MYB Transcription Factors Becoming Mainstream in Plant Roots

Abstract

The function of the root system is crucial for plant survival, such as anchoring plants, absorbing nutrients and water from the soil, and adapting to stress. MYB transcription factors constitute one of the largest transcription factor families in plant genomes with structural and functional diversifications. Members of this superfamily in plant development and cell differentiation, specialized metabolism, and biotic and abiotic stress processes are widely recognized, but their roles in plant roots are still not well characterized. Recent advances in functional studies remind us that MYB genes may have potentially key roles in roots. In this review, the current knowledge about the functions of MYB genes in roots was summarized, including promoting cell differentiation, regulating cell division through cell cycle, response to biotic and abiotic stresses (e.g., drought, salt stress, nutrient stress, light, gravity, and fungi), and mediate phytohormone signals. MYB genes from the same subfamily tend to regulate similar biological processes in roots in redundant but precise ways. Given their increasing known functions and wide expression profiles in roots, MYB genes are proposed as key components of the gene regulatory networks associated with distinct biological processes in roots. Further functional studies of MYB genes will provide an important basis for root regulatory mechanisms, enabling a more inclusive green revolution and sustainable agriculture to face the constant changes in climate and environmental conditions.

1. Introduction

The plant root system provides the interface between plants and the complex soil environment, thereby being crucial for plant survival and crop productivity, such as absorbing nutrients and water from the soil, preventing lodging, and responding to biotic and abiotic stresses. In the past decades, plant root biology has attracted increasing attention as it has important implications for global food security under changing climate and environmental conditions [1,2,3,4,5]. The molecular basis of how the plant root system contributes to plant traits, such as lodging resistance, nutrient stress resistance, and biotic and abiotic stresses resistance are gradually clarified. It is commonly accepted that the root growth and development processes were regulated by complex mechanisms which include a series of transcription factors [6,7], such as AtWOX5 controlling root apical meristem (RAM) division, AtARF7/19 regulating the lateral roots (LRs) initiation, and WEREWOLF (WER/AtMYB66) determining the root hair (RH) cell formation [8,9,10].

The V-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors are commonly characterized by a highly conserved DNA-binding domain repeat (MYB domain) at the N-terminal, accompanied by a variable activation domain at the C-terminal [11,12]. MYB transcription factors (MYB TFs) are widely found throughout eukaryotic organisms and comprise a superfamily in land plant genomes, e.g., they account for ~13% of the 1500 transcription factors in the model plant Arabidopsis thaliana [13]. As compared with the large number in plant genomes, a few MYB TFs are generally present in unikonts, e.g., only three MYB TFs that are associated with cell cycle progression were reported in vertebrate animals including Homo sapiens and Mus musculus [14]; a single MYB TF that functions in cell cycle progression as well was present in invertebrates (such as Drosophila melanogaster and Strongylocentrotus purpuratus) [15]; similarly, only one MYB TF had been identified in cellular slime mold Dictyostelium discoideum [16]. In plants, based on the number of the highly conserved repeats (R) in the MYB domain, MYB proteins fall into four major families, namely MYB-related (R3/R1-MYB), 2R-MYB (R2R3-MYB), 3R-MYB (R1R2R3-MYB), and 4R-MYB (R1R2R2R1/R2-MYB) [17]. In general, the number of 3R-MYB and 4R-MYB families is very limited and conserved across plants, with most species possessing 3 and 2 members, respectively [17]. In contrast, numerous members are observed in MYB-related and 2R-MYB families in plants, especially in angiosperms [14,18]. Consistent with the small number, the functions of 3R-MYB and 4R-MYB families are conserved across plants and even eukaryotes. For example, the members of the 3R-MYB family in Arabidopsis play a similar role with their homologs in animals in cell cycle regulation [19,20,21]. On the contrary, the members of MYB-related and 2R-MYB families have diverse functions in many plant-specific processes [18,22]. To date, the major roles of MYB-related genes characterized are related to organ development, stress response, and circadian clock [18]. For example, CPC-like subfamily homologs are involved in cell fate determination [23], AtMYBL response to ABA and salt stresses [24], and AtCCA1 with its homologs are involved in circadian rhythm [18]. Consistent with its vast number and phylogenetic classification, the 2R-MYB family has diverse functions in plants, which can be briefly summarized into three major biological processes: development and cell differentiation, biotic and abiotic stresses, and specialized metabolism [22]. For instance, the genes in the S15 subfamily are associated with cell fate determination [10], the genes in the S22 subfamily respond to drought and pathogen invasion [25], and those in the S6 subfamily participate in anthocyanin biosynthesis [26], etc. Due to inconvenient visualization, the root traits were generally neglected in plant genetics, biology, etc. Accordingly, to date, the functions of MYB proteins related to shoot attracted much more attention. However, an increasing number of MYB genes (MYBs) have been functionally characterized in roots, such as AtMYB36 controlled LR primordium (LRP) development [27]; AtMYB70 regulated root system development [28]; AtMYB73/77 involved in LRs growth [29], etc. These results raised concerns about the roles of MYB TFs in roots.

