Characteristics and Functions of MYB (v-Myb avivan myoblastsis virus oncogene homolog)-Related Genes in Arabidopsis thaliana

Abstract

The MYB (v-Myb avivan myoblastsis virus oncogene homolog) transcription factor family is one of the largest families of plant transcription factors which plays a vital role in many aspects of plant growth and development. MYB-related is a subclass of the MYB family. Fifty-nine Arabidopsis thaliana MYB-related (AtMYB-related) genes have been identified. In order to understand the functions of these genes, in this review, the promoters of AtMYB-related genes were analyzed by means of bioinformatics, and the progress of research into the functions of these genes has been described. The main functions of these AtMYB-related genes are light response and circadian rhythm regulation, root hair and trichome development, telomere DNA binding, and hormone response. From an analysis of cis-acting elements, it was found that the promoters of these genes contained light-responsive elements and plant hormone response elements. Most genes contained elements related to drought, low temperature, and defense and stress responses. These analyses suggest that AtMYB-related genes may be involved in A. thaliana growth and development, and environmental adaptation through plant hormone pathways. However, the functions of many genes do not occur independently but instead interact with each other through different pathways. In the future, the study of the role of the gene in different pathways will be conducive to a comprehensive understanding of the function of the gene. Therefore, gene cloning and protein functional analyses can be subsequently used to understand the regulatory mechanisms of AtMYB-related genes in the interaction of multiple signal pathways. This review provides theoretical guidance for the follow-up study of plant MYB-related genes.

1. Introduction

The regulation of gene expression controls many important biological processes. This regulatory pathway is often complex and diverse, requiring the involvement of multiple factors, including transcription factors, which have been studied in considerable detail. Transcription factors can recognize specific DNA motifs in gene regulatory regions and, when combined with specific sites on the target gene promoter, regulate transcription [1,2]. The MYB (v-Myb avivan myoblastsis virus oncogene homolog) transcription factors are one of the largest families of transcriptional regulators in plants; they are involved in growth and development, secondary metabolism, signal transduction, and biotic and abiotic stresses [3]. There has been much research and interest in the R2R3-MYB gene, likely due to the wide distribution of R2R3-MYB in plants. However, little attention has been paid to the analysis and systematic induction of AtMYB-related genes. This review analyzes the structure and promoter characteristics of the 59 Arabidopsis MYB-related genes recently identified by Lal et al. [4], and summarizes the functions of these genes. This work will help direct follow-up experiments to verify the functions of these genes in growth, development, and stress responses, and will provide a theoretical basis for the study of MYB-related genes.

2. Survey Methodology

Based on the AtMYB-related genes’ IDs reported by Lal et al. [4], we searched the literature for information on AtMYB-related genes in TAIR (https://www.arabidopsis.org/, accessed on 20 January 2023) and NCBI PubMed [4]. We performed a content review and analysis of the literature, both classic and published within the last 5 years.

The genome sequence file and General Feature Format Version 3 (gff3) file for Arabidopsis thaliana were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html, accessed on 20 January 2023). Sequences of AtMYB-related proteins were extracted using TBtools [5]. The MAFFT version 7 website (https://mafft.cbrc.jp/alignment/server/, accessed on 20 January 2023) was used with default parameters to perform multiple sequence alignment on the AtMYB-related protein sequences [6,7]. Then, a neighbor-joining (NJ) phylogenetic tree was constructed using MAGE version 11 with the following parameters: p-distance, pairwise deletion, and bootstrap analysis with 1000 replicates [8,9]. The conserved motifs (Supplementary Figure S1, Supplementary Table S1) of AtMYB-related genes were predicted by using MEME (https://meme-suite.org/meme/tools/meme, accessed on 21 January 2023) with the following parameters: the distribution of motifs—0 or 1 per sequence; maximum number of motifs to find—8; and other parameters—default values [10]. The intron/exon structure information was obtained from the gff3 file. The sequences of the 2000 base pairs upstream of the AtMYB-related genes were extracted using TBtools. The PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 21 January 2023) was used to predict and analyze the cis-acting elements of the sequence 2000 bp upstream of each AtMYB-related gene [11]. The promoter sequences of AtMYB-related genes are shown in Supplementary File S1. Finally, TBtools was used for visualization.

