Brassinosteroid signal transduction –choices of signals and receptorsZhi-Yong Wang and Jun-Xian He

Department of Plant Biology, Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA

Small signaling molecules that mediate cell–cell com-

munication are essential for developmental regulation

in multicellular organisms. Among them are the ster-

oids and peptide hormones that regulate growth in

both plants and animals. In plants, brassinosteroids

(BRs) are perceived by the cell surface receptor kinase

BRI1, which is distinct from the animal steroid recep-

tors. Identification of components of the BR signaling

pathway has revealed similarities to other animal and

plant signal transduction pathways. Recent studies

demonstrated that tomato BRI1 (tBRI1) perceives both

BR and the peptide hormone systemin, raising new

questions about the molecular mechanism and evolu-

tion of receptor–ligand specificity.

All multicellular organisms have evolved mechanisms toperceive and respond to extracellular chemical signals,including endogenous hormones and external cues fromthe environment, pathogens and symbiotic organisms.Among these signaling molecules, steroids and smallpeptides are widely used in both animals and plants [1,2].Although plant and animal steroids have many simi-larities in biosynthesis and function, the molecularmechanisms of steroid perception and signal transductionappear to be different in the two kingdoms. The plantsteroid hormones brassinosteroids (BRs) are perceived bythe cell surface receptor kinase BRI1 [3]. By contrast, mostanimal steroid responses are mediated by the nuclearreceptor family of transcription factors [4]. Although someanimal steroid responses are mediated by cell surfacereceptors [5], recent cloning of membrane-bound steroidreceptors in fish and mammals indicated that they aresimilar to the G-protein-coupled receptors [6,7] butdistinct from the BR receptors. However, the BR signalingpathway shares features with peptide hormone signalingpathways in animals and plants. Recent studies in tomatodemonstrated that tomato BRI1 (tBRI1) functions as thereceptor for both BR and systemin, a peptide hormone thatmediates systemic responses to wounding by insect pests[8]. These studies raise the possibility of conservedinteraction among the BR signaling, defense responseand peptide signaling pathways. Our aim here is tohighlight the latest findings in BR and related signalingpathways that can provide some insight into the molecularmechanism of BR signal transduction and plant growthregulation.

Genetic studies identified brassinosteroid signaling

components

BRs are a class of plant steroid hormones with importantregulatory roles in multiple developmental and physio-logical processes, including seed germination, stemelongation, leaf expansion, xylem differentiation, diseaseresistance and stress tolerance [9–11]. BR-deficientand -insensitive mutants show various developmentaldefects, including reduced seed germination, dwarfism,dark-green and curled leaves, reduced fertility, delayedreproductive development, and development of light-grown morphology (de-etiolation) in the dark [3]. Similarphenotypes can be caused by BR biosynthetic inhibitors,such as brassinazole (Brz) [12]. By contrast, overexpres-sion of BR biosynthetic enzymes and the BRI1 receptorincreases cell elongation and plant growth [13,14].

Molecular genetic studies of BR response mutants inArabidopsis have led to the identification of a BR receptorand downstream signaling components. Genetic screensfor BR-insensitive mutants have identified multiple allelesof the bri1 [15,16] and bin2 loci [17–19], which led to theidentification of BRI1 as the BR receptor [16] and BIN2 asa negative downstream regulator [20]. BR-insensitivemutants have also been identified in other species,including pea [3], rice [21], barley [22] and tomato [23],and these mutants have been found to contain mutationsin the BRI1 homologs. The det3 mutant also has dwarf andde-etiolated phenotypes, and is partially insensitive toBR. DET3 encodes a subunit of the vacuolar Hþ-ATPase(V-ATPase), suggesting that V-ATPase activity is involvedin the BR response [24].

Mutants that suppress BR-deficient or -insensitivephenotypes have also been identified in various geneticscreens. An activation-tagging screen for suppressors ofthe weak bri1-5 allele identified genes that promote BRresponse when overexpressed. These include the BRS1gene, which encodes a putative serine carboxypeptidase[25], and the BAK1 gene, which encodes a leucine-rich-repeat (LRR) receptor kinase [26]. BAK1 was alsoidentified as a BRI1 interacting protein [27]. Geneticscreens for mutants insensitive to the BR biosyntheticinhibitor Brz and for bri1 suppressors identified thebrassinazole resistant1-D (bzr1-1D) and bri1 EMS sup-pressor (bes1-D) mutants, respectively [28,29]. Cloning ofBZR1 revealed a close homolog in Arabidopsis namedBZR2, and subsequent sequencing of the BZR2 gene in thebes1-D mutant revealed that mutations of the same aminoacid residue in BZR1 and BZR2 were responsible for theCorresponding author: Zhi-Yong Wang (zywang@andrew2.stanford.edu).