In this review, the functions of MYBs in plant roots are described in detail, with a focus on their roles in regulating cell differentiation, coordinating cell cycle, responding to biotic and abiotic stresses, and transducing phytohormone signals. The function characteristics of MYB homologs in roots have been discussed from a subfamily perspective. Finally, the potential research directions of MYB TFs in roots for future work were prospected. To our knowledge, this is the first review of the functions of MYB TFs in roots.

2. MYB TFs Are Regulators of Root Cell Differentiation

The development of plant roots relies on highly precise regulation of cellular differentiation [7]. MYB TFs were widely recognized to regulate the differentiation of root cells, participating in the development processes of primary root (PR), LRs, and RHs.

The roles of MYB TFs in root cell differentiation were represented by WER (2R-MYB) and CAPRICE (CPC, R3-MYB) which act as the core factors in determining root epidermal cell fate. In Arabidopsis, the specification of hair and non-hair epidermal cells are position-dependent, with hair cells arising over clefts between the overlying cortical cells (Figure 1A). In cortical cells, JACKDAW (JKD), a plant-specific zinc finger protein, produces a signal that may bind to the leucine-rich repeat receptor-like kinase (LRR-RLK) SCRAMBLED (SCM) in epidermal cells [30]. Due to the larger contact surface, the epidermal cell located at the cleft between two overlying cortical cells received more signals, resulting in more SCM activation [30]. Subsequently, the activated SCM represses the expression of the WER gene, contributing to more CPC than WER, leading to hair cell (HC) formation (Figure 1A) [31]. On the contrary, the epidermal cell on a single cortical cell has a little activated SCM because of the smaller contact surface, leading to the dominance of WER and thus developed as a non-hair cell (NHC) (Figure 1A) [31]. In NHC, a MYB protein WER, a bHLH protein GLABRA3 (GL3) or its homolog ENHANCER OF GLABRA3 (EGL3), and a WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) form an activator protein complex MYB-bHLH-WD40 (MBW) to activate the expression of the downstream GLABRA2 (GL2) gene that encodes a homeodomain protein [10,32,33,34]; subsequently, the GL2 protein represses a set of downstream genes such as ROOT HAIR DEFECTIVE 6 (RHD6) and RHD6-LIKE1 (RSL1) which are essential to the determination of RHs, leading to an NHC fate (Figure 1A) [35]. In HC, CPC protein competes with WER protein to form a repressor protein complex CPC-GL3/EGL3-TTG1 (MBW), which then inhibits the expression of the GL2 gene [36,37]; thereby, the downstream genes of GL2 (e.g., RHD6 and RSL1) were normally expressed which then promote RH formation (Figure 1A).

The competition between the two types of MYB proteins, WER and CPC, to form the MBW ternary protein complex is attributed to their close evolutionary relationship. The MYB domain of CPC protein and the R3 repeat in the MYB domain of WER protein shared a high degree of sequence similarity. Both of them contain the conserved motif DLx2Rx3Lx6Lx3R in the R3 repeat of the MYB domains that are involved in the interactions between MYB and bHLH proteins [18]. However, as compared to WER, the CPC protein lacks the transcription activation domain causing its opposite function in the RH formation process [38]. Accordingly, the R3 domain of the WER can functionally replace CPC, but the R3 domain of CPC cannot functionally replace that of WER [39]. In addition, the homologs of WER and CPC genes were widely demonstrated to act as similar roles in RH formation process. For example, the AtMYB23 gene has a similar function to the WER gene to reinforce the NHC fate [40]; in Arabidopsis, there are six homologs of the CPC gene, including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ETC2, ETC3, TRICHOMELESS1 (TCL1), and TCL2, which act as positive regulators for RH formation [23,41,42]. In other plant species, the homologs of WER/CPC genes were reported to perform similar functions in RH cell differentiation. For example, overexpressing four homologs of the WER gene in Brassica napus (BnMYB019, BnMYB189, BnMYB231, and BnMYB388) in Arabidopsis could rescue the RH phenotype of the wer mutant to that of WT lines [43]; expressing the Solanum lycopersicum SlTRY gene and Oryza sativa (rice) OsTCL1 gene in Arabidopsis, respectively, both enhanced RH differentiation and thus promoted RH formation [44,45]. These results show a new perspective on subfamily to understand the key roles of WER and CPC homologs in root epidermal cell differentiation.