3. Identification and Characterization of the MYB Genes

The transcription factor MYB was first found in the avian acute myeloblastic leukemia virus in 1941, and in 1982 it was identified and named v-myb [12,13]. In 1987, the first plant MYB transcription factor ZmMYBC1 was identified in maize. Its function is related to anthocyanin synthesis [14]. In 1989, the first fungal MYB transcription factor Bas1 was identified in Saccharomyces cerevisiae, which was found to be necessary to activate the transcription of the histidine dehydrogenase gene HIS4 [15]. The characterization and classification of a gene family is the first step in functional research. Subsequently, increasing numbers of members of the MYB gene family have been identified, and the functions of more MYB genes have been discovered. Chen et al. [16] identified a total of 198 MYB genes in A. thaliana, of which 64 were MYB-related genes. From the phylogenetic analysis, 60 MYB-related genes were divided into five categories: CCA1-like, CPC-like, I-box-like, TBP-like, and R-R type [16]. Subsequently, Katiyar et al. identified 197 MYB genes in A. thaliana, including 52 MYB-related genes [17]. Recently, Lal et al. identified 193 MYB genes in A. thaliana, including 59 MYB-related genes, in a comprehensive analysis of 1R- and 2R-MYBs [4]. There are a total of 122 MYBs in Brachypodium distachyon [18] compared with 171 MYBs in Chinese jujube [19]. Arce-Rodriguez et al. identified a total of 235 MYBs in chili pepper and proposed some candidate genes that may be involved in the regulation of phenylpropane, capsaicin, carotenoid, and vitamin C biosynthesis [20]. In Morus alba, a total of 166 MYBs were identified [21]. A number of identifications and studies have also been published on MYB-related genes in other plants, including soybean [22], potato [23], Brassica napus L. [24], populus [9], pepper [25], and sweet osmanthus [26].

4. Structures and Main Functions of the MYB Gene Family

The N-terminus of the MYB transcription factor contains a conserved specific DNA-binding domain (MYB domain) [27]. This domain generally consists of one to four sequence repeats (R) of about 52 amino acids, each of which forms three α helices. The second and third α helices of each repeat form a helix–angle–helix (HTH) structure [2,27]. Based on the number of domains, MYB can be divided into four categories (Figure 1): a single-repeated MYB with one R sequence, called 1R-MYB or MYB-related; R2R3-MYB with two R sequences; 3R-MYB with three R sequences; and 4R-MYB with four R sequences [27].

1R-MYB, which contains complete or partially repeated proteins, is the second largest subclass of the MYB family and is widely distributed in plants [27]. Phylogenetic and expression analyses revealed the conservation and diversity of MYB-related genes, and functional studies showed that they regulate plant development and stress response [28]. The R2R3-MYB subfamily is the largest group in the plant MYB family. It may have evolved from a 3R-MYB that lost the R1 sequence or from a 1R-MYB that evolved from the copying of the sequence [29,30]. The abundance of 3R-MYB is relatively low in plants. Recent studies have shown that its main function is related to the cell cycle and protein regulation, as well as cell differentiation [29,31]. The smallest member of the MYB family is 4R-MYB. Its function is still unclear and is under investigation [32].

In recent years, with the identification and functional studies of the MYB genes, the number of reviews about the MYB genes has increased. These reviews describe two main points: (1) the biochemical and molecular characteristics of MYB transcription factors [33], including their type, structure, evolution, and function [1,27]; and (2) the role of MYB transcription factors in controlling various biological processes, including stress responses [34,35] and secondary metabolic processes [36]. For example, regarding advances in research on MYB transcription factors in plant stress resistance and breeding [37], Yan et al. discussed the regulatory mechanism of the MYB transcription factors in anthocyanin biosynthesis [32]. Recently, progress has been reported on MYB transcription factors regulating multiple functions in medicinal plants [38]. In addition, studies based on the genome-wide analysis of the structure and evolution of MYB genes have also been published [28].

