Qiushuang Shang,Yaping Wang,Heng Tang,Na Sui,Xiansheng Zhang*,Fang Wang*

a State Key Laboratory of Crop Biology,College of Life Sciences,Shandong Agricultural University,Tai’an 271018,Shandong,China

b College of Plant Protection,Shandong Agricultural University,Tai’an 271018,Shandong,China

c Shandong Provincial Key Laboratory of Plant Stress,College of Life Science,Shandong Normal University,Jinan 250014,Shandong,China

Keywords:Wheat Tillering Genetic control Hormonal regulation Environmental factor

ABSTRACT Tillering contributes greatly to grain yield in wheat.Investigating the mechanisms of tillering provides a theoretical foundation and genetic resources for the molecular breeding of wheat.The regulation of tillering is a complex molecular process that involves a multitude of factors.Little is known about the molecular mechanisms in the wheat genome,although progress has been made in rice.Here we review the developmental characteristics of tillers and summarize current knowledge of the roles of endogenous and environmental factors in wheat tillering.We propose directions for future studies and advanced technologies to be used for gene identification and functional studies.

Contents

1.Introduction .........................................................................................................986

2.Endogenous program regulation of tillering................................................................................987

2.1.Genetic control of tillering ........................................................................................987

2.2.Hormonal control of tillering ......................................................................................987

3.Environmental stimulus control of tillering................................................................................989

4.Future perspectives ...................................................................................................989

4.1.Identifying genes or loci controlling tillering is desirable ...............................................................989

4.2.Investigating the molecular mechanisms of wheat tillering requires more functional studies ..................................989

Declaration of competing interest .......................................................................................990

Acknowledgments ....................................................................................................990

References ..........................................................................................................990

1.Introduction

Wheat(Triticum aestivumL.),one of the first grain crops domesticated by humans,is cultivated worldwide.Wheat development can be divided into vegetative and reproductive periods.During the vegetative period,the repeated production of tillers determines the number of heads produced.Following vegetative development,some tillers enter the reproductive period,generating compound spikes at their tops that bear spikelets that give rise to kernels.Thus,wheat tillering is a critical agronomic trait that determines the number of heads and spikes and consequently affects grain yield[1].Identification of key genes involved in tillering and identifying the molecular mechanisms responsible for the tillering process will advance wheat genetic improvement,potentially providing some insights that can be used by breeders.

Wheat tiller development and leaf emergence on the main stem are typically synchronized.Thus,tillers can be numbered according to the leaves from which they arise [2].In the mature wheat embryo,one axillary bud is generated in the axil of the first leaf primordium.The other,generated in the axil of the coleoptile,is called T0 and usually remains dormant.The tiller growing from the axil of the first leaf primordium is called T1,and starts developing when leaf 3 has fully elongated and leaf 4 is appearing.The secondary tiller emerging from the leaf axil of T1 is consequently called T1.1 and secondary tillers developing from the prophyll of a parent shoot are T1.0,T2.0,and so on (Fig.1).

In wheat,tillers are generated by axillary meristems (AXMs),which are first formed during embryogenesis.Tiller initiation and development in wheat undergo three developmental stages:the generation of an AXM in each leaf axil,the production of leaf primordia from the AXM to form a tiller bud,and consequent growth of the axillary bud to form a tiller[3].The regulation of tillering is a complex process in which numerous interacting factors work together.The establishment of AXMs and the formation of the axillary buds are controlled mainly by genetic factors,whereas bud growth is regulated mostly by a complex network consisting of genetic,hormonal,and environmental factors [3].In this review,the genetic and hormonal control of tillering is classified as endogenous program regulation (Fig.2).

2.Endogenous program regulation of tillering

2.1.Genetic control of tillering

Tillering is quantitatively inherited and influenced by major and many small-effect loci.Quantitative trait locus (QTL) mapping is the main approach to identifying genetic loci underlying this complex trait.As shown in Table 1,several QTL regulating tiller number in wheat have been identified [4–10].

