**2. Axillary Meristem Formation**

The first step of branching is the development of axillary meristems in leaves. In recent years, a series of transcription factors that regulate the initiation of leaf axil meristems have been found in *Arabidopsis*, rice, maize and tomato. The AM is formed in the center of the frontal boundary zone of the leaf base. This region is not only a boundary but also plays an important role in the maintenance of meristem and organ development [9].

The origin of AMs is a controversial topic. There is a major view that AMs originate from meristem cell groups that become detached from the shoot apical meristem (SAM) as leaves form and never lose their meristem identity [10]. Early AM development depends on the maintenance of the specificity and meristem ability of axillary cells. In *Arabidopsis* (Figure 2), *WUSCHEL* (*WUS*) is a homologous domain transcription factor that is expressed in the center of the SAM and specifies the fate of meristem cells in this region. The *wus* mutation leads to an inability to maintain stem cell division ability [11]. ARABIDOPSIS RE-SPONSE REGULATOR 1 (ARR1) is a transcription factor downstream of CK that promotes *LAS* expression by binding to its promoter, promoting AM initiation [12,13]. Cytokinins also activate *WUS* expression through ARR1, enabling stem cell differentiation and axillary bud formation [14].

*MERISTEMLESS* (*STM*) is another important factor for maintaining branch organization. *STM*, a KNOX gene, is expressed in the whole SAM but is excluded from the organ primordium that maintains the function of undifferentiated cells in the SAM [15]. The molecular markers of the AM include concentrated and strong expression of *STM* in the center of the boundary region [16]. Once cells begin to differentiate, *STM* is downregulated by the MYB family transcription factor ASYMMETRIC LEAVES1 (AS1) and the LBD family

transcription factor AS2 [17]. This indicates that the cells in the border region maintain the ability to recover to the meristem stage within a limited period of time. During the developmental stage, AMs began to form [18].

**Figure 2.** The pattern of transcription factors involved in regulating axillary meristem formation in *Arabidopsis thaliana*. The rectangular box represents a transcription factor, the same color represents the same family, and ellipses represent genes other than transcription factors. The arrow and rough line represent positive and negative regulation, respectively.

Before axillary bud formation, REVOLUTA (REV) upregulates *STM* expression and promotes AM initiation. Subsequently, CK reactivates *WUS* expression to establish the AM [1]. Preliminary evidence shows that REV acts upstream of *STM* and *WUS* and that Ls/LAS acts upstream of *STM* to activate expression [19,20]. However, the upstream region of *REV* is regulated by LAS. As an HD-ZIP transcription factor, REV itself is necessary for all lateral meristem formation. In addition to RNA accumulation in other modes, *REV* is expressed in the near-axis position of the developmental leaf primordium in a region similar to *RAX1*, which can produce the position signal of *RAX1* expression and control the radial mode [21,22]. The AP2 family transcription factor DORNRÖSCHEN (DRN) also plays a role in embryonic meristem and lateral organ development. Although AM initiation is affected in the *drn-1* mutant, the *drn-1 drnl-1* double mutant shows more serious defects in axillary bud formation than the single mutant, indicating that *drn* and *drnl* have important redundant functions in AM initiation [1]. Further studies have shown that DRN and DRNL preferentially affect the initiation pathway of the AM at the early nutritional stage rather than at late and reproductive stages. DRN, DRNL and REV can directly activate *STM* expression by binding to the same promoter region. In summary, DRN and DRNL redundantly promote AM initiation at the vegetative growth stage, and DRN/DRNL and REV synergistically upregulate *STM* transcription in mature leaf axils [23].

REGULATOR OF AXILLARY MERISTEM FORMATION (ROX) encodes direct homologs of bHLH transcription factors, namely, LAX1 and BA1. In these mutants, axillary bud formation during vegetative bud development is damaged, and their combination with REGULATOR OF AXILLARY MERISTEMS1 (*rax1*) and *las* mutations enhance these branching defects [24], indicating that ROX regulates AM formation by cooperating with RAX1 and LAS [18]. In the nutritional and reproductive development process, orthologous bHLH transcription factors seem to be involved in the formation of boundary regions in eudicotyledons and Gramineae plants, as delayed growth is needed. In subsequent studies, ABA was found to significantly inhibit the expression of the *RAX1* and *LAS* genes, thereby affecting the growth of axillary buds. RAX1 is a member of the largest MYB TF R2R3-MYB family in *Arabidopsis*. *RAX2* and *RAX3* genes function in the early stages of AM

