Journal of Experimental Botany, Vol. 59, No. 1, pp. 67–74, 2008

Transport of Plant Growth Regulators Special Issue

doi:10.1093/jxb/erm134 Advance Access publication 28 August, 2007

SPECIAL ISSUE REVIEW PAPER

Hormonal control of shoot branching

Veronica Ongaro and Ottoline Leyser*

Department of Biology, University of York, PO Box 373, York YO10 5YW, UK

Received 27 March 2007; Revised 4 May 2007; Accepted 15 May 2007

Abstract

Shoot branching is the process by which axillary buds,

located on the axil of a leaf, develop and form new

flowers or branches. The process by which a dormant

bud activates and becomes an actively growing

branch is complex and very finely tuned. Bud out-

growth is regulated by the interaction of environmental

signals and endogenous ones, such as plant hor-

mones. Thus these interacting factors have a major

effect on shoot system architecture. Hormones known

to have a major influence are auxin, cytokinin, and

a novel, as yet chemically undefined, hormone. Auxin

is actively transported basipetally in the shoot and

inhibits bud outgrowth. By contrast, cytokinins travel

acropetally and promote bud outgrowth. The novel

hormone also moves acropetally but it inhibits bud

outgrowth. The aim of this review is to integrate what

is known about the hormonal control of shoot branch-

ing in Arabidopsis, focusing on these three hormones

and their interactions.

Key words: Auxin, cytokinin, DAD, MAX, RMS, shoot

branching.

Introduction

The plant shoot system is derived from the primary shootapical meristem, which is established during embryogen-esis. The meristem consists of a group of undifferentiatedcells that initiates the leaves of the plant at its flanks andthe elongating stem at its base. The point at which eachleaf connects to the stem is called the node. In the axil ofeach leaf, at the base of the leaf petiole, one or moresecondary axillary meristems can form. The primary shootis formed by the repetition of a stem segment with a nodecarrying a leaf and one or more axillary meristems.

The axillary meristems have the same developmentalpotential as the primary shoot apical meristem, and eachcan therefore form an entire secondary shoot. However,they frequently form only a few leaves before arresting toform a dormant axillary bud. The buds can subsequentlyreactivate, producing a branch. This flexibility in axillarymeristem activity makes possible substantial variation inshoot system architecture, allowing the plant to adapt itsarchitecture to the prevailing environmental conditions.Hence it is not surprising that axillary bud activity isregulated by a wide range of environmental inputs such asthe levels and quality of light (Snowden and Napoli, 2003;Cline, 1996), and nutrient availability (Cline, 1991).These environmental signals are likely to be relayedthrough the action of plant hormones. Of particularimportance are auxin and cytokinin, as well as a newcarotenoid-derived hormone which has yet to be chemi-cally defined, but the existence of which is supported bythe analysis of a series of shoot branching mutants inArabidopsis, pea, and petunia (Beveridge, 2000; Bookeret al., 2005; Simons et al., 2007). This review focuses onthe understanding of the control of shoot branching bythese three hormones.

Auxin

Auxin was the first hormone to be linked to the regulationof shoot branching, and it has been in the spotlight formore that 100 years. It was known that the apex of theplant inhibits axillary bud outgrowth in some waybecause, when the apex is removed, axillary buds thathave been dormant activate and the plant starts branching.The first experiment that linked this phenomenon withauxin was carried out by Thimann and Skoog (1934).They showed that auxin, applied to the top of a decapitatedplant mimics the effect of the removed apex, preventingbud outgrowth.Since Thimann and Skoog, many researchers over the

years have contributed to the understanding of how auxin

* To whom correspondence should be addressed. E-mail: hmol1@york.ac.uk

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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represses bud outgrowth and controls shoot branching(Leyser, 2003, 2005). Indole-3-acetic acid (IAA), the mostabundant type of auxin in plants, has been shown to beabundantly synthesized in the shoot apex and youngexpanding leaves (Ljung et al., 2001); thus removal ofthe shoot apex removes a major auxin source. The auxinis transported basipetally down the shoot in a polarmanner by active transport in the polar transport stream inthe vascular parenchyma (Blakeslee et al., 2005). Severalprotein families are involved in active auxin transport,such as the influx facilitators AUXIN INFLUX CARRIERPROTEIN 1 (AUX1)/LIKE-AUX1 (LAX) proteins (Parryet al., 2004), the p-glycoprotein auxin efflux carriers(PGP) (Geisler and Murphy, 2006), and the PIN-FORMED auxin efflux carriers (PIN) (Paponov et al.,2005). In the Arabidopsis main shoot PIN1 appears to beparticularly important for polar auxin transport, since lossof function of this single gene results in a substantialreduction in transport (Okada et al., 1991). Consistentwith this role, the PIN1 protein is polarly localized in theplasma membrane at the base of xylem parenchyma cells(Galweiler et al., 1998).Although it is clear that apical auxin moving in the