Table 1 QTL for tiller number in wheat.

Tiller inhibition lines or mutants are ideal materials for the study of the molecular mechanisms of wheat tiller development.To date,four genes(tin1,tin2,tin3,andftin)for tiller bud initiation have been mapped,to the respective chromosomes or chromosome arms 1AS,2A,3A,and 1AS [11–15].

Several genes regulating tiller formation in wheat have been identified and characterized by homologous cloning.TaMOC1is a GRAS transcription factor that is likely a functional homolog of riceMOC1,a key regulator of axillary meristem initiation.TaTB1has been shown [16] to coordinate the formation of axillary spikelets during the vegetative-to-floral transition.Gene function is broadly conserved between wheat and rice,but detailed developmental and phenotypic effects are likely to be species-specific.In rice,MOC1is expressed mainly in axillary buds and not in the shoot apical meristem (SAM).TheOsTB1gene negatively regulates lateral tillering and is not detected inmoc1mutants[17].MOC1 interacts with MOC3 and increases MOC3 activation to upregulate the expression ofFON1[18].In contrast,TaMOC1expression has been detected in the epidermal cells of leaf primordia and is subsequently expressed in axillary buds,SAM,and young leaves.TheTaMOC1gene is involved primarily in spikelet development [19].Rice tillering regulatory networks may serve as a reference for those in wheat.

2.2.Hormonal control of tillering

In most grasses,initiated AXMs far outnumber fully developed tillers and bud growth is normally under hormonal regulation,in which auxin and cytokinin play an important role.Auxin is synthesized at the apices and is transported down the polar base to repress tiller bud growth,whereas cytokinin promotes this process[20,21].The growth of wheat tiller buds is regulated by the concentration of IAA and zeatin(ZT)as well as the ratios of IAA to ZT and abscisic acid(ABA)to ZT in tiller nodes[22].External application of kinetin also stimulates wheat tiller bud growth [23].

A third class of hormones that affect tiller number is strigolactones,which negatively regulate tiller number inArabidopsisand rice.WheatTaD27-Bcontrols tiller number by affecting the synthesis of strigolactones[24].In wheat,miR156-TaSPLs regulates tillering by interacting with TaD53,which is a repressor of strigolactones signaling [25].These studies confirm that strigolactones control tillering in wheat.Strigolactones may also interact with auxins to regulate shoot branching [26].There are two hypotheses about the interaction between auxin and strigolactones in branching regulation.The first is that auxin regulates branching by inducing the synthesis of strigolactones,which then act as a second messenger [27].The second is that strigolactones act systemically by modulating the transport of auxin [28].Auxin synthesis and the expression of genes expressed in its signaling pathways are altered inTaD27-RNAi plants,providing further evidence that strigolactones affect the signal transduction and biosynthesis of auxin in wheat [24].

Fig.1.Diagram (left) and photograph (right) of wheat tillering structure.C,coleoptile;L,leaf;T,tiller.

Fig.2.Model of factors regulating tillering in wheat.(a) Endogenous factors.(b) Environmental factors.Auxin,Brassinolides (BRs),Strigolactone (SLs),population density(PD),high temperature(HT)and salinity inhibit tillering,and cytokinin(CKs),gibberellin(GA),Abscisic acid(ABA),nitrogen,and low temperature(LT)promote tillering.The solid and open blue boxes represent respectively identified and predicted genes involved in tillering regulation.

Gibberellin (GA) is especially important in regulating plant height.Tiller number and plant height are normally negatively associated.Plants defective in GA biosynthesis or signaling usually have massive tillers and short culms.Paclobutrazol,an inhibitor of GA synthesis,promotes the onset of tiller buds [29].The RHT-B1 gene is a gain-of-function allele caused by an N-terminal truncation near the DELLA domain and RHT-B1b plants produce much more productive tillers [30].However,the molecular mechanism by which GA influence wheat tillering remains unclear.One report[31] has indicated that GA regulate the biosynthesis of strigolactones and suggested that the two hormones cooperate in the regulation of rice tillering.