Notably, a series of feedback exists in this process to enhance root epidermal cell fate specification. In NHC, in addition to the downstream GL2 gene, the WER-GL3/EGL3-TTG1 complex also positively regulates the expression of the competitors of the WER protein, CPC and TRY, and even the homologs of WER, AtMYB23 (Figure 1A) [40,46]. However, the WER-GL3/EGL3-TTG1 complex negatively regulates the expression of SCM, GL3, and EGL3 genes in NHCs as well [47]. At the same time, the GL3 and EGL3 proteins can move from HC to NHC through plasmodesmata (PD) [47], resulting in more WER-GL3/EGL3-TTG1 complex in NHC that can upregulate more GL2 and CPC/TRY protein formation (Figure 1A). Subsequently, the GL2 protein controls the NHC formation, whereas the CPC and TRY proteins can move from NHC to HC through PD to promote HC formation [37,48]. Moreover, TRY in HC can upregulate the expression of the SCM gene, contributing to the HC fate [49]. Recently, phytosulfokine receptors (PSKRs) which belong to the LRR-RLK family and an O-fucosyltransferase protein, SPINDLY (SPY), were reported to participate in position-dependent root epidermal cell fate determination as well [50,51]. Together, both the position signals and various feedback pathways determine the dominance of different MBW complexes in HCs and NHCs which consequently specify the root epidermal cell fate.

Another 2R-MYB gene, AtMYB36, is a key regulator of cell differentiation in Arabidopsis roots as well [27,52]. In endodermal cells, the AtMYB36 gene is activated by SCARECROW (SCR) and then activates a series of genes such as Casparian strip proteins (CASPs), Peroxidase 64 (PER64) and Enhanced Suberin 1 (ESB1), as well as represses JKD and MAGPIE (MGP) involved in proliferation, resulting in Casparian strip formation that represents a successful transition from proliferation to differentiation in endodermal cells (Figure 1C) [53]. Notably, the JKD-involved formation of the Casparian strip in the endodermal cells is independent of its function in epidermal patterning [47]. Moreover, AtMYB36 plays a similar role in LRP, as the size and shape of LRP are determined by the balance between cell proliferation and differentiation as well (Figure 1B). In this biological process, AtMYB36 is expressed in the cells surrounding LRP where it activates a subset of peroxidase genes such as PER9 and PER64 to regulate the ROS balance, consequently promoting the transition from proliferation to differentiation at later stages of LRP development [27]. Together, AtMYB36 regulates the formation of the Casparian strip in endodermal cells and LRP development by promoting the transition from proliferation to differentiation in a similar manner.

Taken together, the roles of MYBs in cell differentiation in RH development were well-known in many plant species, whereas their roles in other root cell differentiation processes remain to be explored in the future.

3. MYB TFs Regulate Root Growth and Development through Cell Cycle

Root growth and development is a dynamic balance between cell proliferation and cellular differentiation, which rely on the positive and/or negative regulation of cell cycle progression. Another well-known role of plant MYB TFs in roots is controlling the cell cycle.