polar transport stream inhibits bud outgrowth, its mech-anism of action in this process is still unclear. It is knownthat radiolabelled auxin applied apically does not enter thebud in any quantity (Prasad et al., 1993; Booker et al.,2003), and in fact the levels of auxin in buds frequentlyincrease when the buds activate (Gocal et al., 1991). Thisis perhaps not surprising given that active shoot apices areprolific in auxin synthesis. Furthermore, direct auxinapplication onto the bud does not inhibit outgrowth(Brown et al., 1979). Thus the auxin moving in the stemmust act indirectly.

Cytokinin

Another hormone that is involved in shoot branching iscytokinin (Ck) (Cline, 1991). In contrast to the indirectinhibitory action of auxin, Cks directly promote budgrowth. Exogenous Ck applied to buds promotes theiroutgrowth (Sachs and Thimann, 1967; Miguel et al.,1998) and Ck levels increase in buds as they activate(Emery et al., 1998).Three different cytokins are the most abundant in higher

plants: isopentenyladenine (iPT), zeatin (Z), and dihydro-zeatin (DZ). Cks are synthesized in both the root andshoot (Chen et al., 1985; Nordstrom et al., 2004) and canmove acropetally through the plant in the transpirationstream in the xylem.

A novel branch-inhibiting hormone

One approach that enables us to understand the complexprocess of shoot branching regulation is the analysis of

mutants. In Arabidopsis, the more axillary growth mutants(max1, max2, max3, and max4); in pea, the ramosusmutants (rms1, rms2, rms3, rms4, and rms5), and inpetunia, the decreased apical dominance mutants (dad1,dad2, and dad3), have each been described (Beveridgeet al., 1994, 1996, 1997; Napoli, 1996; Morris et al.,2001; Stirnberg et al., 2002; Booker et al., 2004, 2005;Sorefan et al., 2003; Snowden et al., 2005; Simons et al.,2007). Loss of function at any of these loci produces morebranching shoots compared with wild-type (WT) (Fig. 1).Furthermore, for a subset of the mutants, including rms1,rms5, dad1, dad3, max1, max3, and max4 the branchingphenotype of the mutant shoots can be restored to wild-type by grafting to wild-type rootstocks, suggesting thatthe mutants lack a graft transmissible branch inhibitor(Napoli, 1996; Morris et al., 2001; Turnbull et al., 2002;Sorefan et al., 2003; Simons et al., 2007). The productionof this inhibitor can occur either in the shoot or in theroot, because wild-type shoots grafted to mutant roots alsohave wild-type branching.In Arabidopsis, double mutant analysis and reciprocal

grafting experiments with max1, max2, max3, and max4demonstrate that the four genes act in the same pathway,with MAX1, MAX3, and MAX4 necessary for theproduction of a proposed novel hormone, and MAX2

Fig. 1. Branching phenotype of 35-d-old WT Arabidopsis and max4mutant.

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involved in its signal transduction (Booker et al., 2005).The reciprocal grafting results suggest that MAX1 acts ona mobile intermediate downstream of MAX3 and MAX4,which are predicted to have immobile substrates.All the MAX genes have been cloned (Stirnberg et al.,

