factor, Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium. Int. J. Mol. Sci.; 2022; 23, 12186. [DOI: https://dx.doi.org/10.3390/ijms232012186]
85. Wang, W.; Hu, S.; Yang, J.; Zhang, C.; Zhang, T.; Wang, D.; Cao, X.; Wang, Z. A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi. Int. J. Mol. Sci.; 2022; 23, 15452. [DOI: https://dx.doi.org/10.3390/ijms232415452]
86. Li, Y.; Chen, X.; Wang, J.; Zou, G.; Wang, L.; Li, X. Two responses to MeJA induction of R2R3-MYB transcription factors regulate flavonoid accumulation in Glycyrrhiza uralensis Fisch. PLoS ONE; 2020; 15, e0236565. [DOI: https://dx.doi.org/10.1371/journal.pone.0236565]
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