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bzr1-1D and bes1-D mutants, respectively [28,29]. Thesestudies identified BZR1 and BZR2/BES1 as two homolo-gous nuclear proteins that play overlapping yet distinctroles in BR signaling [28,29] (Figure 1).

Brassinosteroid receptors

BRI1 is an LRR receptor-like kinase (LRR-RLK) located onthe cell surface [16]. BRI1 has an extracellular domaincontaining 25 LRRs, a transmembrane domain, and acytoplasmic serine/threonine kinase domain [30,31]. It hasbeen shown that BRI1 immunoprecipitates with BRbinding activity and BR induces autophosphorylation ofBRI1 in vivo [13]. Furthermore, BR binding and kinaseactivation is abolished by a mutation in the extracellulardomain of BRI1 [13]. These experiments demonstrate thatBRI1 perceives the BR signal through its extracellulardomain and initiates a signal transduction cascadethrough its cytoplasmic kinase activity [13,32].

BAK1 is potentially another component of the BRreceptor complex [26,27,33]. BAK1 interacts with BRI1in vitro and in vivo, and they phosphorylate each otherin vitro. Results of both gain-of-function and loss-of-function experiments support a positive role for BAK1 inBR signaling [33]. The molecular mechanism by which BRactivates the receptor kinases is unclear. Although in vivointeraction between BRI1 and BAK1 has been detected,the effect of BR on this interaction is not known. Thus, thehypothesis remains to be tested that ligand binding might

activate the kinases by inducing receptor heterodimeriza-tion, as known for the receptor tyrosine kinases and thetransforming growth factor b receptor kinases in animals[26,33]. Because BR binding activity was not detected forrecombinant BRI1 proteins expressed in non-plant cells[13] and BR treatment did not increase receptor phos-phorylation nor association between BRI1 and BAK1 co-expressed in yeast cells [27], it is believed that eitherproper modification of the receptor kinases or additionalproteins are required for BR binding and signaling. TheBRS1 serine carboxypeptidase has been proposed toproteolytically process an extracellular component of theBR receptor complex [25]. Biochemical purification of allproteins associated with the BRI1–BAK1 receptor kinasecomplex should provide further insight into the molecularmechanism of the BR receptor function.

Downstream brassinosteroid signaling

Although the direct substrates of BRI1 and BAK1 areunknown, components further downstream have beenidentified. BIN2 encodes a cytoplasmic protein kinasehomologous to the Drosophila SHAGGY kinase and themammalian glycogen synthase kinase 3 (GSK3) [20].Genetic studies indicated that BIN2 is a negative regu-lator in the BR signaling pathway, similar to mostSHAGGY/GSK3 kinases in animal systems [18,20]. Twonuclear proteins, BZR1 and BES1, were recently identifiedas positive regulators of the BR signaling pathwaydownstream of bin2. The accumulation of BZR1 andBES1 is increased by BR treatment and by the samemis-sense mutations in bzr1-1D and bes1-D, whichsuppress the bri1 and bin2 mutants [28,29]. BZR1 andBES1 are mostly in phosphorylated forms, and BRtreatment induces dephosphorylation and accumulationof the proteins [28,29].

Biochemical studies indicate that BIN2 directly phos-phorylates and destabilizes BZR1 and BES1 [29,34,35].BIN2 interacts with BZR1 and BES1 in yeast two-hybridassays and phosphorylates them in vitro. In the gain-of-function bin2 mutant, the accumulation of BZR1 andBES1 is decreased and their BR-induced dephosphoryla-tion is attenuated [29,34]. Phosphorylated BZR1 appearsto be degraded by the 26S proteasome, because treatmentof seedlings with the proteasome inhibitor MG132 prefer-entially increased the accumulation of the phosphoryl-ated BZR1 protein [34]. MG132 treatment did not alterthe kinetics of BR-induced BZR1 dephosphorylation,suggesting that BZR1 might be dephosphorylated by aphosphatase [34].