initiation and development [22,24,25]. In addition, RAX1 promotes the early stage of AM formation; it also negatively regulates the gibberellin level in shoot tips to modulate AM formation and affect the timing of development phase transition [26]. Studies have shown that RAX1 is involved in the determination of axillary meristems by generating a tissue environment conducive to the establishment of meristems to control the spatial pattern of AM development [22]. In sunflower, the rax-like gene R2R3-MYB2 was also found to play a key role in AM formation to establish or maintain the leaf axillary stem cell niche [24]. The three genes are expressed at the regional boundary between the shoot apical meristem and leaf primordium prior to the establishment of axillary meristem [22]. Further evidence shows that LAS and RAX1 can replace ROX to some extent and regulate axillary meristem formation [27].

LATERAL ORGAN FUSION1 (LOF1) and LOF2 encode MYB transcription factors that play roles in lateral organ separation and axillary meristem formation, partly through interaction with CUC2, CUC3 and STM [16,28]. *LOF1* is expressed at organ boundaries and acts upstream of *RAX1*, *LAS* and *CUC* [16,29,30]. The *lof1* mutant exhibits defects in organ separation, which is the result of abnormal cell division and amplification during early boundary formation. In addition, low concentrations of BRs in the border area promote specific expression of the *CUC* gene and initiation of the AM [21].

The NAC domain proteins CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 are involved in the initiation of the *Arabidopsis* SAM via *STM* expression [16,22,27,28]. The number of axillary buds in its mutants is significantly decreased [31]. Among the three CUC family members, CUC3 plays a major role in determining the formation and location of axillary meristems [30–32]. Further studies have shown that CUC1 and CUC2 control the development of the axillary meristem by regulating LAS and that CUC3 may play a role independent of LAS [30]. *CUC2* is also regulated by RAX1 in early AM development to establish or maintain the stem cell niche formed by the AM [22,31]. In particular, transcription of *CUC2* is continuously downregulated in the *raxl* mutant, indicating that RAX1 affects AM initiation by regulating the expression of *CUC2* [21]. Thus, RICE FLORICULA/LEAFY (RFL) promote AM specificity through an effect on *LAX1* and *CUC* [33]. *RFL* is expressed in the vegetative axillary meristem and very young tillering buds, and its expression pattern is similar to that of STM, which may be related to the maintenance of the meristem cell zone [34].

Guo et al. [35] identified the *EXCESSIVE BRANCHES1* (*EXB1*) gene, which encodes a WRKY transcription factor previously known as WRKY71 that is mainly expressed in tissues around the AM initiation site. The functional *exb1-D* mutant displays an obvious increase in branching, which is due to the combined effect of excessive AM priming and increased bud activity. Quantitative data show that EXB1 controls the initiation of the AM by positively regulating the transcription of *RAX1*, *RAX2* and *RAX3*. Subsequent data indicate that *EXB1* may be located upstream of the *RAX* gene and regulate AM formation [21,36]. EXB1 also regulates branching in *Arabidopsis* through negative regulation of auxin signaling [37]. Auxin is a well-known bud growth inhibitor, and AM initiation requires minimal auxin [28,38]. In the presence of apical buds exist, auxin is transported from top to bottom in the axilla and inhibits the growth of axillary buds. This phenomenon is called apical dominance [39]. WRKY proteins in the EXB1 clade regulate auxin pathways. Similarly, overexpression of rice *WRKY72* in *Arabidopsis* also increases bud branching, suggesting that the role of EXB1 in bud branching may be evolutionarily conserved between monocots and dicots [40]. Furthermore, a key factor in the establishment of the AM boundary region in *Arabidopsis* is the transcription factor *LATERAL ORGAN BOUNDARYES1* (*LOB1*), which induces *PHYB ACTIVATION TAGGED SUPPRESSOR1* (*BAS1*) expression and encodes a protein that brassinosteroid-inactivation capacity. In leaf axils, BR accumulation is negatively regulated by LOB1, an important boundary-specific transcription factor [18]. LOB1 directly upregulates *BAS1* to produce low BR concentrations to reduce cell division and expansion in border areas [21]. Unlike all other known regulators, AGAMOUS-LIKE 6 (AGL6) specifically promotes stem branching only at the leaf axils of stem leaves in *Arabidopsis* [41].