Brassinosteroids(BRs)are also involved in the regulation of tiller number.In a recent study [32],BRs and strigolactones antagonistically regulated rice tillering throughFC1(TB1)under control of the D53-OsBZR1 complex.TaD53 physically interacts with TaSPL3 and prevents it from upregulating the expression ofTaTB1in wheat[25].It is unknown whether TaD53 also interacts with BZR1 to inhibitFC1expression,indicating a similar functional relationship between BRs and strigolactones in wheat.

Daily application of ABA to growing wheat plants increased the number of tillers and leaves [33].However,high ABA concentrations in the stem and tillers inevitably lead to tiller decline[34,35].These studies provide evidence that ABA participates in regulating wheat tillering.

3.Environmental stimulus control of tillering

In wheat,tiller number depends on tiller emergence and tiller survival.Several factors affect the emergence and survival of tillers in wheat:plant population density,nitrogen availability,sugar,temperature,and salinity (Fig.2).

Population density influences tillering cessation via two mechanisms.The first mechanism is the change in the red/far-red ratio(R:FR) in light [36].Supplemental FR illumination reduced tiller emergence in light-grown grass seedlings [37].It has been speculated [38] that light quality affects tillering by acting on the stem base,where tillering is inhibited by supplemental low R:FR light irradiation.Second,the growth of tiller buds is correlated with photosynthetically active radiation (PAR) intensity and leaf mass per unit leaf area (LMA) [39].High-density planting reduces PAR intensity and causes earlier cessation of tiller development [40].

Nitrogen fertilizer affects wheat tiller development and determines spikelet number [41].Nitrogen may regulate tiller bud growth by regulating nitrogen metabolism and is redistributed from senescent and aborted tillers to the remainder of the plant to improve survival [42].Nitrogen supply may also alter IAA and cytokinin production in wheat [43,44].

Sugar may play a dual role in activating bud outgrowth.First,sugar plays a trophic role.The sustained growth of the tiller requires a steady supply of sugars.Early cessation of tillering resulted in reduced tiller number intinmutants [45].Precocious internode development in the main shoot inhibited bud growth intinowing to a reduced level of sucrose [46,47].Second,sugar may be a signaling molecule for bud growth [48].Elimination of apical dominance by decapitation usually promotes axillary bud growth.Palatinose,a non-metabolizable sucrose analog,was confirmed [49] to trigger bud growth.Exogenous sucrose inhibited the expression of theBRC1gene,the key transcriptional regulator of bud dormancy,and led to rapid bud release [50].Sugar was reported [51] to act in combination with auxin,cytokinin and strigolactone to regulate the development of axillary buds.The molecular mechanism that regulates sugar levels,and the signaling pathways that mediate sugar perception in tiller buds,remain to be investigated.

Temperature directly influences tiller formation and development.A constant temperature is optimal for tillering and leaf emergence.The rates of leaf emergence and tiller appearance are greatly increased at temperatures ranging from 22 to 25 °C [52].Too higher temperatures can reduce tiller number and kernel numbers,so increasing average global temperatures caused by global warming will reduce wheat yields [53].

FLOWERING LOCUS T(FT)is an integrator of environmental signals that regulate the floral transition in flowering plants[54].High expression ofFT-B1stimulates plant development and consequently leads to fewer tillers owing to earlier maturity.At high temperature,FT-B1OX wheat plants show greatly reduced tiller numbers,andFT-B1null plants generally produce more tillers[55].Low temperature (via the vernalization pathway) mediates the transition from vegetative to reproductive stage in winter wheat [56].

Flowering-associated genes may influence wheat tiller number.Overexpression ofTaZIM-A1caused delayed heading and increased effective tiller number by regulatingTaFTandVERNALIZATION1(VRN1) expression [57].The photoperiod-sensitivity genePpd-1influences tiller number in wheat [58].The later-headingvrn-A1allele was associated with higher tiller number per plant in cultivar Cappelle-Desprez [4].