2002; Sorefan et al., 2003; Booker et al., 2004, 2005).MAX3 (At2g44990) was identified as a plastidic caroten-oid cleavage dioxygenase (CCD) (Booker et al., 2004;Auldridge et al., 2006). When expressed in E. coli it hasbeen shown to cleave a range of carotenoid substrates(Booker et al., 2004; Schwartz et al., 2004). MAX4(At4g32810) is also a CCD, although substantially di-vergent from MAX3 (Sorefan et al., 2003). In E. coliMAX4 also shows CCD activity (Auldridge et al., 2006)and is able to cleave one of the products of the MAX3carotenoid cleavage reaction (Schwartz et al., 2004).MAX1 (At2g26170) encodes a cytochrome P450 familymember. MAX1 belongs to the Class III cytochromeP450s and the substrate of this class is often generatedby dioxygenases, consistent with the proposed positionfor MAX1 downstream of MAX3 and MAX4 in thesynthesis of the branching-inhibiting substance. Finally,MAX2 (At2g42620) also known as ORE9 (Woo et al.,2001), belongs to the F-box protein LRR family.Members of this family act as the substrate-selectingsubunit of SCF-type ubiquitin–protein ligases, whichcatalyse the polyubiquitination of selected substrateproteins, usually marking them for degradation by the26S proteosome (Ciechanover et al., 2000; Stirnberget al., 2007). This probable biochemical function is alsoconsistent with the predicted action of this gene in signaltransduction of the novel hormone.Double mutant analysis and reciprocal grafting experi-

ments conducted in pea and petunia reveal a broadlysimilar picture, although with some interesting differences(see below). Consistent with this, the molecular analysisof the MAX genes has allowed a demonstration oforthology between some members of the MAX, RMS, andDAD gene families. Specifically MAX4, RMS1, and DAD1are orthologous (Sorefan et al., 2003; Foo et al., 2005;Snowden et al., 2005), as are MAX3, RMS5 (Johnsonet al., 2006), and possibly DAD3 (Simons et al., 2007).MAX2 is orthologous to RMS4 (Johnson et al., 2006), butin addition in pea there is a second gene, RMS3, predictedto act in the signal transduction of the novel hormone(Beveridge et al., 1996). In petunia, the predicted signaltransduction mutant, dad2, does not affect the petuniaMAX2/RMS4 homologue (Simons et al., 2007), andtherefore an attractive hypothesis is that it is orthologousto RMS3. Thus far there are no mutations in the pea orpetunia MAX1 homologues and no branching mutants inArabidopsis that could be equivalent to rms3 or dad2.Recently, mutations in rice orthologues to MAX3/RMS5and MAX2/RSM4 have been described as resulting inincreased tillering (Ishikawa et al., 2005; Zou et al.,

2006), suggesting that the pathway is conserved widely, atleast in seed plants. Database searches reveal closelyrelated genes across the plant kingdom.

Interactions

Interactions between auxin and cytokinin

The indirect mechanism of action of auxin contrasts withthe direct effects of cytokinin on bud growth. Interest-ingly, there is good evidence that auxin can regulatecytokinin biosynthesis. Application of auxin rapidlyreduces flux through the isopentenyladenosine-5#-monophosphate-independent cytokinin biosynthetic path-way (Nordstrom et al., 2004). This effect of auxin appearsto be mediated by the well-characterized auxin signaltransduction pathway involving the AXR1, TIR1, and theother AFB genes (Leyser, 2006; Quint and Gray, 2006;Teale et al., 2006). For example, the effect of exogenousauxin on cytokinin synthesis is reduced in loss-of-functionmutants in the AXR1 gene (Nordstrom et al., 2004). Thisauxin signalling pathway is also required for inhibition ofbranching. The same axr1 mutants have increased branch-ing and their axillary buds are resistant to the inhibitoryeffects of apical auxin (Lincoln et al., 1990; Stirnberget al., 1999). Using tissue-specific promoters and graftingexperiments, it has been shown that the main site of actionfor AXR1 in bud inhibition is the xylem parenchyma and/orin the interfascicular sclerenchyma of the stem (Bookeret al., 2003). These tissues encompass the main site forpolar auxin transport in the stem (Blakeslee et al., 2005),suggesting that auxin levels in the polar transport stream aremonitored by this pathway and may be read out throughregulation of cytokinin synthesis to regulate bud outgrowth.Consistent with this model, various lines of evidence

suggest that apically derived auxin affects cytokininsynthesis both at the node and in the root, and that thelevel of cytokinin from these sources correlates with budactivity. For example, in pea, it has been reported thatauxin reduced Ck synthesis by repressing the expressionof the genes encoding the cytokinin biosynthetic enzymeadenosine-phosphate-isopentenyl transferase (IPT) in thestem (Tanaka et al., 2006). In chickpeas it has beenreported that, after decapitation, Ck from the rootaccumulates in the bud (Mader et al., 2003); and there isevidence in pea and bean that, after decapitation, thelevels of Ck exported from the root increase, but they canbe restored to the levels found in the intact plant byapplication of auxin to the decapitated stump (Li et al.,1995; Bangerth, 1994; Bangerth et al., 2000).Taken together these data suggest that one mechanism

for auxin-mediated bud inhibition is through down-regulation of cytokinin synthesis, limiting cytokininsupply to the bud and reducing bud outgrowth. Consistentwith this idea, basal supply of cytokinin through the