Similarly, in rice, *TILLERS ABSENT1* (*TAB1*) and *WUSCHEL-RELATED HOMEOBOX4* (*WOX4*) are two *WUS* genes that are necessary for AM initiation [31]. *TAB1* is normally expressed only in the anterior portion of the meristem and is not usually expressed in the SAM or mature AM [42]. Interestingly, *wus* mutation does not affect axillary meristem development in *Arabidopsis*, but rice TAB1 seems to control AM formation through mechanisms different from those of WUS in *Arabidopsis* [43]. The other WUS family transcription factor, WOX4 (the close paralogous homolog of TAB1), plays a role in the development of the AM by alternating with TAB1 [44]. However, unlike *TAB1*, *WOX4* is not expressed in the premeristem but contributes to the maintenance of the AM after almost complete AM establishment. TAB1 forms an AM by enhancing the expression of *O. sativa homeobox 1* (*OSH1*) and *WOX4* [31]. During rice AM formation, *OSH1* is preferentially expressed in the AM, and a significant decrease in its expression and a decrease in tillering in its mutant indicate that the gene is necessary for the initiation or maintenance of the fate of undifferentiated cells at the very early stages of AM formation [45]. *MONOCULM 3* (*MOC3*) is a direct homolog of WUS in rice and is necessary for the formation of tillering buds and interacts with key components of the CK pathway controlling rice tillering [46]. CKs antagonize auxin at the top. Even in the presence of auxin provided by the growing stem apex or the apex, CK applied to buds is sufficient to initiate growth [47].

LAX PANICLE1 (LAX1) and MONOCULM1 (MOC1) encode bHLH family and GRAS family transcription regulators, respectively, which are necessary for the initiation and maintenance of the AM in rice panicles [24,48]. *LAX* is expressed on the boundaries of apical meristem- and neomeristem-forming regions, specifically controlling the initiation or maintenance of new meristems [49]. Studies have shown a significant reduction in the number of spikelets in the *lax1* mutant, and AMs cannot be formed during vegetative development. Similarly, the maize *barren stalk1* (*ba1*) mutant cannot initiate AMs at any stage of the life cycle [24,50]. These two genes accumulate at the proximal axis boundary of the axillary meristems formed during nutrition and reproductive development [27]. *MADS34* in rice encodes a MADS-box transcription factor that coregulates the number of primary and secondary branches with LAX1 [51]. *MOC1* (a direct homolog of *LATERAL SUPPRESSOR*, *LS* in tomato and *LATERAL SUPPRESSOR*, *LAS* in *Arabidopsis*) was the first key transcription factor identified [52,53] controlling rice tillering; it is mainly expressed in leaf axils and axillary buds during AM development, positively regulating rice tillering [43,54]. As expression of *MOC1* and *LAX1* is not altered in the *tab 1* mutant, *TAB1* plays a role in an independent pathway or downstream of MOC1 and LAX1. In addition, LAX2, together with LAX1 and MOC1, plays a role in different AM maintenance pathways to control branching at vegetative and reproductive stages [45]. Similarly, disruption of *LAS* in *Arabidopsis* leads to AM loss during vegetative development, causing loss of branching or tillering and indicating that these genes are highly conserved [5]. In conclusion, both *LAS* and *MOC1* are specifically expressed in the initiation region of the AM [21]. However, in the *moc1* mutant, which lacks axillary buds and tillers, *OSH1* expression completely disappears in leaf axils but is not affected in the SAM. Moreover, *MOC1* is expressed earlier than *LAX1*, *LAX2* and *TAB1* during AM formation, indicating that the sequence or independent role of these genes is the cause of axillary bud formation and that multiple pathways contribute to the development of the AM [31].

Similarly, *REGULATOR OF AXILLARY MERISTEM FORMATION LIKE* (*ROXL*) isolated from sunflower is a homologous gene of *ROX/LAX1*. In situ results of a cross-section show accumulation of *ROXL* transcripts at specific points in the boundary region between the apical meristem and the lateral leaf primordium, displaying a similar pattern in *Arabidopsis* [27]. Based on in situ hybridization, *Ha-ROXL* exhibits clear-boundary transcription in vegetative branches, though the expression pattern of *LATERAL SUPPRESSOR LIKE* (*LSL*) is confined to the boundary region because signals can also be detected in other cellular domains of vegetative and reproductive branches. Transcription of *LSL* is also expanded at the early stage of lateral primordium development, indicating that this gene is

involved in the early development of the lateral primordium and in the initiation of the AM [24,55].

