Genetic and environmental control of rice tillering

Yuping Yan,Chaoqing Ding,Guangheng Zhang,Jiang Hu,Li Zhu,Dali Zeng,Qian Qian,Deyong Ren

State Key Laboratory of Rice Biology and Breeding,China National Rice Research Institute,Hangzhou 310006,Zhejiang,China

Keywords: Rice tiller Axillary meristem Tiller bud Genetic and external factors Regulatory mechanism

ABSTRACT Increasing tiller number is a target of high-yield rice breeding.Identification of tiller-defect mutants and their corresponding genes is helpful for clarifying the molecular mechanism of rice tillering.Summarizing research progress on the two processes of rice tiller formation,namely the formation and growth of axillary meristem,this paper reviews the effects of genetic factors,endogenous hormones,and exogenous environment on rice tillering,finding that multiple molecular mechanisms and signal pathways regulating rice tillering cooperate rice tillering,and discusses future research objectives and application of its regulatory mechanism.Elucidation of theis mechanism will be helpful for breeding high-yielding rice cultivars with ideal plant type via molecular design breeding.

1.Introduction

Rice(Oryza sativa),one of the three main food crops,is a staple food for more than half of the world’s population,and increasing its yield is the primary goal of breeding.Dwarf breeding,heterosis utilization,and super-rice breeding has greatly increased the rice yield per unit area in China [1-5].Because rice tillers determine the number of rice panicles,a component of rice yield [6,7],optimizing tiller number is a goal of rice genetic improvement.

The formation of a rice tiller can be divided into two processes:the initiation of new meristem on the leaf axis,namely the axillary meristem (AM),and its growth [8,9].Axillary buds are produced from rhizome nodes below and near the ground,and branches with adventitious roots form from them [10].During the vegetative growth period,tillers occur repeatedly,and after the plant enters reproductive growth,some tillers begin to form spikelets [11].However,not all axillary buds will form productive panicles.Some axillary buds will enter the dormant state,one of the mechanisms by which rice prevents excessive tillering.In particular,axillary buds that form from high tillering nodes will produce ineffective tillers[12,13].Based on the process of tiller formation,rice tillering mutants found in nature are divided into two categories: AM formation defect mutants and tiller bud growth defect mutants [14-26].Rice tillering also determines plant architecture.Crops with ideal architecture produce higher yield.In‘‘Green Revolution”cultivation,grain yield is greatly increased by planting lodgingresistant semidwarf wheat and rice cultivars [1].

Although tillering is controlled mainly by genetic factors,more and more studies in recent years [23,27-37] have shown that the growth of axillary buds is regulated by a complex network of genetic,hormonal,and environmental factors.This review summarizes the key genes regulating rice tillering and the molecular mechanism by which endogenous hormones as signal molecules and environmental stimuli such as light,temperature,and soil fertility affect rice tillering.

2.Development events in rice tillering

Based on morphological observation during the development of the rice tiller[38],its formation can be divided into two main processes: the initiation of a new AM and its subsequent growth and development (Fig.1).At the embryonic stage,the shoot apical meristem(SAM)establishes a spindle that eventually develops into a bud,and then differentiates into aboveground organs including stems,leaves,and branches [39].AM has the same developmental potential as SAM,and the establishment of new meristem harboring stem cells is necessary for the initiation of AM[40].In the‘‘detached meristem”model[41],AM-initiated stem cells are detached from primary SAM and associated with leaf axils.Next,a boundary needs to be established between the main shoot and the leaf primordium to separate the pluripotent meristematic cell population(which remains indeterminate)from the developing organs,a step that is essential for plant organogenesis [42,43].

Fig.1.The formation process of a rice tiller.At tillering stage,AM-initiated stem cells are detached from primary SAM and associated with the leaf axil.After AM is established in the leaf axils,as a new SAM of the secondary growth axis,it differentiates into tiller bud,leaf sheath primordium,and leaf primordium,and after the formation of the second leaf primordium,the tiller bud rapidly elongates to form a tiller with several leaves.The red dot and blue regions indicate axillary meristem and tiller buds,respectively.AM,axillary meristem;TB,tiller bud;SAM,shoot apical meristem;P1-P6,leaf primordia.

After AM initiation,the cells divide and proliferate,first forming the tiller sheath primordium and then differentiating into the first and second leaf primordia in turn [44].The first leaf primordium differentiates into a tiller bud when the second leaf primordium starts to differentiate,and then the third leaf primordium forms and differentiates after the second leaf primordium differentiates into a tiller bud [10].The tiller bud grows rapidly and stretches out the leaf sheath surrounding the main stem to form a tiller until four young leaves and one leaf primordium are formed,with adventitious roots [26].

3.Endogenous regulation of tiller formation in rice

3.1.Genetic factors regulating tillering in rice

The identification of rice tillering mutants and their corresponding genes over the past decades has helped researchers establish the molecular mechanism of tillering regulation,and the mutants have been divided into AM formation defect mutants and tiller bud growth defect mutants [14-26].Although the external environment influences the development process of the rice tiller,genetic factors are the main factors regulating rice growth and development,and also the main theoretical basis for breeding new varieties (Fig.2;Table 1).

Table 1Regulators influencing AM formation.

Fig.2.Molecular mechanism of AM initiation in rice.MOC1,the first gene identified to regulate tillering in rice,coordinates with MOC3,LAX1,LAX2 and their interaction factor MIP1 to promote the formation of rice tillers.MOC1 interacts with TAD1,OsAPC10 and APC/C complex to form E3 ubiquitin ligase complex,which is further degraded.SLR1 binds to MOC1 and protects it from degradation.OSH1,the common downstream gene of MOC1 and MOC3,is strongly expressed in the early stage of AM formation.MOC3 promoted the formation of AM by increasing the expression of OSH1 and OsWOX4,and also interacts with MOC1 to promote the expression of FON1,thus promoting the growth of tiller buds. RFL is negatively regulated by FZP and promotes AM formation by promoting downstream genes OsCUC1 and LAX1. MOC1, MONOCULM1; MOC3,MONOCULM3;OSH1,O.sativa homeobox1;LAX1,LAX PANICLE1;LAX2,LAX PANICLE2;MIP1,MOC1 interacting protein1;TAD1,Tillering and Dwarf1;APC/C,Anaphase-Promoting Complex; OsAPC10, Oryza sativa Anaphase-promoting Complex Subunit10; FON1, FLORAL ORGAN NUMBER1; RFL, RICE FLORICULA/LEAFY; FZP, FRIZZLE PANICLE; OsCUC1, Oryza sativa CUP-SHAPED COTYLEDON1; OsWOX4, Oryza sativa WUSCHEL-RELATED HOMEOBOX4.

3.1.1.Molecular mechanisms controlling AM formation

During AM formation,O.sativa homeobox1(OSH1) was preferentially expressed in AM and its expression decreased with AM initiation [45].OSH1belongs to the KNOX class homeobox gene family.Previous study [15] has shown that KNOX genes are expressed before organ differentiation,being expressed in SAM,AM and other meristems to maintain undifferentiated cell fate,and their expression decreases after differentiation.The markedly reducedOSH1expression and tiller numbers in the mutant suggested thatOSH1initiates and maintains undifferentiated cells at the early stage of AM formation[46].Arabidopsis STMwas strongly expressed in cells with diverse morphologies to form a bump.It was also found [47] thatSTMandOSH1are expressed at similar times during AM initiation,suggesting that the mechanism controlling AM initiation may be partially conserved in monocotyledonous rice and dicotyledonousArabidopsis.