5. Functions of MYB-Related Genes

Although MYB-related genes contain only one or part of the MYB/SANT domain, this domain is still necessary for the function of MYB transcription factors. On the one hand, it plays an important role in overcoming abiotic and biotic stresses by regulating various defense mechanisms in many plants [39]. For example, the MYB-related transcription factor TaLHY plays an important role in resistance to stripe rust in wheat [40]. On the other hand, some MYB-related genes participate in some biological pathways and regulate other related genes. For example, ZmMYB48, OsMYB48-1, and StMYB1R-1 confer drought resistance by regulating the expression of stress response marker genes and controlling physiological functions [41,42,43]. The wheat MYB-related transcription factor TaMYB72 promotes rice flowering by upregulating the Florigen genes HD3A and RFT1 [44]; GmMYB118 improves drought and salt tolerance by inducing the expression of stress-related genes and regulating osmotic and oxidizing substances [22]; an MYB-related transcription factor from Lilium lancifolium L. (LlMYB3) participates in the Arabidopsis anthocyanin biosynthesis pathway and enhances the tolerance of A. thaliana to a variety of abiotic stresses [45]; and the OsMYB-R1 gene regulates resistance to multiple stressors in rice through the interaction between auxin and salicylic acid [46].

Supplementary Table S2 presents information on AtMYB-related genes. The functions of AtMYB-related genes are described in detail below.

6. Light Response and Circadian Rhythm Regulation

The large number of circadian rhythm regulation genes in Arabidopsis reflects the important role of the circadian rhythm in plant growth and development. This is consistent with the fact that the expression of many genes, including those involved in photosynthesis and light signaling, oscillates rhythmically. In addition, many physiological processes are controlled by the circadian rhythm, such as flowering and the movement of cotyledons and leaves [47].

CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are MYB-related proteins that play a role in or close to the central oscillator of Arabidopsis, and have a synergistic effect in regulating the circadian rhythm of Arabidopsis [48]. The MYB-related protein encoded by the CCA1 gene binds to a region of the light-harvesting chlorophyll a/b protein gene (Lhcb1*3) promoter, which can affect the regulation of the phytochrome promoter in vivo. The expression of the CCA1 gene itself can also be regulated by light, leading to the increased transcription of the Lhcbl*3 gene, which is considered to be part of the phytochrome signal transduction chain [49]. A vernalization-responsive cis-element (VREVIN3) was identified in the VERNALIZATION INSENSITIVE 3 (VIN3) promoter, which consists of two known continuous cis elements: a G-box and an evening element (EE). Kyung et al. found that CCA1 and LHY are involved in the transcriptional activation of VIN3 by binding to EE. In addition, the rhythmic expression patterns of CCA1 and LHY also changed after long-term cold exposure. Therefore, it is considered that CCA1 and LHY are part of the signal transduction mechanism to ensure the vernalization of Arabidopsis [50]. CCA1 and LHY also promote the expression of two day-phased genes, PRR7 and PRR9, which in turn are suppressed by these PRRs and their homolog PRR5, forming another negative feedback circuit [51,52]. EARLY-PHYTOCHROME-RESPONSIVE1 (EPR1) leads to the enhanced opening and delayed flowering of cotyledons induced by far-red light. In wild Arabidopsis plants growing under continuous light, EPR1 shows a similar circadian rhythm to CCA1 and LHY. In addition, EPR1 can adjust its expression to form a slave oscillator, which also adjusts the rhythm of Lhcb [53].

The expression of CIRCADIAN1 (CIR1/RVE2) is transiently induced by light and oscillates in a circadian rhythm controlled by the central oscillator CCA1. The constitutive expression of CIR1 (RVE2) inhibits the rhythmic expression of the endogenous CIR1 (RVE2) gene, changes the cycle length of the central oscillator, reduces the amplitude of CCA1 and LHY, and seriously affects the rhythm of the EPR1 and Lhcb genes. In addition, the overexpression of CIR1 (RVE2) delayed photoperiod flowering, decreased the expression of CON-STANS (CO) and FLOWERING LOCUS T (FT), increased hypocotyl elongation, and inhibited seed germination in the dark [54]. In addition, plants overexpressing CIR1 (RVE2) showed an increased expression of the CBF gene and enhanced tolerance to freezing stress before and after cold acclimation. This indicates that CIR1 (RVE2) positively regulates cold response genes and subsequent cold tolerance [55]. RVE2 and REVEILLE1 (RVE1) can promote the primary dormancy of Arabidopsis seeds and inhibit the red/far-red light-mediated germination downstream of phytochrome B (phyB) [56]. RVE1 was first proved to be a clock-regulated transcription factor that promotes the expression of the auxin biosynthesis gene YUCCA8 (YUC8) and is essential for the circadian rhythm of auxin. Therefore, RVE1 can promote the accumulation of free auxin and the elongation of hypocotyl of seedlings in the daytime [57]. In another study, two independent knockout mutants of RVE1 against the background of Col showed enhanced freezing resistance under domestication, which proved that RVE1 was a negative regulator of freezing resistance in Arabidopsis [58]. Recently, it has been found that RVE1 positively regulates the transcription of PORA (protochlorophyllide oxidoreductase A) by directly binding to the EE-box cis-regulatory element of the PORA promoter. PORA can catalyze the reduction of protochlorophyllide to chlorophyll. The analysis of PORA expression in loss-of-function and overexpressing RVE1 Arabidopsis plants showed that RVE1 regulated the transcription of PORA and promoted seedling greening [59].