*BLIND* (*Bl*) is the homologous gene of the *RAX1* gene in tomato and *Arabidopsis* [21,25]. It encodes an R2R3 Myb transcription factor and regulates the early steps of AM initiation. There are fewer axillary buds due to defects in AM initiation caused by the *bl* mutant [31]. Double-mutation analysis has shown that different members of the Bl-related subgroup of the *R2R3 Myb* gene regulate axillary meristem formation in a partially redundant manner [22]. Double mutants of *ls* and *bl* in tomato display an additive phenotype, suggesting that at least two pathways are involved in AM initiation [20,22,27]. It is noteworthy that these genes are expressed in leaf axils during AM development. The homologous compounds of BLIND and RAX do not play a role in AM formation in rice, indicating that the BLIND/RAX pathway is unique to true dicotyledonous plants [31]. It should also be noted that in rice, axillary meristems usually develop into tillers from the basal nodes of plants, forming a typical cluster plant structure [34]. *FRIZZLE PANICLE* (*FZP*) is a very important gene in rice tillering development. Its overexpression inhibits *RFL/PANICLE ORGANIZATION 2* (*APO2*), which seriously suppresses the formation of axillary meristems at the vegetative stage and leads to a significant decrease in tiller number [48]. In maize, *BAD1* transcripts are detected mainly in the AM boundary region and between lateral branches, and its functional deficiency results in organ fusion via a reduction in the number and angle of branches [56]. Nevertheless, homologs of this gene, *COMPOSITUM 1* (*COM1*) and *RETARDED PALEA1* (*REP1*), in barley and rice have no similar functions. In addition, *BELL1-like homeobox 12* (*BLH12*) and *BLH14* play an important role in maintaining axillary meristems and possess the redundant functions necessary for axillary bud development throughout nutritional and reproductive development [19].

In addition to common plants such as *Arabidopsis*, rice and maize, other species are predicted to have transcription factors involved in branching or tillering. For example, it was found that the formation of the AM in *Antirrhinum majus* requires *ERAMOSA* (*ERA*), a gene encoding a GRAS transcription factor (orthologous to *LAS* in *Arabidopsis*). The basic role of *ERA* in AM formation is consistent with that of *LAS/MOC1/ERA* in preventing cell differentiation in the boundary area and in stimulating AM formation [57].

#### **3. Axillary Meristem Outgrowth**

The structure of mature plants is determined by the occurrence of axillary meristems, control of bud growth and subsequent dynamics of branching growth. Changes in these parameters result in the high morphological diversity observed in different plant species and even among individuals of a particular species [18]. After the formation of the AM, its growth as a branch repeats the development pattern of primary branches and endows plants with a branching structure [33]. Although a series of genetic studies have revealed the molecular mechanism and genes involved in SAM formation and maintenance, little is known about the generation and growth control of axillary buds [20].

The transcription factor *BRANCHED1* (*BRC1*) in *Arabidopsis*is homologous to *TEOSINTE BRANCHED 1* (*TB1*) in maize and plays a core inhibitory role in regulating axillary bud growth (Figure 3). *BRC2* is a paralog of *BRC1*, which is also expressed in axillary buds and plays a redundant role in the regulation of axillary bud growth [43]. Similarly, *VvBRC* plays a key and negative role in branching in grape [58]. LAS and REV act upstream of *BRC1*. As expression of *BRC1* is significantly downregulated in the *max2* mutant, the MAX-mediated pathway seems to control the activity of BRC1 [59]. *Homeobox 21* (*HB21*), *HB40* and *HB53* act directly downstream of BRC1 to regulate branching [60]. Moreover, BRC1 was identified as an inhibitor of downstream branches of SL signal transduction [61,62]. SL, a plant hormone synthesized by carotenoid catabolism, moves from root to stem, inhibiting stem branching by preventing the growth of axillary buds [63]. In *Arabidopsis*, another transcription factor involved in the SL pathway, namely, BRI1-EMS-SUPPRESSOR1 (BES1), negatively regulates cambium activity in the SL signaling pathway. Further studies have shown that SL signaling regulates the expression level of *WOX4* through BES1, controlling secondary

growth [64]. CK is another class of hormones that has an important role in regulating apical dominance and axillary bud outgrowth. At the later stage of development, MYB2 reduces the concentration of CK by inhibiting the expression of *IPTs*, suppressing the growth of axillary buds [47]. In functional exploration of the MADS domain factor *FRUITFULL* (*FUL*) in *Arabidopsis thaliana*, it was found that the combination of auxin and BR strongly induced the growth regulator *SMALL AUXIN UPREGULATED RNA 10* (*SAUR10*) to be directly modulated by FUL, thus participating in branching angle regulation [65]. It should be noted that FUL also responds to a decrease in R:FR light by regulating the SAUR10 pathway and affecting *Arabidopsis* branching [65]. The auxin-regulated branching pathway in *Arabidopsis* involves a class of photochrome-targeted transcription factors, *PHYTOCHROME INTERACTING FACTOR 4* (*PIF4*)/*PIF5* [66]. When these transcription factors participate in photoreactions, they inhibit the branching caused by phyB dysfunction and low R: FR. Hence, R:FR plays an important role in branching.