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transpiration stream can release Arabidopsis buds frominhibition imposed by apical auxin (Chatfield et al., 2000).

Auxin and the novel hormone

An intimate connection between the Arabidopsis MAXpathway and auxin has been proposed. Firstly, maxmutant buds are resistant to the inhibitory effects of apicalauxin (Sorefan et al., 2003; Bennett et al., 2006), suggest-ing that the pathway is required for full auxin-mediatedbud inhibition. Furthermore, there is strong evidence tosuggest that the MAX pathway acts by regulating auxintransport capacity in the main stem. This is achieved atleast in part by modulation of the levels of PIN auxinefflux carriers (Bennett et al., 2006). In max mutants,there is an accumulation of PIN1 protein and the tran-scripts of several other auxin transporters have also beenshown to be up-regulated (Lazar and Goodman, 2006).This results in an increase in auxin transport capacity inthe stem of the mutants (Bennett et al., 2006). Theincrease in transport capacity apparently causes theincreased shoot branching since, if auxin transport levelsare reduced back to WT, either chemically with pharma-cological transport inhibitors or genetically in the pin1mutant background, the characteristic bushy phenotype isrestored to WT. Furthermore, transport inhibitors can alsorestore wild-type auxin responses to max mutant buds.These results link high auxin transport to high shoot

branching. This paradox can be resolved by proposinga model in which the apical meristems on the shoot

compete for limited auxin transport capacity in the stem.The assumptions of the model are that active shoot apicalmeristems must be able to export auxin, and that there islimited sink strength for auxin in the main stem ofArabidopsis. The first assumption is based on a strongcorrelation between bud activity and auxin export fromthe bud that has been known for many years (Morris,1977; Li and Bangerth, 1999). Interestingly, recent resultson the role of auxin in pattern leaf initiation at the shootapex (Reinhardt et al., 2003) suggest an explanation as towhy auxin export may be needed for meristem function.Leaves are initiated in the meristem at the convergencepoint of PIN protein orientation in the epidermal celllayer, suggesting auxin movement towards the site of leafinitiation. As the leaf is specified, additional PIN expres-sion is observed in sub-epidermal layers with orientationsuggesting the internalization of auxin. This domain of PINexpression predicts the future vascularization of the leaf. Itis possible that this auxin internalization is required forcontinued meristem activity and that, in turn, it requiressufficient auxin transport capacity in the main stem to beestablished and maintained. The second assumption—that,in Arabidopsis, the auxin transport capacity in the WTstem is limiting is derived from the analysis of the maxmutants, in which increased transport capacity is accom-panied by increased branching (Bennett et al., 2006).Thus this model suggests a second mode of action of

apically derived auxin in the inhibition of bud outgrowththat relies on competition for limited auxin transportcapacity in the main stem (Fig. 2). In the WT situation,

Fig. 2. Model for MAX pathway function through the regulation of auxin transport capacity. In WT stems the MAX pathway down-regulates auxintransporters; thus auxin transport capacity in the stem is limited and saturated by apically-derived auxin. Buds are unable to export auxin; therefore theycannot outgrow. In max mutants there is more auxin transport capacity in the stem, and this means that the buds are able to export auxin and outgrowth(indicated by the yellow arrow). Green squares represent auxin transporters and the arrows represent auxin that is being carried down the stem.