Lax panicle mutants found in rice includelax1andlax2.LAX PANICLE1(LAX1) encodes a bHLH transcription factor that is expressed only in the border region between SAM and newly formed AM,helping establish AM and maintain its growth in rice.The LAX1 protein accumulated transiently at the Plastochron 4(P4)stage and moved to AM for full function [16].Mutation ofLAX1affected AM initiation and led to the reduction of tiller number.Subsequent study[46]found thatlax1 moc1double mutants exhibited fewer tiller numbers thanlax1andlax2,indicating thatLAX1cooperatively regulated rice tillering withLAX2andMOC1.In maize [48],BARREN STALK1(BA1) homologous to riceLAX1[49],controls AM formation during vegetative and reproductive branching,and theba1mutant did not produce tillers,indicating thatLAX1andBA1are functionally conserved during AM formation in grass plants.

TheLAX PANICLE2(LAX2) gene contained plant-specific conserved domains that were essential forLAX1andLAX2interaction,and its expression was observed throughout the process of AM formation in rice [27].In thelax2mutant,OSH1was normally expressed in cells,and these cell populations formed bulges and continued to proliferate to form AM.However,in thelax2 moc1double mutant[46],although bulges composed of cell populations were observed in the early stage of AM formation,the cells could not continue to proliferate to form AM in the later stage,resulting in the mutant’s inability to form tillers.This result indicated thatLAX1,LAX2,andMOC1are functionally redundant in the process of regulating AM formation.In transgenic plants,deletion of any two of these genes strongly inhibited AM formation at all developmental stages except panicle branch formation [16].

As the first identified gene regulating rice tillering[14],MONOCULM 1(MOC1)encodes a GRAS family transcriptional factor that is a key regulator of AM formation in rice.Themoc1mutant failed to form axillary buds and tillers,andOSH1signal completely disappeared inmoc1AMs,but was not affected in SAM.TEOSINTE BRANCHED 1/FINE CULM1(OsTB1/FC1),as a downstream regulatory factor in the tillering regulation pathway,was down-regulated in themoc1mutant,suggesting thatMOC1may be the main regulator in rice tillering [9].InMOC1-OE plants,upper tiller buds could develop into tillers,indicating thatMOC1also promoted the growth of tiller buds [14].Subsequent study [49] identified the Bre1 family geneMOC1 interacting protein 1(MIP1) containing a C3HC4 ring finger domain.Overexpression ofMIP1led to an increase in tiller number,andMOC1-OE plants displayed similar phenotypes,including increased tillers and decreased plant height.However,in themoc1mutant,the expression level ofMIP1was not changed,indicating that the full function of theMIP1must beMOC1-dependent.MIP1in rice shared the highest similarity withHUB1inArabidopsis thaliana.These two genes have been confirmed[50,51] to participate in histone H2B monoubiquitination as E3 ligase,and affect the expression and transcription of downstream genes via histone modification.Investigating the molecular mechanism by whichMOC1regulates tillering,subsequent studies[17,52] found that MOC1 was ubiquitinated by TAD1/TE,the coactivator of the APC/C complex,and then degraded by the 26S proteasome.The DELLA protein SLR1 interacted with MOC1 and inhibited MOC1 degradation independently of the E3 ubiquitin ligase complex [53],suggesting that MOC1 might act in the GA signal pathway regulating rice tillering.InArabidopsis,LASwas homologous toMOC1and was expressed in leaf axils,and the phenotype of lateral branch deletion was also observed inlasmutants [54].The initiation of AM in the tomatolsmutant (homologous toMOC1) was also inhibited,and AM cells could not maintain their meristem capacity,resulting in failure to produce lateral buds during the vegetative phase [55].The functional similarity of these genes in multiple plant species suggested that the functions ofMOC1and its homologs are conserved in monocots and dicots.

Tillering And Dwarf 1(TAD1) encodes a Cdh1-type activator of anaphase-C complex(APC/C),which is a highly conserved E3 ubiquitin ligase in eukaryotes[17].TAD1 interacted with MOC1 to form a complex with OsAPC10 [52].As a coactivator of APC/C,TAD1 recruited MOC1 to the APC/C complex and mediated the ubiquitination degradation of MOC1,leading to the weakening of the promotion of AM initiation and the reduction of tiller numbes[52].The alleleTILLER ENHANCER(TE) was identified in the same year [17].TEandMOC1were coexpressed in leaf axils,and the formed APC/CTEcomplex degraded MOC1 by the 26S proteasome pathway,thereby inhibiting the initiation and formation of AM.

A CLV-WUS negative feedback loop has been found to regulate meristem activity inArabidopsis thaliana,but its function in monocot plants remains elusive.WUSCHEL(WUS) encodes a type of homeobox protein and belongs to the WOX protein family,which maintains stem cell viability,whereas CLAVATA (CLV) signaling repressedWUSexpression [27].In rice,CLV-like genes,known asFLORAL ORGAN NUMBERgenes,such asFON1andFON2,negatively regulate the meristematic activity of tissues.Fewer tillers were found in thefon1mutant,but axillary buds formed normally,indicating thatFON1was not involved in the inhibition of AM formation [9].WUS did not regulate branching inArabidopsis,but the tiller phenotype ofWUS-loss-of-function mutants was changed in rice.MONOCULM 3(MOC3)/TILLERS ABSENT 1(TAB1)/O.sativa WUS(OsWUS) encodes a protein containing a homeodomain,WUS box,and EAR motif [18].A dramatic reduction ofOSH1expression and absence of meristem in thetab1mutant showed thatTAB1was critical to AM initiation by promotingOSH1expression to maintain stem cell division in the premeristem region.But the expression ofMOC1andLAX1was unchanged in thetab1mutant,implying thatTAB1acted in a pathway independent ofMOC1,or downstream ofMOC1andLAX1[45].Later studies[9,56] showed that MOC3 interacted with MOC1 to promote the growth of tiller buds by activating the expression ofFON1in rice,indicating that MOC3 produced a marked effect during AM formation and tiller bud growth.AnotherWUSgene,WUSCHEL-RELATED HOMEOBOX4(WOX4),highly similar toTAB1,functioned redundancy withTAB1in AM formation [45].WOX4was not expressed in the promeristem region but was expressed in SAM and established AM,suggesting thatWOX4maintained its meristem capacity after AM was established.Further study[45,57]revealed thatTAB1promoted AM formation by increasing the expression ofOSH1andWOX4.

