Once a branch/tiller bud is formed, the plant is confronted with a critical decision to stimulate bud sprouting, giving rise to a branch/tiller, or to maintain bud dormancy. Bud outgrowth typically progresses through three discernible stages: dormancy, transition, and sustained growth
. The fate of buds in the transition stage is influenced by the complex interplay of environmental and endogenous cues, ultimately determining whether buds return to dormancy or enter a sustained growth phase
The expression of
TB1, encoding a non-canonical basic helix–loop–helix (bHLH) transcription factor of the TCP family, is negatively correlated with bud growth
[10][11]. This TCP protein family is represented by four founding members: TB1, CYCLOIDEA (CYC), PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1), and PCF2. These family members were identified by their functions in plant development or their DNA binding capacity
[10][12][13][14][15]. The maize (
Zea mays)
tb1 mutant exhibits an uncontrolled proliferation of tillers, resulting in a bushy architecture reminiscent of its ancestor, teosinte
[16]. The inhibitory effect of
TB1 on bud outgrowth is spatially restricted to axillary buds as soon as they become visible
[16]. By contrast, in teosinte,
TB1 is not expressed in axillary buds, allowing axillary bud outgrowth
[16]. The role of
TB1 in suppressing axillary bud outgrowth is conserved in rice, as ectopic overexpression of its ortholog
OsTB1 under the control of the actin promoter leads to reduced tillering
[17]. Conversely, loss-of-function mutants of
OsTB1, such as
fine culm 1 (
fc1), show increased tillering
[17].
TB1 and its orthologs (e.g.,
OsTB1 or
FC1 in rice,
BRANCHED1 (
BRC1) in Arabidopsis,
PsBRC1 in pea [
Pisum sativum], and
SlBRC in tomato) operate in conjunction with other vital genes and plant hormones to regulate bud outgrowth
[18][19][20][21]. These genes and phytohormones are discussed in subsequent sections. The coordinated action of TB1 with these factors has earned it the title “branching integrator”.
2.1.2. SQUAMOSA Binding Proteins Inhibit Bud Outgrowth
SQUAMOSA promoter binding protein-like (SPL) transcription factors, which are specific to plants, mediate various aspects of plant development, including branching
[22]. Different members of the
SPL gene family in Arabidopsis are post-transcriptionally regulated by miR156
[23]. Overaccumulation of miR156 leads to a considerably bushy phenotype
[24][25]. Notably, double mutants of the Arabidopsis paralogs
SPL9 and
SPL15 exhibit an increased branching phenotype, highlighting the crucial role of miR156-targeted
SPL genes in regulating shoot branching
[26] (
Figure 2).
Figure 2. Summary of phytohormones and key genes involved in shoot branching. Blue arrows represent promotion, whilst red flat-ended lines denote inhibition. In this model, BRC1/TB1 acts as an integrator to interact with other genes, such as SPL genes and phytohormones, to mediate shoot branching. Abbreviations: SL, strigolactone; ABA, abscisic acid; CK, cytokinin; GA, gibberellin; PIN1, PIN-FORMED1; SCF, Skp-Cullin-F-box; Aux/IAA, Auxin/Indole-3-Acetic Acid; IPT, ADENYLATE ISOPENTENYLTRANSFERASE; PAT, polar auxin transport; CYP735A, cytochrome P450 monooxygenase 735A; LOG, LONELY GUY; tRNA-IPT, transfer RNA isopentenyltransferase; CKX, cytokinin oxidase; ARR5 and ARR6, RESPONSE REGULATOR5 and 6; NRT1.2, nitrate transporter 1.2; PCNA1, PROLIFERATING CELL NUCLEAR ANTIGEN1; SPL9, SPL14 and SPL15, SQUAMOSA PROMOTER BINDIN PROTEIN-LIKE9,14 and 15; BRC1/TB1, BRANCHED1/TEOSINTE BRANCH 1; NCED3, 9-CIS-EPOXYCAROTENOID DIOXYGENASE3; HB21, HOMEOBOX PROTEIN 21; HB40, HOMEOBOX PROTEIN 40; HB53, HOMEOBOX PROTEIN 53; DELLA, aspartic acid–glutamic acid–leucine–leucine–alanine; D14, DWARF 14; MAX3, 2, 1, and 4, more axillary growth 3, 2, 1, and 4; TPRs, TOPLESS-RELATED PROTEINs; TFs, transcription factors; D53-like SMXLs, DWARF53-LIKE SMAX1-LIKEs; BES1, bri1-EMS-suppressor 1; AM, axillary meristem.