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auxin transport capacity (for example, PIN protein levels)in the main stem is limited, and becomes rapidly fullyoccupied by apically-derived auxin. Thus new auxinsources, i.e. axillary meritesm, are unable to establish anauxin transport stream out into the main stem. By contrast,in the max mutants, PINs and other auxin transportcomponents over-accumulate and the auxin transportcapacity of the stem is not saturated, allowing most of theaxillary buds to export auxin into the stem and conse-quently to develop into new branches, showing thecharacteristic max bushy phenotype.This additional mode of action for auxin is distinct from

that described above in which auxin concentration in thestem is monitored through the AXR1/AFB pathway andread out to regulate cytokinin production. The indepen-dence of these pathways is supported by the observationthat auxin transport capacity in axr1 mutants is notdifferent from wild-type, and axr1 max double mutantshave additive phenotypes, with respect to both branchnumber and bud auxin response (Bennett et al., 2006).Despite the clear distinction between these two auxin-

related pathways, the situation is further complicated bythe fact that the AXR1/AFB pathway interacts moredirectly with the MAX pathway through auxin-regulatedMAX gene expression. In Arabidopsis, auxin is able to up-regulate a MAX4 promoter–reporter GUS fusion in theroot tip and in the hypocotyl, and this up-regulationrequires AXR1 (Bainbridge et al., 2005). Interestingly, inpea, much stronger auxin regulation of RMS1 and RMS5expression has been observed using quantitative real-timePCR (qRT-PCR) in stem segments (Foo et al., 2005;Johnson et al., 2006). For RMS1, the most dramaticeffects come from the reduction in basal expression levelsby decapitation and hence presumably auxin removal.RMS1 transcript levels can be maintained by the additionof auxin to the decapitated stump. This difference mightbe due to differences in the regulation of the networkbetween pea and Arabidopsis, or it might be due to thedifferent sensitivities of the GUS and qRT-PCR systems.Certainly, it is not possible to detect GUS activity inmost tissues in the MAX4 promoter::GUS reporter lines(Bainbridge et al., 2005), so detecting reductions in expres-sion with auxin removal would clearly not be possible.

The MAX pathway and cytokinin

In pea, there is excellent evidence that the RMS pathwayregulates cytokinin levels through a feedback signal thatcan move from the shoot to the roots (Foo et al., 2007).Xylem sap cytokinin is significantly below wild-type inthe rms1, rms3, rms4, and rms5 mutants, and this effectcan be mediated by the shoot, because when rms4 or rms3scions are grafted to WT rootstocks, this low xylem sapCk from the root is still observed (Beveridge, 2000).Recently, it has been shown that a similarly reduced level

of cytokinin is present in max mutant xylem sap (Fooet al., 2007).Interestingly, in the pea rms2 mutant, which shows the

characteristic increased shoot branching of rms mutants,xylem sap Ck levels are normal or slightly elevatedcompared with WT. This has led to the proposal that theRMS2 gene is required in some way for the action of thedownwardly mobile feedback signal. (Beveridge et al.,1997; Beveridge, 2000; Foo et al., 2007).The idea of a basipetally moving feedback signal that

requires RMS2 for its action is further supported byanalysis of the expression of the RMS1 and RMS5 genesin other rms mutant backgrounds (Foo et al., 2005;Johnson et al., 2006). These genes are massively up-regulated in all the other rms backgrounds except rms2,where levels of RMS1 and RMS5 transcript are reduced.In Arabidopsis, this effect could not be detected usingthe MAX4 promoter::GUS fusion except in the hypocotylof max2 mutants (Bainbridge et al., 2005). Here again,this might be due to differences between the systems orto differences in the sensitivity of the techniques used.Interestinly, in petunia the DAD1 gene, a MAX4 ortho-logue, is up-regulated in the stems of dad mutants but notin the roots (Snowden et al., 2005).

The nature of the feedback signal

As briefly outlined above, the detailed analysis of the peaRMS system has clearly demonstrated the existence ofa basipetally moving feedback signal that regulates bothxylem sap Ck levels and RMS1 and RMS5 transcriptabundance. It is possible that this signal is novel, but analternative hypothesis is that this signal is, in fact, auxin.In this hypothesis, the RMS2 gene would be predicted tobe involved in auxin signal transduction in some way,such that candidate Arabidopsis orthologues would in-clude AXR1, AFB etc. Certainly there are strikingsimilarities between the properties of the feedback signaland auxin, and between the phenotype and geneticbehaviours of the rms2 and axr1 mutants.If the feedback signal is auxin, then the hormonal

network in question might resemble the diagram in Fig. 3.In this model, auxin, produced at the active primary shootapex, is transported down the plant in the polar transportstream. In the stem, it has two distinct effects. One is todown-regulate cytokinin biosynthesis via the AXR1/AFBpathway, and one is to occupy limited auxin transportcapacity, preventing establishment of auxin export fromdormant axillary meristems. In rms1/dad1/max4 mutants,the lack of the unknown upwardly mobile signal results inenhanced auxin transport capacity in the main stem,allowing auxin export from the buds. This results in bothincreased bud outgrowth and increased auxin in the polartransport stream. The increased auxin in the polar