Some mutants with defective panicle development also showed changes in the number of tillers.RICE FLORICULA/LEAFY(RFL),orABERRANT PANICLE ORGANIZATION2(APO2),was expressed in the axils of leaves to promote vegetative branching formation in the seedling stage,and was originally identified as a gene associated with panicle development [58-60].Expression ofCUP-SHAPED COTYLEDON 1(OsCUC1),which belongs the NAC transcription factor family and was shown [61] to be involved in embryonic SAM formation and boundary specification,was decreased in therflmutant,resulting in AM formation defects.MOC1andLAX1expressions were also decreased,resulting in blocked AM formation and reduced tiller number,indicating thatLAX1andCUCsact downstream ofRFL[62].In young panicles ofRFL/APO2-RNAi plants,LAX1was downregulated andFRIZZLE PANICLE(FZP) was upregulated.Overexpression ofRFL/APO2resulted in a reduction in tiller number.FZP,a rice homolog of maizeBD1,maintains the transformation from spikelet meristem to floral meristem and participates in the regulation of rice AM formation.InFZP-OE plants,tiller number was reduced by inhibition ofRFL/APO2expression [63,64].

3.1.2.Molecular mechanisms controlling tiller bud growth

After formation of AM,it develops into an axillary bud,which will immediately grow to form tillers or begin its dormant period[65].The different fates of tillering buds ultimately determine the architecture of plants.In recent years,with the use of molecular markers to facilitate the localization of several genes affected tiller number,a major breakthrough has been made in elucidating the molecular mechanism of rice tillering (Fig.3;Table 2).

Fig.3.Molecular mechanism regulating rice tiller bud growth.OsTB1,as the main negative regulator of tillering bud growth in rice,acts downstream in the SL signaling pathway to regulate rice tiller bud growth.The synthesis of SL from Carotenoid is catalyzed by OsMAX1a, OsMAX1e, D17, D27 and D10.As a receptor, D14 is negatively regulated by OsMADS57,interacts with D3,and forms a complex with SCF,which transmits SL signals and causes D53 protein degradation D53 further binds to a DLT-RLA1-OsBZR1 complex to inhibit OsTB1 expression.OsTB1 interacts with OsMADS57 to reduce its inhibition of D14 transcription and regulate the growth of tiller buds in rice.IPA1,a well-known ideal plant type gene,is a target gene of OsCCA1 along with OsTB1,D10 and D14.IPA1 interacts with and is inhibited by OSHI1.These two genes competitively bind to the promoters of OsDEP1 and OsTB1,which inhibit the formation of tillers in rice.OsDRM2,FON1 and Hd3a also promote the growth of tiller buds.OsMAX1a,O.sativa MORE AXILLARY GROWTH1a; OsMAX1e, O.sativa MORE AXILLARY GROWTH1e; D3, DWARF3; D10, DWARF10; D14, DWARF14; D17, DWARF17; D27, DWARF27; D53, DWARF53;OsTB1,O.sativa TEOSINTE BRANCHED1;OsCCA1,O.sativa CIRCADIAN CLOCK ASSOCIATED1;IPA1,IDEAL PLANT ARCHITECTUTRE1;OsSHI1,O.sativa SHORT INTERNODES1;OsDEP1,O.sativa DENSE AND ERECT PANICLE1; DLT, Double-lumen Tube; OsDRM2, O.sativa Domains Rearranged Methyltransferase2; RLA1, Reduced Leaf Angle1; BZR1, BRASSINAZOLERESISTANT1; FON1, FLORAL ORGAN NUMBER1; Hd3a, Heading date 3a; TN1, tiller number 1; TIF1, TN1 interaction factor 1.

Strigolactones (SLs) are a class of plant hormones derived from carotenoids.They are synthesized in roots and moved to the aboveground part[66],and exert an inhibitory effect on the growth of axillary buds [67].Genes involved in SL biosynthesis or the SL signaling pathway function in controlling the growth of axillary buds.MAX3/RMS5/DAD3/D17/HTD1[21]encoding homologous protein carotenoid lyase dioxygenase CCD7,MAX4/RMS1/DAD1/D10[22] encoding homologous protein carotenoid lyase dioxygenase CCD8,MAX1encoding cytochrome P450 monooxygenase,andD27[23] encoding β-carotene isomerase are involved in the synthesis of SLs.In both dicots and monocots,when these genes were mutated,the branch number of mutant plants was altered,indicating that SLs functioned in a conserved regulatory role in plant branches [68].

DWARF3(D3),aMAX2homolog gene identified in rice,encodes a nuclear-localized F-box protein,which functioned in the formation of an SCF complex and interacted withDWARF14(D14) andDWARF53(D53),then ubiquitinating them to promote their degradation and inhibit rice tillering [32].The axillary bud of thed3mutant appeared earlier than that of the wild type (WT),and thed3mutant showed more tillers.Thed3mutant did not respond to treatment with the synthetic SL analog rac-GR24,indicating thatD3acts in SL signal transmission [19].

HIGH TILLERING DWARF1(HTD1) encodes carotenoid cleavage dioxygenase,a homolog ofArabidopsis MAX3that inhibited the growth of axillary buds and subsequent tiller formation [20].The beneficial allele ofHTD1came from the Green Revolution rice cultivar IR8 and its parent Peta and is present in mostIR8-derived cultivars.Combining the increased lodging resistance conferred by the GA signal pathway geneSEMIDWARF1(SD1) and the increased high-yield tillering conferred by the SL signal pathway geneHTD1produced IR8 and its derived cultivars [21].

DWARF10(D10)/CAROTENOID CLEAVAGE DIOXYGENASE8(OsCCD8)is orthologous toArabidopsis MAX4and encodes a carotenoid cleavage dioxygenase that is involved in the synthesis of SLs[22].Thed10mutant showed dwarf culm and multiple tillers,and the inhibitory effect of apical dominance on branching was weakened [22].InD10-RNA interference (RNAi) transgenic plants,the reduced expression of mostOsPINs in culms resulted in reduced auxin transport capacity.Shoot apices of the mutantd10showed higher auxin levels than those of the WT.Exogenous auxin increased the expression ofD10in shoot nodes,suggesting that auxin might regulate rice tillering partly by inducingD10expression [69].The expression of the cytokinin (CK) synthesis genesOsIPT4,OsIPT5,andOsIPT7was also reduced.However,whenD10-RNAi plants were decapitated,the expression of these genes increased,indicating that auxin inhibited tillering by indirectly inhibiting the expression of CK synthesis genes.Given that the tiller number of aD10-RNAi transgenic plant did not decrease with CK content,the inhibition by SL of rice tillering did not depend on inhibition of CK by auxin.But the reduction of CK inD10-RNAi plants did not lead to a reduction in the number of tillers.The multi-tillering phenotype showed [69] that the inhibition of tillering by SL was not caused by the inhibition of CK synthesis by auxin.

Five rice orthologs ofMORE AXILLARY GROWTH(AtMAX1):OsMAX1a,OsMAX1b,OsMAX1c,OsMAX2d,andOsMAX1e[65],have been identified,encoding cytochrome P450 monooxygenases,which are involved in SL biosynthesis.Among them,OsMAX1aandOsMAX1ewere highly expressed in rice tissues [70].These two genes responded to phosphate deficiency and response to various phytohormones,in particular SLs.Given thatOsMAX1a-RNAi andOsMAX1e-RNAi plants had more tillers than the WT,it could be concluded thatOsMAX1aandOsMAX1eparticipated in SL biosynthesis and inhibited rice tillering.