Many metabolic, physiological, and behavioral processes in plants are controlled by the biological clock [60,61], for instance, the response of plants to auxin. Recently, it has been reported that CCA1 and LHY are necessary for the control of the auxin response [62]. LHY has also been found to be involved in the regulation of the ABA pathway. The genome-wide analysis of its binding targets has shown that LHY can bind to the promoters of many ABA biosynthesis and signal transduction genes. Under drought stress, plants with LHY overexpression accumulated lower levels of ABA, and LHY mutants were more sensitive to ABA treatment during seed germination [63]. Similarly, the biosynthesis and signal genes of most brassinosteroids (BRs) are also controlled by clocks [64]. The BR-activated transcription factor bri1-EMS-suppressor 1 (BES1) regulates the expression of CCA1 and LHY and then transmits the BR signal to the clock oscillator. In the presence of BRs, BES1 binds the CCA1 and LHY promoters and inhibits their expression, especially at night. This BES1-CCA1/LHY module subtly adjusts the circadian rhythm oscillation to ensure that the BR signal is activated acutely at a specific time of the day [65]. Lei and Zhu-Salzman reported that both CCA1 and LHY are necessary for the circadian regulation of indole glucosinolate biosynthesis, and contribute to plant defense against aphids [66]. Furthermore, CCA1 and LHY inhibited the expression of the dehydration-responsive element (DRE) binding protein 1 (DREB1) under non-stress and were degraded rapidly and specifically under cold stress. Therefore, as transcriptional suppressors, CCA1 and LHY can indirectly regulate the cold-induced expression of DREB1 [67].

The functional deletion mutation of REVEILLE5 (RVE5) decreased the expression of the circadian gene EARLY FLOWERING 4 (ELF4) in Arabidopsis, and promoted the growth of hypocotyls under warm conditions [68]. REVEILLE8 (RVE8) can bind directly to the promoter of TIMING OF CAB EXPRESSION1 (TOC1) and promote the expression of histone H3 by increasing the acetylation level of histone H3, while CCA1 inhibits the expression of TOC1 by reducing the level of histone acetylation [69]. RVE8 can directly activate the clock and output genes containing EE. The loss of RVE8 and its homologs, RVE4 and RVE6, causes a delay and reduction in levels of evening-phased clock gene transcripts and a significant lengthening of the clock pace [70]. In addition, Perez-Garcia et al. showed that RVE8 could directly bind to the promoters of anthocyanin-biosynthesis-related genes and regulate the expression of these genes in response to diurnal fluctuations. NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) and RVE8 can collaboratively control the anthocyanin metabolic pathway [71]. Based on these findings, RVE8 can be considered a regulator of anthocyanin biosynthesis in plants [72]. In another study [73], RVE3 and RVE5 promoted only the clock speed together with RVE4, RVE6, and RVE8, and their roles in the clock function were subtle. However, RVE3 and RVE5 only played a secondary role in the adjustment of the clock function [73].