Among the MYB superfamily, the 3R-MYBs were well-known to regulate the cell cycle in both animals and plants. In animals, the MYB family is composed of three members, MYB (C-MYB), MYBL1 (A-MYB), and MYBL2 (B-MYB), which play important roles in controlling the cell cycle. The B-MYB gene is ubiquitously expressed in all cell types and activates the transcription of CDC2 and cyclin B1 in the G2/M phase; the A-MYB and C-MYB exhibit a tissue-specific expression profile and are also involved in the regulation of cell cycle [54]. In Arabidopsis, four of the five 3R-MYBs are demonstrated to regulate cell cycle progression, except AtMYB3R2 which is associated with circadian rhythms rather than cell cycle [19,20,55]. Among them, AtMYB3R1 and AtMYB3R4 (AtMYB3R1/4) act as transcriptional activators which are expressed in proliferating tissues, such as root tips and LRPs (Figure 2A). In atmyb3r1/4 double mutants, the expression of G2/M-specific genes was significantly reduced, leading to short roots and defective cytokinesis in root epidermal cells [20,56]. In contrast, AtMYB3R1, AtMYB3R3, and AtMYB3R5 (AtMYB3R1/3/5) act redundantly as transcriptional repressors in both proliferating and differentiated cells by inhibiting the expression of G2/M-specific genes directly (Figure 2A). In atmyb3r1/3/5 triple mutants, the expression of G2/M-specific genes was significantly upregulated, leading to an increased size of root meristems and a longer length of PR [19]. Commonly, the above AtMYB3Rs all bind to the cis-acting mitosis-specific activator (MSA) element (“AACGG”) in the promoter regions of several G2/M-specific genes (e.g., AtKNOLLE, AtCYCB1, and AtCDC20) to regulate the cell cycle (Figure 2A) [19,20]. However, the precise mechanism of how the AtMYB3Rs activate or repress the expression of downstream genes is still unclear. An explanation was that the AtMYB3Rs proteins can recruit different types of the DREAM (DP, RBR, E2F, and MuvB) complex which is well-known in the regulation of cell cycle [19]. In this process, the AtMYB3Rs indirectly recruit different E2F isoforms (E2FB or E2FC) through MuvB to form distinct DREAM complexes, such as AtMYB3R4-MuvB-E2FB and AtMYB3R3-MuvB-E2FC (Figure 2A,B) [57]. Consistently, E2FB functions as an activator while E2FC acts as a repressor in the regulation of cell cycle genes [58,59]. Notably, AtMYB3R1 has dual roles in controlling the cell cycle, acting as an activator or a repressor in this process. In the atmyb3r1 mutant, the expression of cell cycle genes underwent little change. However, enhanced downregulation and upregulation of G2/M-specific genes were observed when introducing atmyb3r1 into atmyb3r4 single mutant or atmyb3r3/5 double mutants, respectively [19], demonstrating the dual roles of AtMYB3R1 in the cell cycle. In RAM, the transcription of AtMYB3R1 is downregulated by TSO1 (a core subunit of MuvB) rendering the cells unable to enter the differentiation process [60]. In other plants, 3R-MYB homologs are demonstrated to perform a similar role in cell cycle progression. For instance, three Nicotiana tabacum 3R-MYBs (NtmybA1, NtmybA2, and NtmybB) and one rice OsMYB3R-2 gene were reported to regulate the expression of cell cycle genes through binding to the MSA cis-element as well [61,62]. Recently, 225 3R-MYBs identified from 65 plant species appeared to be enriched for the MSA cis-element within their upstream promoter region, indicating a conserved functional involvement in cell cycle regulation [63]. Similarly, in animals, B-MYB is repressed by the DREAM complex in the G1/S phase while interacting with MuvB to activate the G2/M cell cycle genes [64,65]. These results suggest that 3R-MYBs have undergone functional specialization during the evolution of plants.

The 2R-MYBs also regulate the cell cycle in roots, and their regulatory mechanisms appear to be spatiotemporally specific that is distinct from the broad regulation manner of 3R-MYBs. For example, the AtMYB59 gene is specifically expressed in roots and functions in the S phase of the cell cycle progression (Figure 2A) [66]. In AtMYB59-overexpressing lines, about half of the mitotic cells in root tips are at metaphase, leading to shorter PR than wild-type (WT); in contrast, the atmyb59 mutants show the opposite phenotype, indicating that AtMYB59 inhibits root growth by disturbing the cell cycle [66]. Different from AtMYB3Rs, AtMYB59 upregulates the expression of cell cycle genes (such as AtCYCB1;1) by binding to the MRE (“AACC”), MRE2 (“TATAACGGTTTTTT”), and ERE (“ATTTCAAA”) cis-elements in the promoters instead of the MSA cis-element [66]. On the contrary, the AtMYB56 (BRAVO) gene negatively regulates the expression of cell cycle genes (e.g., AtCYCD2;2 and AtCYCD3;3) in quiescent center (QC) cells to ensure a low dividing activity (Figure 2B) [67]. Ectopic expression of AtMYB56 resulted in the inhibition of root growth and the failure of root regeneration upon damage of stem cells [67]. Recently, the AtMYB56 protein has been reported to interact with the WOX5 protein to form a complex in QC cells (Figure 2B) [68]. In addition, an atypical 2R-MYB gene, AtCDC5 (cell division cycle 5), is well-known for its role in regulating the cell cycle. This gene is predominantly expressed in proliferating cells and participates in the G2/M transition by upregulating the expression of the AtCDKB1 gene (Figure 2A) [69]. Its binding site is the “CTCAGCG” motif in the promoters of target genes [69]. The root growth of the AtCDC5-RNAi plants was severely inhibited, and the atcdc5 mutant was embryonic lethal, suggesting that AtCDC5 is essential for cell cycle progression [69]. Moreover, these 2R-MYBs commonly belong to the early derived subfamilies of the 2R-MYB family in plants, indicating that the ancient and important role of 2R-MYBs in the cell cycle may be derived from 3R-MYBs. To date, the knowledge regarding the roles of 2R-MYBs in cell cycle progression is focused on the studies in Arabidopsis. However, as mentioned above, given the functional conservation characteristic of MYB homologs in diverse plant biological processes, it is foreseeable that 2R-MYBs have an important role in root cell cycle progression in other plant species as well.