DWARF27(D27)encodes an iron-containing chloroplast protein and is expressed in several organs [23].Thed27mutant showed defects in SL biosynthesis,thereby inducing more tillers.Thed27 d10double mutant showed similar phenotypes tod10[22].On treatment ofd27,d10,andd27 d10double mutants with various concentrations of polar auxin transport (PAT) inhibitor NPA,the double mutant showed a similar response tod10,strongly suggesting thatD27participated in the MAX pathway likeD10.Exogenous treatment with rac-GR24 inhibited axillary bud growth and tiller emergence in thed27mutant.These results showed thatD27regulated rice tillering via the MAX pathway and was involved in SL biosynthesis [65].The PAT was much stronger in thed27mutant than in WT plants,and the exogenous PAT inhibitor NPA rescued the multi-tiller phenotype of the mutant,indicating thatD27inhibited rice tillering by inhibiting polar auxin transport,leading to auxin accumulation in apical tissue [23].

DWARF14(D14),also known asHIGH TILLERING DWARF2(HTD2)andDWARF88(D88),is an esterase gene that senses SL signal and negatively regulates the growth of rice axillary buds [71].Under the action of SL,D14 and F-box protein D3 formed a SCFD3-D14 complex,which interacted with the nuclear-located D53 protein,leading to the degradation of D53 by ubiquitination,thereby resulting in tiller inhibition [28].Study [72] showed that the precursor mRNA (pre-mRNA) formed afterD14transcription was spliced by a monocotyledon specific hnRNP like protein DWARF AND HIGH TILLERING1(DHT1),and then further translated to form a protein.The stability and RNA binding activity of the DHT1 protein was impaired whenDHT1was mutated,resulting in a splicing defect ofD14pre-mRNA and the reduction ofD14expression,leading in turn to a SL signal defect.A tiller phenotype similar to that ofd14was observed in thedht1mutant,and showing that the regular splicing ofD14pre-mRNA ensured SL inhibition of rice tillering.

D53,as a transcription factor negatively regulating rice tillering,encodes a protein that is structurally similar to the class I Clp ATPase and is a substrate for the SCFD3ubiquitination complex,degraded by the proteasome pathway [70].The gain-of-function mutantd53was not sensitive to exogenous SL treatment and displayed more tillers and higher yield than the WT owing to an increase in upper tillers.Exogenously applied rac-GR24,led to up-regulation ofD53expression and faster degradation of D53 protein in the WT because the D53 protein inhibited its gene expression [33].However,thed53mutant did not respond to GR24 treatment,indicating that D53 as a negative regulator acted in the SL signaling pathway [24,32].Subsequent study [34] showed that the CKs content in the shoot ofd53mutant was increased.Further experiments showed thatCK OXIDASE/DEHYDROGENASE 9(OsCKX9) was a CK metabolism gene and a primary responder to SL signaling whose response9to SL required the complete function ofD53.

OsMADS57encodes a MADS-box protein.InOsMADS57-OE transgenic plants [35],tiller number was increased,and inOsMADS57-RNAi plants it was decreased.OsMADS57was negatively regulated by miR444a,andD14,the target gene of transcription factorOsMADS57was down-regulated inOsMADS57-OE plants[35].OsMADS57interacted withOsTB1,alleviating the inhibitory effect ofOsMADS57onD14transcription and thereby coregulating rice tillering.OsMADS57-RNAi plants were not sensitive to rac-GR24 [36].These results suggested thatOsMADS57directly inhibitedD14expression and promoted axillary bud growth and subsequent tiller formation via SL signaling.

OsTB1/FC1,a transcription factor of the TCP family,acts downstream of the SL signaling pathway to inhibit lateral bud extension and negatively regulate tiller number in rice.The tiller number ofOsTB1-OE transgenic plants was reduced and that in the loss-offunction mutantfc1was increased[37,73].FC1controls rice lateral branches.In the studies[33,36,37]of the molecular mechanism of Circadian clock,brassinosteroid (BR),SL,IAA,abiotic strains and other factors regulating rice tillering,it was found thatFC1acts as a key regulatory factor.Rac-GR24 inhibited tiller bud growth in WT but not thefc1mutant,suggesting thatFC1may act downstream of SLs [36].Genetic and biochemical experiments [33]showed that both SL and BR signaling pathways control rice tillering by regulating the transcriptional complex of D53 and OsBZR1-RLA1-DLT modules.This study [33] revealed that D53 interacted with OsBZR1,which directly bound to theOsTB1promoter and recruited D53,thus regulatingOsTB1transcription.These findings shed light on the mechanism by which SLs and BRs coordinate the regulation of rice tillering via the early response geneOsTB1.

IDEAL PLANT ARCHITECTUTRE1(IPA1) encodes the squamosalike promoter binding protein SPL14 and is regulated by miR156[25].OsSPL14controls rice tillering during the vegetative growth period [74].In the reproductive growth stage,high expression ofOsSPL14promoted panicle branching [74].In theosspl14mutant,tiller number was decreased,but grain number per panicle,1000-grain weight,lodging resistance,and yield were increased[74].Accordingly,OsSPL14was inferred to be a target gene for constructing the ideal rice plant type.IPA1bined with promoter regions of several genes regulating plant architecture,includingOsTB1,OsDEP1,andWRKY45,thereby increasing rice yield[41,75].OsSHI1,as a gene cloned from the tillering reduction mutantosshi1,interacted withIPA1,and acted upstream ofIPA1,with the function of directly combining the promoters of two target genes ofIPA1,OsTB1andOsDEP1.OsSHI1inhibitedIPA1transcriptional activation activity by affectingIPA1binding toOsTB1andOsDEP1promoters,thereby synergistically regulating rice plant type [76].

CIRCADIAN CLOCK ASSOCIATED1(OsCCA1) is a core control element that adjusts the internal clock to match the external light/-dark cycle.It has the dual functions of conferring early and delayed heading under differing day lengths,negatively regulates rice tillering,and functions in ear development [77].Observations of theoscca1mutant showed that this gene affected the growth rather than the formation of axillary buds.Predicted targets ofOsCCA1included the SL signaling pathway genesOsTB1,D10,D14,andIPA1.Genetic analysis showed thatOsTB1,D14andIPA1acted downstream ofOsCCA1[78].Analysis of SL content and rac-GR24 response in theoscca1mutant andOsCCA1overexpressing plants suggested thatOsCCA1might affect SL signal transduction.The study[78]found that sugar also participates in phytohormone signaling pathways Photosynthetic sugar inhibited the expression ofOsCCA1in tiller buds and promoted its growth.The promotion effect of photosynthetic sugar on rice tillering disappeared whenOsCCA1mutated,indicating thatOsCCA1acted as a sensor of photosynthetic sugar to regulate the growth of tiller buds [79].

Hd3ais a gene with high similarity toFTinArabidopsis thaliana,and affects the heading date of rice under varying temperatures and light conditions.It has the function of forming florigen activation complex(FAC),which affects rice flower formation.It also promotes rice axillary bud growth [80-82].Axillary bud formation inHd3a-RNAi plants did not differ from that in the WT,but dormant axillary buds were promoted for growth.Experiments [81] has shown that Hd3a protein produced in the phloem was transported to axillary buds,where it interacted with 14-3-3 protein and the bZIP domain transcription factor OsFD1 to promote tiller bud growth.However,the expression and transport of various genes involved in regulating SL biosynthesis and signal transduction such asFC1,D10andHd3ain transgenic plants were not affected,indicating that the mechanism of regulation of axillary bud growth mediated by this gene was independent of the SL pathway [82].