These studies illustrate a BR signal transductionpathway leading from the cell surface receptors to thenucleus (Figure 1). BR activation of the BRI1–BAK1receptor kinases inhibits BIN2 through an unknownmechanism, allowing accumulation of unphosphorylatedBZR1 and BES1, which in turn regulate BR target genes inthe nucleus [29,34]. In the absence of BR, BIN2 kinaseinhibits downstream BR responses by phosphorylatingBZR1 and BES1, and targeting them for degradation bythe proteasome (Figure 1) [34]. The regulation of BZR1 andBES1 degradation by BIN2 phosphorylation is similar toseveral signaling pathways in both animals and plants.

Figure 1. A diagram of the brassinosteroid (BR) signal transduction pathway in

Arabidopsis. BR is perceived by the receptor complex containing BRI1 and BAK1,

which are leucine-rich-repeat receptor-like kinases (LRR-RLKs) that interact with

each other. Activation of the receptor kinases by BR binding leads to the depho-

sphorylation and accumulation of the nuclear proteins BZR1 and BZR2/BES1, poss-

ibly by inhibiting the negative regulator BIN2. In the absence of BR, the BIN2

kinase phosphorylates BZR1 and BZR2/BES1, and targets them for degradation by

the ubiquitin-dependent proteasome pathway. BZR1 and BZR2/BES1 regulate BR-

target genes differently; these targets include the BR biosynthetic gene CPD, which

is feedback inhibited by BR through BZR1, and genes encoding enzymes for cell

wall synthesis that are probably regulated by both BZR1 and BZR2/BES1. The

vacuolar Hþ-ATPase (V-ATPase) is also a mediator of certain BR responses. The

serine carboxypeptidase BRS1 is proposed to process an unknown extracellular

factor that contribute to the activation of the BR receptors.

TRENDS in Plant Science

BRI1

BR

BZR1BZR2/BES1

BRsynthesis

BR

Growth response

CPD

BIN2

BZR1–BZR2/BES1–+

Degradationby proteasome

BR-regulatedgene expression

BAK1

?

V-ATPase

BRS1?

Cell wall enzymes

PP

P

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Particularly, the structural homology between BIN2 andGSK3 highlights the similarity to the Wnt signalingpathway in animals [36]. However, regulated degradationof nuclear factors by the proteasome has been observed invarious plant signaling pathways and has emerged as acommon theme of signal transduction in plants (Table 1).

The biochemical function of BZR1 and BES1 has yet tobe determined. It is unclear whether BR regulatestransport of BZR1 and BES1 into the nucleus [28,29,35].Both bzr1-1D and bes1-D mutants have altered expressionof BR-regulated genes [28,29], but it is not known whetherBZR1 and BES1 directly bind to DNA or regulate geneexpression by interacting with DNA-binding proteins. Thedifferent phenotypes of light-grown bzr1-1D and bes1-Dsuggest that the two proteins have overlapping yet distinctfunctions and thus should have different downstreamtargets. A better understanding of the functions of theBZR1 and BES1 proteins should be achieved by identifyingtheir interacting proteins, which could include thephosphatase that dephosphorylates them, the ubiquitinligase that targets the phosphorylated BZR1 and BES1 forubiquitination and degradation, and possibly transcrip-tion factors that interact with BR-regulated promoters.

Tomato BRI1 has dual functions as receptor of

brassinosteroid and systemin

A similar BR signaling mechanism is apparently conservedin other plants because mutations in BRI1 homologs areresponsible forBR-insensitivemutantphenotypes inpea [3],rice [21], barley [22] and tomato [23]. Interestingly, recentstudies in tomato demonstrated that tBRI1 is not onlyrequired for BR response but also functions as the receptorfor systemin [37], which is a peptide hormone that mediatessystemic wound responses in tomato partly through induc-ing jasmonic acid synthesis [38].

Using radiolabeled systemin, Justin Scheer et al.identified a 160-kDa plasma membrane protein thatbound systemin with high affinity [39], and purified aputative systemin binding protein using photoaffinitylabeling [37]. Surprisingly, the identified protein, SR160,was most homologous to BRI1 in Arabidopsis [37], andmutations in this gene were later found in the tomatoBR-insensitive mutants altered brassinolide sensitivity1(abs1) and curl3 (cu3) [23], indicating that SR160 is theBRI1 ortholog tBRI1 [23].