Light morphogenesis in plants is often regulated by a variety of hormone signal pathways. The MYBS3 homologous gene in Arabidopsis, MYBH, was induced in the dark, which enhanced the expression of auxin-related genes, such as PIF4 and PIF5, and then induced the accumulation of auxin, thus increasing the elongation of the hypocotyl. At the same time, it was found that the transcription level of the auxin biosynthesis gene YUCCA8 (YUC8) was also increased in MYBH-overexpressing seedlings [74]. Additionally, researchers [75] have observed that, in transgenic Arabidopsis, the overexpression of MYBH enhanced the gene expression of SAUR36, a key regulator of auxin-induced leaf senescence, and accelerated leaf senescence induced by ABA and ethylene. In this study, it was also found that darkness and aging could activate the activity of the MYBH promoter [75]. The biosynthesis of anthocyanins is often regulated by many factors, such as light, auxin, and cytokinin. Light and cytokinin can stimulate the expression of MYBD, a homologous gene of MYBH. MYBD inhibits the expression of MYBL2 and promotes the biosynthesis of anthocyanin by directly binding to the MYBL2 promoter. It was further observed that ELONGATED HYPOCOTYL 5 (HY5) directly binds to the MYBD promoter, especially in the G-box-containing region, resulting in anthocyanin accumulation. It can be seen that MYBD and MYBH have opposing roles in the process of plant photomorphogenesis [76]. The expression of the MYBL2 gene is not only inhibited by MYBD but also by HY5. Furthermore, the translation of MYBL2 is suppressed by microRNA MIR858a. MIR858a is the direct target of HY5 and displays light-responsive expression in an HY5-dependent manner [77]. Thus, AtHY5-AtMYBD-AtMYBL2 and AtHY5-miR858a-AtMYBL2 work together to form a control system for anthocyanin regulation [36]. Recently, AtGLK1 (GOLDEN2-LIKE 1) was reported to regulate sucrose-induced anthocyanin synthesis upstream of MYBL2 [78].

7. The Development of Trichomes and Root Hairs

CAPRICE (CPC) was first reported to be involved in the development of trichomes and root hairs [79]. Studies have shown that CPC reduces the formation of the TRANSPARENT TESTA GLABRA1 (TTG1)-(E) GL3-GL1 complex by competing with R2R3-MYB GL1 for bHLH binding at the initiation of trichomes, thus inhibiting trichome formation [80]. Then, it was found that CPC is a positive regulator of stomatal formation [81]. In addition, in the overexpression of CPC in plants, the accumulation of anthocyanin is negatively correlated with the level of CPC. It was also found that the regulation of anthocyanin biosynthesis in Arabidopsis is inhibited by competition with R2R3-MYB for the binding site of the bHLH protein, which prevents R2R3-MYB and bHLHs from forming an active complex of anthocyanin biosynthesis, thus negatively controlling anthocyanin biosynthesis [82]. According to previous studies, CPC and its six homologs—TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3 (ETC3)/CAPRICELIKE MYB3 (CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2 (TCL2)—can induce root hair differentiation and inhibit trichome formation [83,84,85,86,87,88,89,90,91]. These CPC family genes lead to root hair formation mainly by inhibiting the expression of GLABRA2 (GL2) [92]. The overexpression of the MYBL2 gene inhibits root hair development in transgenic Arabidopsis. The synergistic effect of GL3 gene function and the overexpression of MYBL2 inhibit hair formation by negatively regulating the expression of GLABRA2 (GL2) [93]. Among the homologs, only CPL3 has multiple effects on flower development and epidermal cell size by regulating internal replication [86]. It was reported that, under the condition of phosphate (Pi) deficiency, the expression of the ETC1 and ETC3 genes is enhanced, which can promote root hair formation by inhibiting the expression of GL2 [94]. Another study found that the rice gene OsTCL1 could inhibit the formation of trichomes and promote the formation of root hairs when expressed in Arabidopsis. However, the expression of OsTCL1 in rice had no effect on the formation of root hairs and trichomes, and the expression of the OsGL2 gene was increased rather than decreased. This shows that rice regulates the formation of trichomes and root hairs differently from Arabidopsis [95]. Recently, it has been reported that different climates and genomic structures lead to trichome diversity [96].