Overall, the regulatory roles of 3R-MYBs in root cell cycle progression are widely recognized in many plants showing a conserved mechanism across different plant species, while the roles of 2R-MYBs in root cell cycle are still poorly understood, especially in non-model plants.

4. MYB TFs Function in Root System Architecture in Response to Biotic and Abiotic Stresses

Due to the immobility, plants face diverse volatile environments during the life cycle, whereas the root system plays a key role and shows strong plasticity in response to abiotic stress. To date, a mass of studies has demonstrated that plant MYB TFs are involved in diverse abiotic stress responses, such as drought, salt stress, nutrient stress, gravity, and light (

Table 1. MYB gene functions in roots under biotic and abiotic stresses.

Species	Genes	Function Description	References
Arabidopsis thaliana	AtMYB2	Positively regulate LRs and RHs under P starvation	[70]
	AtMYB12	Maintain root growth under salt and drought stress	[71]
	AtMYB20	Promote PR growth under salt stress	[72]
	AtMYB30	Regulate root growth and development under salt stress	[73,74]
	AtMYB42	Protect root from ion toxicity under salt stress	[75]
	AtMYB59	Modulate RSA under nutrient stress and participate in the response to nematode infestation	[76,77,78]
	AtMYB60	Promote root growth under mild drought	[79]
	AtMYB62	Negative regulators of LRs growth under P starvation	[80]
	AtMYB73/77	Mediate the inhibition of LRs under UV-B light	[29]
	AtMYB96	Negatively regulate LRs under drought	[81]
	AtMYB88/124	Participate in PR and LRs gravitropism	[82]
	AtPHR1	A central regulator of P starvation	[83]
	AtPHL1/2	Dimerize with AtPHR1 to regulate P starvation responses	[84,85]
	AtHRS1	Regulate RHs and PR under P starvation	[86]
	AtHHO2	Promote LRs under P starvation	[87]
	CPC/TRY/ETC1	Positively regulate RHs development under P starvation	[88]
Glycine max	GmMYB84	Promote PR elongation under drought	[89]
Leymus chinensis	LcMYB2	Positively regulate root growth under drought	[90]
Malus×domestica	MdFLP	Promotes ARs in response to gravity	[91]
Oryza sativa	OsMYB1	Regulate LRs elongation under P starvation	[92]
	OsMYB2P-1	Positively regulate root growth under P starvation	[93]
	OsMYB4P	Promote PR growth under P starvation	[94]
	OsPHR1/2/3/4	A central regulator of P starvation	[95,96]
Panax notoginseng	PnMYB2	Regulate the resistance against the root rot	[97]
Populus ussuriensis	PuMYB40	Promote ARs formation under P starvation	[98]
Thinopyrum intermedium	TiMYB2R-1	Enhance the resistance to take-all disease	[99]
Triticum aestivum	TaMpc1-D4	Negative regulators with reduced root growth under drought	[100]
	TaPIMP2	Contribute to wheat resistance to root rot	[101]

PR, primary root; LRs, lateral roots; ARs, adventitious roots; RHs, root hairs; RSA, root system architecture; UV-B, ultraviolet B; P, phosphorus.

). Most of these processes were achieved by regulating root growth and/or development, root morphology, and root system architecture (RSA).