2.1.3. Auxin Indirectly Inhibits Sustained Bud Outgrowth
A principal function of auxin, as observed in various studies, is mediating apical dominance, as lateral bud outgrowth is inhibited by auxin. For instance, the
auxin-resistant 1 (
axr1) mutant of Arabidopsis exhibits a lower sensitivity to auxin than wild-type plants, resulting in weak apical dominance and an increased number of branches, due to the release of axillary buds, highlighting the role of auxin in inhibiting axillary bud outgrowth
[27].
Moreover, apical dominance largely depends on polar auxin transport (PAT) mediated by PIN-FORMED (PIN) proteins in the stem
[28]. Studies involving RNA interference (RNAi)-mediated knockdown and overexpression of
OsPIN1 have demonstrated the negative effect of
OsPIN1 on tillering in rice
[29].
In addition to the crucial role of PAT in regulating branching/tillering, the auxin signaling pathway also influences shoot branching/tillering via SKP1-CULLIN1-F-box (SCF)-mediated protein degradation. Auxin receptors, including TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and closely related family numbers (AUXIN SIGNALING F-BOX [AFB])
[30][31][32], bind to auxin to stabilize the interactions between TIR1/AFBs and members of the Aux/IAA (Auxin/INDOLE-3-ACETIC ACID INDUCIBLE) family of transcriptional repressors
[33]. Their interaction with TIR1/AFBs leads to the degradation of Aux/IAA, permitting auxin-mediated upregulation of transcription
[34][35][36]. By contrast, loss of function of
IAA12 in Arabidopsis leads to auxin-resistant stabilization of the SCF complex and, thus, constitutive suppression of target auxin-upregulated genes
[32], resulting in a bushy phenotype. Overexpressing
OsMIR393, whose mature miRNA product OsmiR393 targets and downregulates the transcripts of the auxin receptor genes
OsTIR1 and
OsAFB2, leads to increased tiller production
[37].
SLs are a collection of carotenoid-derived lactones secreted by plants. These phytohormones are primarily known for their roles as rhizosphere signals used by root-parasitic plants to detect their hosts
[38] and as cues for mycorrhizal fungi to form symbiotic associations
[39]. Importantly, SLs also inhibit the outgrowth of axillary buds. This inhibitory effect was initially observed in
ramosus (
rms) mutants in pea, which exhibit excessive branching, as well as
decreased apical dominance (
dad) mutants in petunia (
Petunia hybrida) and
more axillary growth (
max) mutants in Arabidopsis
[40][41][42][43][44][45][46][47].
SL biosynthesis involves several enzymes. DWARF27 (D27) is responsible for isomerizing all-
trans-β-carotene at the C-9 position to form 9-
cis-carotene
[48][49]. Carotenoid cleavage dioxygenase 7 (CCD7, also named MAX3) and CCD8 (also named MAX4) then cleave 9-
cis-carotene to produce carlactone, a key endogenous SL precursor
[40][50]. The conversion of carlactone to SLs is catalyzed by MAX1, an Arabidopsis cytochrome P450 that acts downstream of MAX4 and MAX3
[51]. MAX1 converts carlactone to carlactonoic acid, which is further converted to methyl carlactonoate, an SL-like compound (
Figure 2)
[52].
SLs are perceived by MAX2, an ortholog of D3 from rice, which plays a crucial role in regulating plant branching. Disruption of
. Moreover, the perception and signaling roles of SLs in rice require their interaction with D14, a putative SL receptor. D14 interacts with D3, an F-box protein of the SCF E3 ubiquitin ligase complex, to form an SL-induced D14-D3 complex
. This complex targets proteins for ubiquitination and degradation, resulting in changes in plant branching/tillering
).