**Figure 3.** Patterns of transcription factors involved in regulating axillary meristem growth in *Arabidopsis thaliana*. The rectangular box represents transcription factors, the same color represents the same family, and ellipses represent genes other than transcription factors. The arrow and rough line represent positive and negative regulation, respectively.

Overall, the interaction between ABA and auxin may mediate the effect of PIF4/PIF5 on branching [67]. Mohammad et al. [68] reported another transcription factor, ERF BUD ENHANCER (EBE), that affects cell proliferation, axillary bud growth and branching in *Arabidopsis*. The gene encodes the AP2/ERF transcription factor and is strongly expressed in proliferating cells. Moreover, overexpression of *EBE* promotes cell proliferation, shortens the cell cycle in growing calli and stimulates the formation and growth of axillary buds [68].

*TB1*, also known as *FINECULM1* (*FC1*), is a TCP family transcription factor that negatively regulates rice tillering and inhibits the subsequent growth of axillary buds [69]. This gene encodes a TCP TF that is expressed at the base of these buds and SAMs. Its overexpression leads to a significant reduction in tiller number, whereas the *tb1* mutant exhibits an increase in tiller number [31]. In addition, TB1 may be a common target for the CK and SL pathways and act downstream of SL [70,71]. *MADS57* is a transcription factor of the MADS domain family that participates in the regulation of axillary bud growth in rice. RNA in situ hybridization analysis has shown that MADS57 is mainly expressed in meristems and axillary buds. Additionally, its expression is higher in the tillering and stem elongation stages than in other stages of rice growth [70]. Interaction of TB1 with MADS57 reduces the negative regulatory activity of MADS57 on *D14* expression, allowing MADS57 to affect tillering [71].

In a study of branching-related genes in chrysanthemum, *CmERF053* was found to be rapidly upregulated in axillary buds when apical dominance was relieved, which may be related to the growth of lateral branches. The gene belongs to the AP2/ERF family and is mainly expressed in stem and root organs. Further transcriptome analysis showed that CK-mediated control of stem branching may be related to the transcription level of *CmERF053* [26]. *IDEAL PLANT ARCHITECTUREL1* (*IPA1*), also known as *SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14* (*SPL14*), is another key regulator that determines plant structure but not meristem activity, and upregulation of *IPA1* expression leads to fewer tillers in rice [72]. Furthermore, *IPA1* overexpression lines display a reduced tillering phenotype, whereas tillering in the *ipa1* mutant increases [73]. This gene acts as a direct downstream component of D53 in regulating tiller number and SL-induced gene expression. In fact, *D53* is one of the only known transcription targets of SL, and D53 inhibits *IPA1* upregulation [74]. IPA1 directly binds to the *D53* promoter and plays a key role in the feedback regulation of SL-induced *D53* expression. In summary, *IPA1* may constitute a long-term transcription factor that acts with D53 to mediate SL-regulated rice tillering development [75]. In later vegetative axillary bud growth, OsMADS57 enhances axillary bud growth and subsequent tillering through SL signal transduction via direct inhibition of expression. OsMADS57 directly inhibits *D14* transcription to regulate tillering during organogenesis [70].

The SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SPL13) gene encodes an SBP transcription factor that is mainly expressed in meristems and is essential for regulating branching and vegetative growth in *Medicago* [63]. Overexpression of *SPL13* inhibits axillary bud growth, thereby reducing lateral branches. In this process, *MYB112* is targeted and downregulated by SPL13. Compared with WT, *MsMYB112* RNAi plants show more branches, which confirms that MYB112 itself also inhibits the lateral branch growth of alfalfa [63,76]. Furthermore, MdWUS2 in apple can regulate branches by inhibiting the expression of *MdTCP12* [77].

*Related to ABI3 and Viviparous 1* (*RAV1*) is a circadian rhythm gene in *Castanea mollissima* that is homologous to the *TEM* gene in *Arabidopsis*. It reaches its peak expression at noon under vegetative growth conditions and is highly expressed during winter dormancy and in response to low temperature [78]. When the CsRAV1 protein was overexpressed in hybrid poplar, high sylleptic branching was induced during the same growing season as lateral bud formation.