4.1. Drought

Drought is one of the most serious abiotic stresses affecting root growth. Under drought stress, the elongation of PR and the formation of LRs are adjusted to sustain the viability of plants. For example, as a typical adaptive response to environmental stress, PR growth can be promoted to absorb water deeper underground by sacrificing LRs development under drought conditions. MYB TFs have been reported frequently in drought resistance through modulating the RSA, including the length of PR and LRs as well as the number of LRs [25]. For example, in Arabidopsis, the AtMYB96 gene was reported to negatively regulate LRs development and enhance drought resistance [81]; its overexpression lines showed significantly reduced LRs and enhanced drought resistance [81]. AtMYB60, a paralog of AtMYB96, promotes both PR and LRs growth to increase water uptake under mild drought stress [79]. In other plants, MYB TFs also regulate the RSA in response to drought. For instance, in Glycine max, GmMYB84-overexpressing lines exhibited enhanced drought resistance with a longer PR by controlling reactive oxygen species (ROS) balance [89]; in Leymus chinensis, LcMYB2 improved plant drought resistance by promoting root growth as well [90]. However, MYB TFs play a negative role in drought response as well, e.g., overexpression of a wheat MYB gene TaMpc1-D4 reduced the root length and repressed the expression of stress-related genes under drought stress [100].

4.2. Salt Stress

Salt stress is a major abiotic stress that adversely affects plant growth and development, significantly reducing crop productivity. Elevated soil salinity mainly causes ion toxicity and oxidative stress to roots, whereas MYB TFs are involved in these processes to respond to salt stress. AtMYB30 enhances salt tolerance by improving alternative respiration which can maintain the root cellular redox homeostasis [73]. Moreover, AtMYB30 links the reactive oxygen species (ROS) signaling and root cell elongation, and its mutant shows an increased cell length in root under H₂O₂ treatment [74]. Similarly, AtMYB12 can upregulate the expression of ROS scavenging genes to maintain the root cellular ROS balance under both drought and salt stress conditions [71]. AtMYB42 has been proven to participate in the regulation of ion toxicity. In this process, AtMYB42 directly activates the expression of salt overly sensitive 2 (SOS2), which plays a crucial role in regulating Na⁺:K⁺ homeostasis, resulting in the root system avoiding ion toxicity [75]. AtMYB20 enhances salt tolerance by downregulating the expression of type 2C serine/threonine protein phosphatases (PP2Cs) that plays a negative role in ABA signaling [72]. The AtMYB20-overexpressing lines exhibit enhanced salt tolerance with a longer PR [72]. These results suggest that MYB TFs are involved in the regulation of diverse physiological processes to alleviate and even avoid the effects of salt stress, thereby ensuring root growth and development.

4.3. Nutrient Stress

Soil nutrient limitations, such as phosphorus (P), nitrogen (N), and potassium (K) starvation, are major abiotic stresses that affect plant growth and development and crop production. Consequently, roots have evolved a set of mechanisms to enhance nutrient acquisition, including changing RSA to enlarge root surface area to uptake nutrients from soil. Recently, an increasing number of MYBs has been reported to be involved in nutrient stress response processes by regulating the RSA, especially in response to P starvation. In Arabidopsis, a MYB-like gene P Starvation Response 1 (PHR1), and its homologs AtPHR1-like 1 (AtPHL1) and AtPHL2 are recognized as central regulators of P starvation response (PSR) by directly regulating various P starvation-induced (PSI) genes, consequently affecting the P uptake and transport as well as modulating the RSA [83,84,85,102]. In Oryza sativa, four orthologs of AtPHR1 (OsPHR1/2/3/4) function redundantly in a highly conserved manner to that of AtPHR1 [95,96]. Two target genes of AtPHR1, AtHRS1, and AtHHO2 (HRS1 Homolog 2) are MYB-like genes that participate in the regulation of RSA to enhance the adaptability to P starvation as well [103]. Under P starvation, AtHRS1-overexpression lines exhibited enhanced RHC development and shortened PR length, while AtHHO2-overexpression lines showed augmented LRs development [86,87]. Moreover, several R3-MYBs, CPC, TRY, and ETC1 were well-known to play key roles in P starvation response by positively regulating RH development [88].