Recently [83],researchers conducted a genome-wide association analysis of tillering number per plant in multiple rice germplasm materials from a wide range of sources,combined with haplotype and linkage analysis,and identified a candidate gene,TN1(TILLER NUMBER 1).Compared with the wild-type Japanese rice,the mutanttn1showed significantly increased tiller numbers,andTN1overexpressing lines decreased numbers,indicating thatTN1negatively regulates tiller growth and thus affects the number of tillers in rice.As a gene interacting withTN1,TIF1synergistically regulates downstream gene expression and negatively regulates rice tillering.The expression ofD14,D3,andOsCCA1was positively regulated by TN1 and TIF1.

DNA modification also affected rice physiological activities.OsDRM2encodes DNA methyltransferase,and its regulation of DNA methylation is crucial for rice development during vegetative and reproductive growth stages.The loss-of-function mutantosdrm2showed decreased tiller number,growth retardation,and delayed flowering [26],indicating thatOsDRM2-regulated DNA methylation functioned in regulating tillering and other growth and development processes in rice.

3.2.Regulation of plant hormones in rice tillering

Studies [84-90] on mutants with abnormal tillering showed that plant hormones acted in controlling axillary bud growth and tiller number (Fig.4;Table 3).Auxin,SLs,and gibberellins (GAs)inhibited the growth of rice tiller buds and negatively regulated the formation of rice tillers,whereas BRs and CKs promoted tiller bud growth and positively regulated tiller formation.

Table 3Genes in hormone regulation pathway.

Fig.4.Molecular regulatory network of endogenous hormones regulating tillering in rice.The main endogenous hormones regulating tillering in rice are auxin,CK,GA,SL and BR.Auxin,GA and SL inhibit tillering,whereas CK and BR promote tillering.TDD1 functions in auxin synthesis.OsTIR1,as an auxin receptor,regulates the expression of downstream AUX/IAA genes and further regulates the transcription of OsTB1 to inhibit rice tillering.OsPINs are responsible for auxin polar transport and inhibit tillering by maintaining the apical dominance.Auxin inhibits CK synthesis by down-regulating the expression of OsIPTs,and the regulatory genes OsTB1 and D53 in the SL signaling pathway inhibit CK degradation by inhibiting OsCKXs expression,thereby regulating endogenous CK content and cooperating to regulate rice tillering.GA binds to GID1 receptor and interacts with SLR1 to promote the degradation of SLR1 by upstream gene GID2,thus inhibiting tillering in rice. T20 and MHZ5 are genes that synthesize carotenoids,the precursor substances of SL.After synthesis of SL,they can inhibit the polar transport of auxin and suppress tillering of rice by transmitting signals.D2 and D11 participate in the synthesis of BR,and the DLT-RLA1-OsBZR1 complex operates downstream in the BR signaling pathway,which cooperates with SL signaling pathway to regulate tillering in rice.CK,cytokinin;GA,gibberellin;SL,strigolactone;BR,brassinolactone;PAT,Polar Auxin Transport; TDD1, Tryptophan Deficient Dwarf1; THIS1,High Tillering, Reduced Height, And Infertile Spikelet; D10, DWARF10; D17, DWARF17; D27, DWARF27; D53, DWARF53; OsTB1, O.sativa TEOSINTE BRANCHED1; OsCKX2, O.sativa Cytokinin Dehydrogenase2; OsCKX4, O.sativa Cytokinin Dehydrogenase4; OsCKX9, O.sativa Cytokinin Dehydrogenase9; OsTIR1, O.sativa TRANSPORT INHIBITOR RESPONSE1;OsAUX1,O.sativa AUXIN1;OsIAA6,O.sativa Indoleacetic Acid6;OsIAA20,O.sativa Indoleacetic Aci20;OsIPT,O.sativa adenosine phosphate-isopentenyl transferase;DLT,Doublelumen Tube;OsDRM2,O.sativa Domains Rearranged Methyltransferase2;RLA1,Reduced Leaf Angle1;BZR1,BRASSINAZOLE-RESISTANT1;GID1,GA-INSENSITIVE DWARF1;GID2,GAINSENSITIVE DWARF2;SLR1, SLENDER RICE1;OsGSK2, O.sativa Glycogen Synthase Kinase 2;D2, DWARF2;D11, DWARF11.

3.2.1.Role of auxin in control of tillering

In plants,apical dominance maintained by auxin inhibits the growth of lateral branches.Lateral branches were activated after a plant was decapitated,but inhibited again when auxin was reapplied to the apical site[91].In an auxin-insensitive mutant,axr1inArabidopsis,that displayed a multi-branched phenotype,there was no difference between the mutant and WT in the number of axillary buds formed,suggesting that auxin affected the growth and not the formation of axillary buds [92].Auxin was formed in the apical and young tissues of the plant and was transported down the main stem by polar auxin transport(PAT).Given that in isotope labeling experiments,labeled auxin did not enter the axillary bud,auxin may inhibit the growth of the lateral bud indirectly [84].It was also found [29] that auxin regulates tillering in rice.The rice genome contains 12PINgenes that encoded auxin efflux carriers,including fourPIN1and onePIN2gene [93].OsPIN1,as a polar auxin transporter,acted in the process of rice tillering regulated by auxin.OsPIN1-RNAi transgenic plants produced more tillers owing to interference with auxin-mediated inhibition of axillary bud growth [29].The same phenotype was also found inOsPIN2andOsPIN3t-overexpressing plants[94,95].However,tiller number was decreased inOsPIN5b-overexpressing plants [96].TDD1encodes anthranilate synthetase β,participating in the first step of tryptophan synthesis and acting upstream of auxin synthesis.In thetdd1mutant,auxin content was decreased,and the plant showed dwarfism and fewer tillers [97].OsTIR1,as a rice auxin receptor,is a target gene of miR393 and is negatively regulated by it [30].After being perceived and bound by OsTIR1,auxin has a higher affinity for Aux/IAA protein interactions[30].The degradation of Aux/IAA proteins led to auxin-induced gene expression,leading to auxin signal transmission,regulating plant physiological activities [86].Overexpression ofmiR393promoted the development of tiller buds and the growth of tillers [30].The expression levels ofOsAUX1andOsTIR1were decreased,reducing the perception and transport capacity of auxin and thereby positively regulating tiller growth and development [30].In theOsIAA6knockout mutant,OsPIN1andOsTB1expression was down-regulated and tiller number increased.The phenotype of increased tillers was also found inOsIAA10-overexpressing plants [98,99].Thus,auxin synthesis,polar transport,and signal transduction exerted indirect effects on rice tillering.