Scheer et al. have recently confirmed that tBRI1/SR160is the systemin receptor by expressing the tomatosystemin in tobacco and analysing systemin response inthe cu3 mutant [8]. Systemin is present only in members of

the Solaneae subtribe of Solanaceae family, includingtomato and potato, and is absent from tobacco, a member ofthe Nicotianae subtribe. Wild-type tobacco has neitherbinding activity nor response to tomato systemin. How-ever, tobacco cells transformed with the tomato tBRI1/SR160 gene showed systemin binding activity and asystemin-induced alkalinization response, similar to thatof tomato cells [8]. These results indicate that tBRI1/SR160 is sufficient to confer systemin responsiveness ontobacco and that the downstream components of thesystemin signaling pathway are present in tobacco [38].Furthermore, the BR-insensitive tomato line cu3 hasgreatly reduced response to systemin [8]. Although BRdoes not inhibit systemin binding to tBRI1 in suspension-cultured tomato cells [37], it reversibly antagonized sys-temin response in tomato leaves [8]. These studies haveestablished that tBRI1/SR160 functions as receptor for bothBR and systemin [8]. This exciting conclusion raises manyinteresting questions. For example, how can one receptorkinase perceive two hormones with such different physio-logical functions [40], andhow is specificityachieved for bothligand recognition and downstream signaling? Further-more, why does systemin use BRI1 among the hundreds ofLRR-RLKs in plants? Is tBRI1 a special case or are BRreceptors of other plants also bifunctional, perceiving bothsteroidal and peptide ligands?

One receptor for two signals

The only other receptor known to perceive two types ofligands is the mammalian oxytocin receptor (OTR), amember of the G-protein-coupled-receptor family. OTRbinds to both the peptide hormone oxytocin and the steroidhormone progesterone [41]. In this case, oxytocin andprogesterone antagonistically regulate similar physiologi-cal responses by competing for binding to the samereceptor [41]. The regulation of BRI1 by BR and systeminappears to involve a mechanism different from theoxytocin–progesterone systems. First, loss-of-functionmutation of tBRI1 inactivates both BR and systeminresponses, indicating that BRI1 is a positive regulator forboth pathways and should be activated by both ligands [8].Second, BR does not reduce systemin binding [37] but doesinhibit the systemin response [8], suggesting that BRbinds to different ligand-binding sites and inhibitssystemin response by recruiting tBRI1 from the systeminpathway into the BR signaling pathway (Figure 2).

If systemin competes with BR for tBRI1, one mightexpect systemin to act as an inhibitor of growth. Interest-ingly, the transgenic tomato plants that overexpress the

Table 1. Common theme in plant signal transduction – signal for degradation of key nuclear regulators by the proteasome

Signals Nuclear factor and functiona E3 ligase Effect of the signal Refs

Brassinosteroid BZR1 (þ) Unknown Accumulation [34]

Auxin Aux and IAAs (2 ) TIR1 Degradation [55]

NAC1 (þ) SINAT5 Accumulation [56]

Gibberellins GAI, RGA and SLN1 (2 ) SLY1 Degradation [57,58]

Abscisic acid ABI5 (þ) AFP?b Accumulation [59]

Jasmonic acid Unknown COI1 Unknown [60]

Light Hy5 (þ) COP1 Accumulation [61]

aActivation of plant signal transduction pathways often leads to degradation of repressors (2) or accumulation of activators (þ ) in the nucleus by regulating the interaction

between the nuclear protein and specific ubiquitin E3 ligases that promote its ubiquitination and degradation by the proteasome.bThe biochemical function of AFP is unknown.

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pro-systemin gene (35S-Prosys) have significantly longerhypocotyls than wild-type plants [42], suggesting thatsystemin promotes seedling growth. However, the long-hypocotyl phenotype of 35S-Prosys plants is unlikely to becaused by direct activation of the BR signaling pathway.Mutants that suppress the accumulation of proteaseinhibitors also suppress the long-hypocotyl phenotype of35S-Prosys tomato. These include the suppressors ofprosystemin-mediated responses 2 (spr2) mutant, whichis blocked in jasmonic acid biosynthesis [43], suggestingthat prosystemin promotes growth through a jasmonicacid-dependent pathway. Jasmonic acid might affectgrowth directly or by feedback regulating systemin andtBRI1 activity. Jasmonic acid feedback activates prosys-temin [38,44] and systemin binding activity [39] to amplifyand spread the systemic signals efficiently. The specificityof downstream responses might vary with developmentalstages, as known for the dual-functional Drosophila Tollreceptor (see below).