Wheat has moderate tolerance to salinity,and it has been reported that salinity reduces tiller number per plant [59].The effect of salinity on plant growth and yield has been ascribed to osmotic effect,ion toxicity and nutritional imbalance [60].The osmotic effect reduces total leaf area and eventually leads to reduced tiller number.Studies [61,62] in salt-sensitive and salttolerant wheat genotypes show the accumulation and exclusion of ions under the saline conditions,respectively.The primary and secondary tillers may alleviate salt tolerance by accumulating more sodium and chlorine ions than mainstem tillers.The primary and secondary tillers of a salt-tolerant genotype displayed greater exclusion of harmful ions during the reproductive growth stage than did those of the salt-sensitive genotype [63].The molecular mechanism by which salinity affects tiller development is unknown.QTL for tiller number were associated with theVRN-A1gene on the long arm of chromosome 5A in both saline and control soils [4,64].

4.Future perspectives

4.1.Identifying genes or loci controlling tillering is desirable

Despite great efforts to elucidate the control of wheat tillering at the molecular level,only a few QTL or genes have been identified to date in the complex wheat genome (Table 1).A challenge is to identify more QTL and determine the responsible genes.For early QTL mapping analyses in wheat,the problem was the scarcity and low coverage of molecular marker[65].The rapid development of wheat genome sequencing has led to the construction of highdensity SNP chips,which contain abundant DNA markers.Genome-wide association study (GWAS) has emerged as a new approach for high-throughput gene identification in wheat.Transcriptome-wide association studies (TWAS) offer increased power for confirming functionally relevant loci by leveraging expression quantitative trait loci,which can identify candidate genes at loci identified previously by GWAS[66].We propose that the integration of TWAS,GWAS,and gene expression datasets will accelerate the identification of gene–tillering associations.

Laser capture microdissection (LCM) technology can harvest a collection of target cells from microscopic regions in tissue samples.This technology can be deployed for the collection of special cells from wheat axillary bud sections.Combined with RNA sequencing techniques,it will be a powerful tool for examining genome-wide expression profiles of tiller development in wheat.

N6-methyladenosine (m6A) modification is concentrated mainly in meristems and functions in regulation of many agronomic traits [67].m6A modification may thus influence the tiller generation process.Single-cell RNA sequencing can identify the transcripts expressed in specific types of cells[68].The emergence and development of single-cell sequencing methods make it possible to obtain m6A-modified dynamic maps at the cellular level during the tiller generation process.

4.2.Investigating the molecular mechanisms of wheat tillering requires more functional studies

As an allohexaploid plant,common wheat has three copies of most genes in its genome.There may be functional redundancy among homoeologous genes.RNA interference (RNAi) is a useful tool for studying gene function in wheat.We can effectively silence homoeologous genes with a single RNAi construct.In contrast to RNAi technology,CRISPR-based editing allows targeting not only a single gene,but all three homoeologs and tandem gene clusters.Mutants created via CRISPR-based editing can be directly used for crop production or as prebreeding materials [69].CRISPR-based editing is powerful for the analysis of key tillering-related genes.

In conclusion,although our knowledge of wheat tillering is still limited,advanced technologies such as GWAS,LCM,and CRISPRbased editing may lead to the identification of genes that control wheat tillering.As more regulatory genes are identified and studied,the genetic network that controls wheat tillering will be revealed,and it will facilitate the improvement of wheat cultivars by improving wheat tillering.

CRediT authorship contribution statement

Fang Wang and Xiansheng Zhang:planned and designed the manuscript.Heng Tang and Na Sui:drew figures.Fang Wang,Qiushuang Shang,and Yaping Wang:wrote the manuscript.

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 Major Research Plan of the National Natural Science Foundation of China (91935302),the National Natural Science Foundation of China (31971812),and Major Basic Research Project of Shandong Natural Science Foundation (ZR2019ZD15).