2R-MYBs participate in PSR as well. Under P starvation, AtMYB2-overexpression lines exhibited more and longer LRs and denser RHs [70]; the overexpression of OsMYB2P-1 and OsMYB4P both enhanced the P starvation tolerance with enhanced root growth [93,94]. These three genes act as a positive regulator in PSR by activating the expression of downstream PSI genes, such as miR399 and P transporter (PHT) genes. In contrast, AtMYB62 acts as a negative regulator of the PSR by repressing the expression of PSI genes, such as AtPHT1 and AtACP5 [80]. Overexpression of the AtMYB62 gene significantly decreased LRs length [80]. Rice 2R-MYB gene, OsMYB1, coordinately regulates the maintenance of P homeostasis and root development under P starvation conditions [92]. Moreover, MYB TFs were involved in adventitious root (AR) formation under P starvation, e.g., in Populus ussuriensis, PuMYB40 controlled the P starvation resistance by promoting AR formation [98]. In addition, MYB TFs also participate in responding to other nutrient stress processes through modulating the RSA. Recently, AtMYB59 has been reported to modulate the RSA in response to low K⁺, low NO₃⁻, or low calcium (Ca) stresses [76,77], suggesting its multiple roles in nutrient stress responses.

4.4. Light and Gravity

Light is an important signal that regulates root growth and development, especially ultraviolet B (UV-B) light which is a part of sunlight that markedly affects root morphology [104]. Recently, AtMYB73 and AtMYB77 genes were reported to directly interact with the UV-B photoreceptor UVR8 (UV Resistance Locus 8) to regulate root growth under UV-B light in Arabidopsis [29]. In this process, AtMYB73 and AtMYB77 proteins interact with auxin response factors to promote LRs growth and development; however, UVR8 can interact with AtMYB73/77 proteins in a UV-B-dependent manner to inhibit their DNA-binding activities, consequently inhibiting the LRs development [29]. The root system can change the growth direction in response to gravity stimulation, and MYBs are involved in this process. It was reported that AtMYB88 and AtMYB124 (AtFLP) performed a redundant role in response to gravity stimulation by regulating the temporal–spatial expression patterns of PIN-FORMED 3 (PIN3) and PIN7 genes in gravity-sensing cells of primary and lateral roots [82]. After gravity stimulation (reorientation of 90°), the curvature of PR in the atflp mutant exhibited a defective gravity response. Moreover, AtMYB88 and AtFLP functioned complementarily in establishing the gravitropic set-point angles, with the former functions in later stages while the latter in earlier stages [82]. Similarly, in Malus×domestica, MdFLP, an ortholog of AtFLP, regulated ARs gravitropism by directly binding to the promoters of MdPIN3 and MdPIN10 genes [91].

4.5. Biotic Stress

To date, many MYBs had been demonstrated to have an important role in biotic stress resistance in aerial parts, e.g., AtMYB30 and AtMYB72 participated in the response to pathogen attack in Arabidopsis leaf [105,106]; AtMYB15, AtMYB34, AtMYB51, and AtMYB75 were involved in the resistance against insect herbivores [107]. In recent years, a few studies revealed the roles of MYBs in response to biotic stress in roots as well, such as pest and disease stress responses. In wheat, the TaPIMP2 gene plays a positive role in defense responses to fungal pathogen Bipolaris sorokiniana infection by regulating the expression of defense-related genes, contributing to the host resistance to common root rot [101]. In Panax notoginseng, the PnMYB2 gene has a positive role in root rot resistance caused by Fusarium solani pathogen through regulating JA signaling, disease-resistance-related genes, and photosynthesis [97]. The Thinopyrum intermedium TiMYB2R-1 gene exhibits enhanced resistance to the take-all disease in wheat by upregulating the expression of defense-related genes [99]. In Arabidopsis, the AtMYB59 gene was found to play an important role in the response to Heterodera schachtii infestation in roots [78]. Recently, based on transcriptome and co-expression network analyses in Brassica rapa, several MYBs were speculated to be involved in clubroot resistance caused by the soil-borne protist Plasmodiophora brassicae [108]. These studies demonstrated that MYBs are also involved in biotic stress resistance in roots, which is an important direction for future gene functional research on this gene family in plants.

5. The Regulatory Roles of MYB in Roots Are Mediated by Various Phytohormones

Phytohormones are involved in many biological processes in roots, such as root growth and development, root morphogenesis, stress response, and signal transduction. In fact, it was demonstrated that the roles of MYB TFs in roots are widely mediated by various phytohormones, including indole acetic acid (IAA), abscisic acid (ABA), gibberellin (GA), cytokinin (CTK), and brassinosteroid (BR). Meanwhile, MYB TFs are also involved in phytohormone response processes to regulate root growth and development and stress response by activating downstream phytohormone-related genes and/or acting as response factors to regulate various physiological biochemical processes in roots (Figure 3).