8. Telomere Metabolism

In Arabidopsis, AtTRB1-5 is a plant-DNA-binding protein [97,98]. The C-terminus of AtTBP1 can bind sequence-specific DNA with plant double-stranded telomere DNA, which may play an important role in plant telomere function in vivo [99]. AtTBP2 (TRB3) and AtTBP3 (AtTRB2) have MYB-like domains at the N-terminus. They do not affect telomerase activity in vitro, but are similar to other MYB-like telomere-binding proteins and can indirectly participate in the regulation of telomere metabolism [100]. The TRFL family 1 proteins have a highly conserved region in the C-terminus of the MYB domain, called MYB expansion (MYB-ext), which is necessary for binding to plant telomere DNA and does not exist in the TRFL family 2. The TRFL family 1 includes TBP1, TRP1, TRFL1, TRFL2, TRFL4, and TRFL9, while the TRFL family 2 contains TRFL3, TRFL5-8, and TRFL10. In vitro, the C-terminal fragment of the TRFL family 1 protein binds specifically to the double-stranded plant telomere DNA. TRFL family 2 proteins cannot bind to plant telomere DNA in vitro. TRFL family 2 proteins may bind to telomeres in vivo through protein interactions, similar to human Rap1. The TRFL family 1 protein is not the only factor that can specifically bind to double-stranded plant telomere DNA in vitro [101]. Telomere binding proteins are not only considered to be essential components of the telomere structure but are also important components of the telomere metabolism involved in telomere length regulation and telomere protection [102]. For instance, a double-stranded telomeric repeat binding factor in Nicotiana tabacum, NgTRF1, is involved in the maintenance of telomere length and stability [103]. In another study, RICE TELOMERE BINDING PROTEIN1 (RTBP1) was also involved in controlling telomere length and telomere stability in rice [104]. We found that previous studies of these telomere-binding proteins in plants focused mainly on their interactions in vitro, and the physiological role of these proteins in plants is still unclear.

9. Plant Hormone Response

The RAD-like family in Arabidopsis consists of at least four members: RADILAS-LIKE SANT/MYB1-4 (RSM1, RSM2, RSM3, and RSM4). RSM1 (RL2) is closely related to the HLS1 gene in early morphogenesis [105]. HLS1 was originally identified as an important regulator implicated in the formation and maintenance of the apical hook of dark-grown etiolated seedlings in response to ethylene [106,107]. In addition, RSM1 and HY5/HYH may converge on the ABI5 promoter and independently (or possibly, dependently) regulate ABI5 expression and ABI5-targeted ABA-responsive genes, thereby modulating ABA and abiotic stress responses [108]. At present, it is known that these four genes in the RAD-like family are highly homologous to the Antirrhinum RAD genes, although the functions of RSM2, RSM3, and RSM4 in Arabidopsis have not been studied [105]. DRMY1 controls cell expansion in vegetative and reproductive organs and is strongly expressed in developing organs. Its expression is inhibited by ethylene and induced by ABA. DRMY1 plays an important role in organ development by directly affecting the cell wall structure and cytoplasmic growth, or by indirectly regulating cell expansion through ethylene or ABA signaling pathways [109]. DRMY1 focuses the spatiotemporal signaling patterns of the plant hormones auxin and cytokinin, which jointly control the timing of sepal initiation. DRMY1 ensures sepal size uniformity by coordinating the timing of sepal initiation [110]. As a transcription factor, NID1 binds directly to the CHL1 promoter under a low-nitrate condition, activates an unknown pathway through the CHL1 receptor, and promotes ABA accumulation, thereby inhibiting root growth [111].

10. Promoter Analysis of AtMYB-Related Genes

In order to gain a comprehensive and in-depth understanding of the functions of MYB-related genes during plant development, we divided these genes into seven subgroups according to the phylogenetic tree (Supplementary Figure S2) and predicted the promoter sequences of AtMYB-related genes (Figure 2, Supplementary Table S3). Except for the core promoter element and common cis-acting element in the promoter (CAAT-box and TATA box), the 26 cis-elements detected could be divided into three types: growth and development response, hormone response, and stress response. Among the response elements related to plant growth and development, the number of light-responsive elements was the largest, including TCT-motif, G-box, GT1-motif, and AE-box. The distribution of light-responsive elements was also the widest. All AtMYB-related genes contained light-responsive elements, indicating that AtMYB-related genes played a role in light-response-mediated regulation. In addition, cis-acting elements involved in circadian rhythm control were detected in nine genes. Of course, this response element was not detected in many genes, but this does not mean that they do not play a role in the regulation of circadian rhythm because they may play a role through indirect effects with other related genes, such as CCA1. CAT-box and HD-Zip1 are related to gene expression in the meristem and the differentiation of palisade mesophyll cells, respectively, and may be related to specific expression. Ten cis-acting elements are involved in the reactions of abscisic acid (ABA), auxin, gibberellin (GA), salicylic acid (SA), and jasmonic acid (JA), including ABRE, TGA-element, GARE-motif, TCA-element, and the TGACG-motif. Stress response elements include MBS and DRE, which are related to drought and low temperature. There were 30 drought-responsive elements (MBS) in 23 AtMYB-related genes, while DRE was predicted only in TRFL7. The genes containing these cis-elements in the promoter region may be involved in the adaptation of Arabidopsis to different environmental conditions.