3.2.2.Role of second messenger of auxin in control of tillering

In view of the indirect regulation of auxin on rice tillering,researchers believe that there are second messengers to transmit auxin signals to axillary buds.The plant hormone originally found[100] to be the second messenger of auxin was CK.In contrast to auxin,CK promotes the growth of axillary buds.Exogenous auxin did not inhibit axillary bud growth of s,but exogenous CK accelerated it,as did an increase in endogenous CK [87,101].

Auxin regulates the growth of rice axillary buds by affecting the content of CKs.Auxin reduced CK content by down-regulating the expression ofAdenosine phosphate isopentenyltransferase(IPT)[31].As a rate-limiting enzyme,IPTcatalyzesthe first step of CK biosynthesis,and 8IPTgenes have been found in rice [88].Increasing expressions ofIPTgenes could promoted tiller bud growth of and inhibited root growth [102].Auxin also promoted the expression ofcytokinin oxidase/dehydrogenase(CKX) and reduced the level of CK.CKXis a regulator of endogenous levels of CK.In rice,11CKXgenes have been identified [103].OsCKX2encodes a CK oxidase,which degrades CK in plant tissues,and controls the growth of axillary buds and tillering by inhibiting the accumulation of CK in buds.OsCKX2-RNAi plants formed more,andOsCKX2-overexpressing plants fewer,tillers than the WT.The expression levels ofD53andOsTB1,the two negative regulators of axillary bud growth,were unchanged inOsCKX2overexpressing plants.OsCKX2was concluded to inhibit axillary bud growth via a pathway independent ofD53[104].RLB/OSH15encodes a KNOX-type homeobox protein.In therlbmutant,plant height was reduced,effective tiller number increased,and CK content was reduced relative to the WT.RLBbound directly to theOsCKX4promoter and inhibited its expression,thereby inhibiting the degradation of CK and promoting rice tillering [89].

OsCKX9is also a CK oxidase,and the phenotype of increased tillers was found in both its loss-of-function mutant and overexpressing plants [34].OsCKX9did not respond to exogenous CK treatment,in contrast to the other CKX genes,but responded to exogenous SL treatment via aD53-dependent pathway [89].Thus,OsCKX9may be a regulator of the interaction between CK and SL signaling pathway to affect rice tillering [34].

SLs and auxin also cooperate to control rice tillering.Auxin promoted SL biosynthesis,whereas SLs inhibited auxin polar transport[89].In thed27mutant,comparison of auxin contents at the base and top of the mutant and WT showed that polar auxin transport was increased in thed27mutant [22].Exogenous auxin increased the expression ofD10andD17in plants,increased SL synthesis,and inhibited plant branching [21].More tiller buds in athis1mutant grew and formed more effective tillers,indicating thatTHIS1promotes axillary buds.The expression of the auxinresponse geneOsIAA20and SL-synthesis geneD10changed inthis1mutant.These results suggested thatTHIS1may regulate rice tillering formation by affecting the signal pathway of auxin and SL[105].

As a new class of hormones that inhibit plant branching,SL is synthesized in the root and transported into the stem,and then enters axillary buds to regulate their growth directly[66].A series of genes acting in SL biosynthesis or signal transduction have been identified indwarf(d)mutants,with their changed tillering phenotype.They areD10,D17/HTD1andD27involved in SL biosynthesis,andD3,D14/HTD2andD53involved in SL signal transduction[9,14,49,53].Carotenoids are precursors of SLs and ABA,and mutations in the genes involved in its biosynthesis caused failure of synthesis of SLs and ABA,thus affecting plant growth and development [106].T20encodes ζ-carotene isomerase (Z-ISO)and functions in a key step in the carotenoid biosynthesis [13].In thet20mutant,tiller number was increased,and the plant height and SL and ABA contents were decreased.SLs promoted ABA biosynthesis in the stem base,whereas ABA inhibited SL biosynthesis in roots and the growth of tillers [107].Followup study [108]showed that the rice Z-ISO homologHTD12regulated chloroplast development and photosynthesis,thus affecting plant architecture.OsCRTISO/MAO HUZI5(MHZ5)encodes a carotenoid isomerase.Themhz5mutant showed excessive tillers,smaller spikelets,reduced panicle branches,shorter internodes,longer and narrower grain,shorter roots,fewer adventitious roots,and increased lateral roots,further indicating that carotenoid biosynthesis genes too could regulate plant growth and development [108,109].

3.2.3.Role of GA and BR in control of tillering

Gibberellins (GAs) are a large family of tetracyclic diterpenoid plant hormones [110].Breeding by changing GA signaling advanced the crop Green Revolution [90].Plant branches were increased in GA-synthesis or GA signal pathway-defective mutants[1,7,90].In these mutants [1,7,90],plant height was generally inversely proportional to tiller number,giving rise to the phenotype of dwarfism with multiple tillers.There are 10 genes possibly encoding GA2 oxidases in rice with the function of catalyzing the inactivation of active GA.A semi-dwarf phenotype and increased tillers were observed[111]in plants overexpressing GA2 oxidases,another finding confirming the role of GA in inhibiting rice tillering.As a GA receptor,GA-INSENSITIVE DWARF 1(GID1) interacts with DELLA protein,an inhibitor of GA,and acts in the GA signaling pathway.GID1-overexpressing plants showed reduced tillers and increased plant height [112].SLENDER RICE 1(SLR1) encoded a DELLA protein.When GA signal was sensed and received byGID1,it formed a GID1-GA-DELLA complex with DELLA protein,which was further ubiquitinated by E3 ubiquitin ligase SCFSLY1/GID2complex and then degraded by 26S proteasome [113,114].SLR1-RNAi plants showed reduced tillers and greater plant height,whereasSLR1overexpressing plants showed more tillers and shorter stems.Further study [53] showed that GA signal triggered SLR1 protein degradation,leading to the degradation of MOC1 protein and thus inhibiting the formation of rice tillers.This is also the route by which the Green Revolution geneSD1mutation produced dwarfism and multiple tillers,and accounts for the common appearance of the dwarf and multiple-tiller phenotype in GAbiosynthesis and GA-signal transduction defective mutants.

The signal network of brassinolide(BR)regulating plant growth was first established inArabidopsis.Some genes homologous toArabidopsishave also been found in rice,such asBRASSINOSTEROID INSENSITIVE 1/DWARF 61(OsBRI1/D61),GLYCOGEN SYNTHASE KINASE 2(OsGSK2),andBRASSINAZOLERESISTANT1(OsBZR1) [115,-118].However,inArabidopsis thaliana,BR signaling has not been found to regulate plant branching,although changes in tillering traits have been found in some BR-related rice mutants.In rice BR-deficient mutants [33],ebisu dwarf(d2) anddwarf 11(d11),plants with inactivation of the BR signaling pathway,bri1,OsGSK2-OE,OsBZR1-RNAi,dwarf and low-tillering(dlt),andreduced leaf angle 1(rla1),displayed fewer tillers than the WT.Instead,transgenic rice plants with increased BR signaling,includingOsGSK2-RNAi andOsBZR1-OE,all showed multiple tillers [33].Thus,BRs promote rice tillering.