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transport stream would have several consequences. Firstly,it would result in a down-regulation of cytokininsynthesis, and secondly it would result in the up-regulation of RMS1/DAD1/MAX4 expression; both effectsof the predicted feedback signal. In the axr1 mutant, auxinresponse is reduced, such that Ck levels are elevated, andauxin is unable to up-regulate MAX4 expression. Theeffects on branching and bud auxin response are additivewith max mutants (Bennett et al., 2006). These results aresimilar to those observed in the rms2 mutant (Beveridgeet al., 1997; Foo et al., 2005, 2007).Various arguments against the feedback signal being

auxin have been raised, such as rms2, which lacksfeedback regulation, and has higher IAA levels than theother rms mutants (Beveridge et al., 1996; Morris et al.,2001). However, if rms2 is an auxin signalling mutant, likeaxr1, this characteristic would be expected. In addition,measured auxin levels in the rms mutants are near WT(Beveridge, 2000). However, if high auxin content wasrestricted to the polar transport stream as suggested for

Arabidopsis (Bennett et al., 2006), then whole tissuedifferences might be obscured. Auxin transport rates arenot affected in rms or max mutants, but capacity appears tobe affected in both (Beveridge, 2000; Bennett et al., 2006).However, in axr1 and rms2 the transport capacity issimilar to WT.It is worth mentioning that one of the differences

between axr1 and rms2 mutants is that shoot branching inrms2 shoots is restored to wild-type when WT roots aregrafted to the mutant scion (Morris et al., 2001). Incontrast, with axr1, mutant scions cannot be rescued byWT roots (Booker et al., 2003). This difference, andothers such as apparently WT auxin responses of rms2mutants in some assays, demonstrate that there aresignificant differences between the two mutants.

Future directions

The chemical identity of the novel branch-inhibitinghormone is still unknown and it is one of the priorities in

Fig. 3. Model for the hormonal control of shoot branching in Arabidopsis. Auxin is transported down the stem in the polar transport stream. Thecapacity of the stream is regulated by the MAX pathway, in which MAX3, MAX4, and MAX1 act in the synthesis of a novel upwardly mobilehormone that regulates PIN1 levels through MAX2. For bud activation, buds must be able to export auxin and thus MAX-limited auxin transportcapacity in the stem prevents bud outgrowth. In addition, the concentration of auxin in the polar transport stream is monitored by the AXR1/AFBpathway to regulate cytokinin synthesis. High auxin down-regulates cytokinin synthesis, inhibiting bud activation. The model predicts, ratherparadoxically, that because of the increased capacity for auxin transport in the stem of max mutants, there will be high auxin and thus low cytokininin the stem, but none the less increased branching, because of the high transport capacity (see Fig. 2). Orthologues of the MAX genes in pea (RMS)and petunia (DAD) are written in brackets.

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this research area. The analysis of this pathway inArabidopsis, pea, and petunia shows similarities betweenthem, but there are also differences. The analysis of thepathway in a wider range of species, such as rice,Medicago, etc. will allow a better understanding of thepathway and its evolution. Another challenge ahead is theunderstanding of how the environment, such as nutrientsand light quality, feed into the system.

Conclusions

The process of shoot branching is an important determi-nant of the plant’s shape. After buds are formed, decisionsare made within the plant as to whether to produce newbranches or not. These decisions are based on environ-mental signals received, and endogenous signals produced.Many of the molecules that act and interact in this finelytuned network of communication are already known, andnew findings are being incorporated in order to builda complete picture of the control of the shoot branchingprocess. The unlocking of the signal transduction pro-cesses involved is the challenge that was set more thana century ago, and we are still being driven by it today.

Acknowledgements

VO is funded by a BBSRC grant to OL. Thanks to Patrick Crozierfor proofreading the manuscript.

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