11. Discussion

The analysis of cis-acting elements of AtMYB-related gene promoters and the review of previous functional studies can not only help us better understand the functions these genes already possess, but can also provide a direction for future research on the functions of these genes. We found that genes belonging to the same subgroup have similar structures and functions. For example, genes with known functions in the S1 subgroup are involved in the regulation of light response or circadian rhythm. Therefore, it is speculated that those genes with unknown functions in the S1 subgroup may also be involved in the regulation of related functions. In addition, the activity of the RVE1 promoter is regulated by the clock [57]; the expression of RVE2 can be rapidly induced by light [54]; and the activity of the MYBH promoter is regulated by light [74]. All of these findings are consistent with the fact that their promoter regions contain light-responsive elements. It is not difficult to see that CCA1 and LHY are involved in many biological processes, complex pathways, and interactions with many other transcription factors. For example, ATAF2 (ANAC081) is an NAC (NAM, ATAF, and CUC) transcription factor (TF). CCA1 physically interacts with ATAF2 and inhibits ATAF2 expression through promoters that bind to CBS motifs. CCA1 and ATAF2 were reported to synergistically inhibit the light morphogenesis of seedlings [112]. Genes belonging to the S3 subgroup are all involved in the regulation of trichome or root hair development. TRB1, TRB2, TRB3, and the genes of TRFL subsets belonging to the S2 subgroup all bind to telomere DNA, but their physiological roles and developmental regulation are still unclear. The function of DRMY1 in the S6 group is to regulate cell expansion, but the function of its paralog gene, DP1, is still unknown. RAD-like genes in the S4 subgroup are related to the growth and development of Arabidopsis, but their specific function is not clear. In particular, although NID1 belongs to the S1 subgroup and its promoter contains cis-acting elements for circadian control, it has not been reported that it is involved in the regulation of circadian rhythm, so further study is needed. Therefore, the known functions of these genes partly verify the results of the promoter analysis, but the functions of these genes are often diverse. For example, CPC not only participates in the development of trichomes and root hairs but also negatively regulates anthocyanin accumulation and positively regulates stomatal formation. The same is true of MYB-related genes in other plants. For example, the overexpression of the rice MYB-R1 gene can increase tolerance to drought, salt, and chromium stresses simultaneously [113].

Of course, in recent years, there have also been reports that AtMYB-related genes are involved in both biological clock and abiotic stress responses. For example, the genome-wide analysis of LHY binding sites shows that LHY directly controls the expression of genes related to the biosynthesis of ABA and the rhythmic accumulation of this hormone. Furthermore, LHY also regulates the expression of ABA signal modules and downstream response genes to enhance some ABA responses and inhibit others. This reveals the complex coupling between the biological clock and the ABA pathway, which may make an important contribution to plant performance under drought and osmotic stress [63]. Therefore, these genes not only play a role in one pathway, but also interact with each other in multiple signaling pathways.

12. Conclusions and Outlook

This review summarized the advancement in research on the functions of MYB-related genes in A. thaliana and analyzed the promoter characteristics of AtMYB-related genes using bioinformatics. However, the functional studies of most reported AtMYB-related genes are relatively simple, and the research on their regulatory mechanisms in the interaction of different signal pathways is not sufficiently extensive. Moreover, the functions of some AtMYB-related genes, such as TKI1, ALY3, and DIV1, are still unknown. There are even some unnamed AtMYB-related genes with functions that have not yet been separately reported. Moreover, there are few studies on these gene promoters. On the one hand, it is important to identify the upstream regulators that interact with the promoters of these genes. On the other hand, as transcription factors, it is crucial to identify the downstream target genes to which they bind. Therefore, there is still a long way to go to map complete complex pathways. First, the unidentified MYB-related transcription factors’ biological roles can be anticipated by studying their protein structures and promoter cis-acting elements. Then, we can use gene cloning and protein functional analysis to study their mechanisms in growth, development, and stress responses. Second, a study of the regulatory mechanism of AtMYB-related genes in the interactions of multiple signal pathways is also very important for understanding their function. Finally, these studies can be applied to the development and utilization of related genes in other plants, especially crops, to improve their yield through crop breeding.