The receptorOsBRI1receives the BR signal and activates a receptor complex,inhibiting OsGSK2 phosphorylation,thereby regulating the expression of downstream genes includingOsBZR1,DLTandRLA1[117,118].DLTencodes a GRAS-family protein.In thedltmutant,plant height and tiller number were decreased,and were insensitive or less responsive to exogenous BRs,indicating thatDLTwas involved in BR signal transduction [119].The transcript accumulation of BR biosynthetic genes in thedltmutant indicated thatDLTfeedback inhibited BR biosynthesis,while exogenous BRs also negatively regulatedDLTexpression.OsBZR1,a key regulator of BR signal,binds to the promoter ofDLT,thereby affectingDLTexpression and regulating rice growth and development [117,119].The interaction between theSMOS1/RLA1andSMOS2/DLT-associated auxin signal pathway with the BR signal pathway leads to their jointly regulating the expression of BR signaling genes,thus influencing rice plant height,cell proliferation and other physiological activities [120,121].

4.Effect of environmental factors on rice tillering

Suitable environmental conditions,including light,temperature,water,and fertilizer,are required for rice growth and development at all stages [122-126].Biological and abiotic stresses in the external environment also affect the normal growth and development of rice.The main environmental factors affecting plant branching include light,temperature,water,and soil fertility.(Fig.5;Table 4).

Table 4Environmental response genes.

Fig.5.Molecular mechanism of rice tillering regulation by exogenous environmental factors.Light activates phyB while inhibiting phyA photoreceptors,thereby inhibiting OsPILs expression to promote tillering in rice.OsPILs bind to the miR156 promoter to inhibit its regulatory effects on downstream genes,releasing IPA1 and inhibiting tillering.Both ammonium and nitrate nitrogen in soil can be used by plants.OsNiR,OsNR2 and NLP4 increase rice nitrogen use efficiency and NLP4 promotesexpression of OsNiR by binding to the NRE motif on the OsNiR promoter,thus promoting tillering. OsTCP19 gene expression is affected by nitrogen content,and it interacts with DLT,a downstream gene of the BR signaling pathway,to inhibit tillering.Photosynthetic sugar inhibits rice tillers by inhibiting OsCCA1 expression and regulating downstream genes.OsPIL11, O.sativa PHYTOCHROME-INTERACTING FACTOR-LIKE11; OsPIL12, O.sativa PHYTOCHROME-INTERACTING FACTOR-LIKE12; OsNiR, O.sativa Nitrite Reductase; OsNR2, O.sativa Nitrate Reductase2; NLP4, NIN-LIKE PROTEIN4; NRE, Nitrate Responsive Cis-element; DLT, Double-lumen Tube; IPA1, IDEAL PLANT ARCHITECTUTRE1; D10, DWARF10; D14,DWARF14; OsTB1, O.sativa TEOSINTE BRANCHED1; miR156, miRNA156; OsCCA1, O.sativa CIRCADIAN CLOCK ASSOCIATED1.

4.1.Effects of light,temperature,and water on rice tillering

Light not only provides energy for plant photosynthesis,but also acts as a signaling molecule to influence the plant to adapt to the external environment.The mechanism by which far red light(FR) and red light (R) as signals regulate plant growth has been studied [123,124].The R/FR ratio in the natural environment will change with the alternation of day and night,the change of seasons and the planting density in the field [125].Plants adapt to these changes by adjusting their morphology.

Phytochrome is the main photoreceptor of R and FR,and there are two forms of absorption: red light (Pr) and far red light (Pfr)[123].Phytochrome transfers light signals via interaction with transcription factors,kinases,phosphatases,E3 ligases,and other light signaling components to regulate plant growth and development [126,127].One of the reasons why planting density affects yield is that the R/FR is changed.When plants are shaded by one another,apical dominance will increase,leading to rapidly upward growth with reduced branching.Auxin accumulated in plants when shading occurred,indicating that light signal regulated plant branching via the auxin signaling pathway [124,128].

InArabidopsis,PhyB was inactivated when shading occurred,leading to the accumulation of phytochrome interaction factors(PIFs) [129].These PIFs bound to the promoter ofmiR156and inhibited its expression,reducing the inhibition by miR156 ofSQUAMOSA promoter-binding protein like(SPL) genes.The releasedSPL9/15transcription factors inhibited branch formation.In rice,twoIPHYTOCHROME-INTERACTING FACTOR-LIKE(PIL) family transcription factors,OsPIL11andOsPIL12,inhibited rice tillering[130].OsPIL11-OE andospil11showed altered tiller number andOsTB1expression.It was confirmed[18]thatOsSPL14exerted transcriptional activation activity onOsTB1and that OsPIL11 interacted with OsSPL14,further explaining how light regulates rice tillering[130].

At the tillering stage,rice plants are more sensitive to water loss than before.If plants are stressed by lack of water,they will respond with various stress reactions,eventually obstructing physiological activities and affecting material transport,energy transfer,and functions of organs,affecting the normal production of tillers [131].An ambient temperature within the range of 30-32 °C is most suitable for rice tillering.Temperatures lower than 16°C or higher than 38°C did not favor tillering[132].At temperatures was lower than 15°C,tillers stopped growing.Research[36]showed thatOsMADS57andOsTB1coordinated the transcription of its target genesOsWRKY94andD14under cold stress,thus changing rice activity from organogenesis to cold-adapted defense.OsMADS57bound to the promoter ofOsWRKY94,a cold-stress defense gene,and activated its transcription,but inhibited its activity at normal temperature.OsWRKY94was directly targeted and inhibited byOsTB1at both normal and low temperature,butOsTB1strengthened the binding ofOsMADS57to theOsWRKY94promoter.However,at low temperature,OsMADS57directly promotedD14transcription to inhibit tillering,whereasD14was inhibited to promote tillering under normal conditions [37].

4.2.Effect of soil fertility in rice tillering

Soil contains nutrient elements,and nutrition is also one of the factors influencing plant branching.High-nitrogen conditions not only promote plant growth and increase tiller numbers,but also break the dormancy of tiller buds and promote the growth of dormant tiller buds to form effective tillers [133].However,under a low phosphorus environment,the number of tillers was reduced,and SL content was increased [133,134].It was suggested that nitrogen,phosphorus,and other nutrients may regulate plant branching by regulating the content of phytohormones such as CKs and SLs.

Soil application of nitrogen fertilizer increases rice tiller numbers.Nitrogen use efficiency (NUE) is one of the factors affecting rice tillering.In recent years,changes in tillering phenotype have been found in many mutants of high NUE genes,providing a new idea for rice high-yield breeding.OsNR2encodes NAD (P) Hdependent nitrate reductase,which has a strong nitrate absorption capacity and high nitrogen utilization rate.In theindicarice 9311,the number of effective tillers ofOsNR2-overexpressing plants was increased,whereas the number of effective tillers ofOsNR2-RNAi plants was decreased,as was plant height.OsTB1,the gene negatively controlling tiller bud formation and elongation,was negatively regulated byOsNR2,another finding suggesting thatOsNR2indirectly participates in the mechanism of regulation by the SL signaling pathway of tillering [135].NIN-LIKE PROTEIN4(OsNLP4)belonging to the NLP family of transcription factors,regulates NUE and the expression of multiple genes in the processes of nitrogen absorption,assimilation,and signal transduction.It is central to the regulation of rice nitrogen metabolism and signal pathways,increasing rice yield and nitrogen utilization at varying nitrogen content.In theosnlp4mutant,a reduced-tillering phenotype was observed,whereas inOsNLP4-overexpressing plants increased tillering was observed,suggesting thatOsNLP4positively regulates rice tillering[136].OsNLP4could bind to the NRE motif of theOsNiRpromoter,and an OsNLP4-OsNiR cascade reaction increased nitrogen assimilation efficiency,tiller number,and rice yield [137].OsNiRencodes a nitrite reductase,which is responsible for reducing nitrite to ammonia [52].InOsNiR-overexpressing plants,tiller number was increased.When the number of NRE motifs in theOsNiRpromoter was increased,its transcription could be readily activated byOsNLP3,increasing nitrogen assimilation and promoting tiller bud growth.InOsNiRtransgenic plants with increased numbers of NRE motifs,the expression of genes involved in SL biosynthesis and signal transduction was decreased,suggesting that a tandem effect of nitrogen and SLs on rice tillering [137].