The IAA-mediated MYB signaling transduction pathways are widely involved in the regulation of diverse developmental and stress response processes in roots. In most cases, MYBs directly regulate the expression of IAA-related genes to modulate the RSA. For instance, in Arabidopsis, AtMYB88 and AtMYB124 participate in root gravitropism by regulating the transcription of PIN3 and PIN7 genes that encode auxin transporter proteins [82]; AtMYB96 upregulates the expression of the GH3 gene which encodes the auxin-conjugating enzyme to negatively regulate LRs growth [81]. In turn, IAA could induce the expression of MYBs to perform their biological function in roots. For example, the expression of AtMYB96 could be induced by IAA leading to reduced LRs and enhanced drought resistance in Arabidopsis [81]; AtMYB93 is a novel auxin-induced negative regulator in LRs development [109].

ABA is well-known for its key role in abiotic stress. Many MYB TFs are widely involved in the ABA signaling pathway to modulate RSA. For example, AtMYB30 regulates root cell elongation depending on the ABA signaling [110]; AtMYB20 modulates the RSA by downregulating the expression of ABI1 and PP2C genes which encode the key regulators of the ABA signaling pathway [72]; AtMYB12 modulates the RSA under salt and drought stresses by upregulating the ABA biosynthesis genes (e.g., AtZEP, AtNCED, AtABA2, and AtAAO) [71]. In most cases, the roles of MYBs in roots were addressed by integrating signaling of different phytohormones, such as ABA-IAA. For instance, AtMYB70 regulates the RSA through activating the expression of the GH3 gene under ABA signaling [28]; AtMYB44/73/77 promotes LRs growth by activating the auxin-responsive genes under ABA signaling [111]; AtMYB96 is a molecular link that integrates ABA and IAA signaling in LRs growth under drought stress [81].

MYBs can regulate root development through other phytohormone signal pathways, such as GA, CTK, and BR. AtMYB62 and OsMYB1 function in the modulation of RSA under P starvation by changing the GA metabolism and signaling [80,92]. AtMYB11, AtMYB12, and AtMYB111 positively regulate root growth under the presence of GA signaling by affecting the IAA content, suggesting that AtMYB11/12/111-mediated GA signals are integrated into the IAA signaling pathway [112]. In addition, CTK promotes the nuclear localization of AtMYB3R4 protein which is essential for cell division in both SAM and RAM [113]; AtMYB56 acts as a switch to modulate the QC cells division by interacting with the BR-regulated transcription factor BRI1-EMS SUPPRESSOR 1 (BES1) [67]. Thus, the roles of MYB TFs in roots were generally mediated by phytohormone-mediated signaling pathways.

6. Conclusions and Prospects

The root system plays an essential role in plant anchorage, water, nutrient acquisition, and stress response. Therefore, genetic improvement of the root system has been recognized gradually as an effective strategy, which contributes to improving crop productivity under diverse growing conditions and thus enables a more inclusive green revolution. In the past decade, an increasing number of MYBs have been experimentally demonstrated to play critical roles in nearly all areas of root systems, such as PR, LRs, RHs, and ARs growth and development, and responding to diverse biotic and abiotic stresses. Among the MYB superfamily, 3R-MYBs maintain their cell cycle regulatory roles in roots, whereas 1R-MYBs and 2R-MYBs function in root regulation in various ways, including cell cycle, cell differentiation, biotic and abiotic stresses, and phytohormone signaling pathways. The current understanding shows the fact that MYB TFs are becoming mainstream in plant roots, showing important application potential in molecular and genetic manipulation of root traits.

The MYB gene family is one of the largest transcription factor families in plant genome, with hundreds of members in most angiosperms. However, compared with its larger number, relatively limited members have been functionally characterized to date. The current knowledge regarding the functions of plant MYBs is mainly based on the studies in model plant Arabidopsis, these in non-model plants, especially in crops, remain rather unclear. Fortunately, an increasing number of sequenced genome and transcriptome data are available, which enabled genome-wide identification, systematic analysis, and overview of this gene family in diverse plant species. Consequently, the MYB superfamily has been widely identified and functionally verified in many plants, such as maize, soybean, and rapeseed. Based on the genome-wide expression analyses, a large proportion of MYBs have been proved to be preferentially and even specifically expressed in roots. Undoubtedly, much wider roles of MYBs in plant roots will be continuously recognized in the near future based on the efforts from both of the traditional and modern disciplines, such as Molecular Biology, Genetics, Cell Biology, Genomics, multi-omics, etc.