Teosinte branched 1,Cycloidea,and Proliferating cell factor(TCP)-domain proteins are plant-specific regulators of growth and organ patterning.OsTCP19encodes a Class-I TCP transcription factor and belongs to the TCP transcription factor family.InOsTCP19-overexpressing plants,tiller number was decreased,whereas inOsTCP19-RNAi plants it was increased,indicating thatOsTCP19negatively regulated rice tillering.OsTCP19and its target geneDLTwere regulated by nitrogen in soil.As a gene transcribed in the BR signal transduction pathway,DLTforms a OsTCP19-DLT module withOsTCP19,integrates nitrogen and BR signaling pathways,and regulates rice growth and development by transmitting environmental nitrogen stimuli.The haplotypeOsTCP19-H has the potential to increase rice NUE,which is conducive to plant growth,promoting the formation and growth of tillers [125].

4.3.Effect of sugar on rice tillering

Sugar is the principal energy source for plant growth.The nutrition hypothesis [138] revealed the influence of sugar on the process of plant branching,holding that the axillary buds can be regarded as a source organ and need sugar input to maintain normal metabolic activities and growth.The growth of axillary buds is often accompanied by starch accumulation in the stem and increases in the activity of sugar metabolism enzymes,the amount of sugar absorbed by the axillary buds,and the content of soluble sugar in the juice of axillary buds and xylem,suggesting that axillary bud growth needed carbohydrates to provide energy[138,139].Sugar was also a signal molecule regulating rice tillering.After plants were decapitated,the transport dynamics of isotope labeling in axillary buds revealed that sucrose moved faster than auxin,suggesting that sugar rather than auxin acted as the initial signaling molecule to activate axillary bud growth [125].

In roses,peas,Arabidopsis,and rice,exogenous sugars promoted axillary bud growth,and sugar,as a signal molecule,participated in the regulation of plant branching by SLs[139,140].In roses,exogenous sucrose supply reduced the expression of genes that inhibited bud growth via the SL signaling pathway [140].,thereby integrate sugar signals into the regulatory pathway of biological clock regulating plant growth and development [141,142].In rice,sugars inhibited the expression ofOsCCA1andOsTB1in tiller buds and promoted bud growth dependently onOsCCA1,indicating thatOsCCA1integrates the sugar signaling,biological clock regulation,and SL signaling pathways to affect rice tillering [78].Treatment of plants with exogenousrac-GR24 inhibited tillering,whereas exogenous application of high-concentration sucrose antagonized the inhibition by SLs of rice tillering and rescue the rice tillering phenotype [143].High-concentration sucrose inhibited the degradation of D53 protein,thereby alleviating the inhibition by SLs of tillering.Sucrose increased the level of D53 protein and inhibited the expression ofOsTB1in thed3mutant without exogenous SL,indicating that there was at least one pathway independent ofD3involved in the process of sucrose-induced tillering [143].

5.Conclusions and prospects

Rice tillering has been widely studied by scientists Many genes involved in rice tillering regulation have been identified,functioned at multiple levels (genome,transcriptome,translatome,protein interaction) [27,41,43,68].Great progress has been made in identifying the molecular mechanisms of plant hormone biosynthesis and hormone and other signal transduction pathways,revealing the response of plants to environmental conditions(such as nitrogen and light),[7,10,72,126].The joint regulatory network and control of rice tillering has become more and more clear and comprehensive,and a regulatory network of rice tillering has been established [13,107,119,123,130,133].This paper reviewed the process of tiller formation(Fig.1),and the regulatory role of genes in AM formation and axillary bud elongation (Figs.2,3;Tables 1,2),as well as the molecular mechanism by which hormone signaling molecules and environmental stimuli control tillering (Figs.4,5;Tables 3,4).But our current understanding of rice tillering regulation mechanism is still limited.

Although Chinese rice yields are among the highest in the world,new breakthroughs are needed to continue to improve them.Molecular design breeding is a new technological system and approach to ensure food security,achieving a shift from experiential breeding to targeted,efficient,and precise breeding[144,145].Cloning functional genes is a prerequisite for stepping into the era of molecular design breeding [146].The following advances are needed: (1) The approach of analyzing mutants that can isolate only a single gene has become inadequate,and a effective technology is needed to identify QTLs.Association mapping provides a promising tool for identifying QTLs by detecting DNA molecular marker-trait associations [147-149].Genome-wide association study (GWAS) has emerged as a new approach for high-throughput gene identification in rice.We believe that the identification of tillering related genes can be accelerated by integration of GWAS,association mapping,and gene expression measurement.(2) The pan-genomes from large populations can help researchers facilitate pinpointing of lineage-specific haplotypes for trait-associated genes,and accelerate the process of functional gene mining [150].(3) The discovery of synergistic regulation of tillers by genes and synergistic or antagonistic regulation of tillers by hormones(Fig.4),suggests that we can establish a network that regulates plant architecture by studying the crosstalk between signaling pathways.(4) In addition to identifying the crosstalk between hormones in regulating rice tillering,recent studies have shown that the external environment not only acts as a signal to regulate rice tillering,but also acts together with hormone signaling pathways to regulate downstream gene expression to control tillering.The biological clock,light,sugar,and nitrogen have been found to work together with hormone signal pathways to regulate rice tillering.These studies can lead the way to controlling rice tillering by changing cultivation measures.(5) Panicle number,as a determinant of rice yield,depends directly on tiller number.However,an increase in tiller number does not mean an increase in yield[74].The discovery of rice mutants with increased tiller number and increased yield will make a significant contribution to future production.(6)Research on the molecular mechanism of tillering regulation should be applied to practical production as theoretical study to achieve a transformation from experiential breeding to precision breeding.

CRediT authorship contribution statement

Yuping Yan:Writing -original draft,Visualization.Chaoqing Ding:Writing -original draft,Visualization.Guangheng Zhang:Validation.Jiang Hu:Validation.Li Zhu:Validation.Dali Zeng:Validation.Qian Qian:Conceptualization,Funding acquisition,Project administration.Deyong Ren:Conceptualization,Visualization,Writing -review &editing,Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32071993,32188102,31971872,31861143006,U2004204)and Key Agricultural Technology Project(NK2022010302).