One receptor for two responses

The dual function of tBRI1 is similar to that of theDrosophila receptor Toll, which also has an extracellularLRR domain structurally similar to that of BRI1. Toll isessential for establishing dorsoventral patterning inembryos as well as for innate immune defenses to fungiand bacteria in adult flies [45]. During embryo develop-ment, binding of Toll by its ligand Spaetzle leads toactivation of the Pelle kinase, which is evolutionarilyrelated to the kinase domain of BRI1 [46], and nucleartranslocation of the transcription factor Dorsal. Theasymmetric generation of Spaetzle results in a gradient

of Dorsal nuclear localization, leading to embryonicpolarity [47]. In innate immunity of adult flies, recognitionof bacteria and fungi by the peptidoglycan-recognitionproteins triggers a proteolytic cascade that ultimatelycleaves inactive Spaetzle into a shortened activated form,which activates Toll and leads to the production ofantimicrobial peptides that mediate defense [48]. Inaddition to Spaetzle and Toll, some downstream com-ponents (Tube, Pelle and Cactus) are required for bothdevelopmental and defense responses [45]. Active Spaetzlebinds to Toll directly with high affinity and with astoichiometry of one Spaetzle dimer to two receptors,thus activating Toll by inducing receptor dimerization[49]. The dual function of tBRI1 in developmental anddefense responses is thus similar to Toll. Unlike Toll, whichis activated by the same ligand Spaetzle, tBRI1 apparentlyperceives both BR and systemin. Thus, tBRI1 appears tobe the only receptor known to be activated by two signalsthat lead to two distinct responses.

Perspectives and prospects

LRR-containing receptors are conserved in plants, insectsand mammals for mediating innate immunity [50,51].Homologs of Toll (Toll-like receptors, TLRs) have beenfound in mammals to mediate both innate and adaptiveimmune responses. Mammalian TLRs perceive not onlyexogenous molecules from microorganisms but alsoendogenous agonists such as the degradation products ofmacromolecules, products of proteolytic cascades andintracellular components of ruptured cells [52]. It hasbeen proposed that TLRs represent an ancient mechanismof perceiving environmental challenges and cellular

Figure 2. The similarities between the brassinosteroid (BR) and systemin signaling pathways in tomato and the Toll signaling pathways in Drosophila. (a) The Toll signaling

pathway controls both embryonic development and innate immunity in Drosophila. Toll is a large transmembrane receptor with an N-terminal extracellular leucine-rich-

repeat (LRR) region similar to that of the BRI1 receptor kinase in plants and a C-terminal intracellular (TIR) domain. Toll is activated by binding of its ligand Spaetzle. Spaet-

zle is synthesized as an inactive precursor, which is processed and activated by proteases generated either during embryo development or upon pathogen invasion of adult

flies. The binding of Spaetzle to Toll induces dimerization of the receptor and activation of the downstream kinase Pelle, a serine/threonine kinase evolutionarily related to

the kinase domain of BRI1. Activation of Pelle leads to dorsoventral polarity gene expression during embryo development and defense gene expression in innate immune

responses of adult flies. (b) BR signaling (left) and systemin signaling (right) pathways in tomato. tBRI1 perceives both BR and systemin signals. BR interacts with the extra-

cellular domain of BRI1 and activates the kinase activity of BRI1, initiating a signaling cascade that leads to BR-regulated gene expression and growth responses. The tBRI1

receptor complex might also contain BAK1. Systemin is produced from a precursor protein prosystemin by proteolytic processing upon wounding. Systemin binds to the

tBRI1 to initiate a signaling pathway that leads to systemic defense responses by inducing jasmonic acid (JA) synthesis. Jasmonic acid also feeds back to induce systemin

and tBRI1, further amplifying and spreading the signals. BR inhibits systemin responses, suggesting that the two signals compete for the receptor to regulate developmen-

tal or defense responses.

TRENDS in Plant Science

BR

Cell wall synthesisGrowth responses

BAK1?

tBRI1?

Systemin

Wounding

BR

JA

Prosystemin

Defense response

(b) Tomato(a) Drosophila

Plasmamembrane

Pro-Spaetzle

Spaetzle

Toll

Pelle

Defenseresponse

tBRI1

Embryopolarity

Pathogen ordevelopmentalsignals

damage [52]. Whereas most animals have fewer than adozen LRR receptors, plants have evolved with anexpanded family of more than 500 LRR proteins. Theseinclude ,210 Arabidopsis LRR-RLKs containing a cyto-plasmic kinase domain related to Pelle [46], some of whichare known to function in disease resistance [53] anddevelopmental regulation [54]. The dual function of tBRI1might represent an evolutionarily conserved mechanism.It has been proposed that BRI1 might have a defensive rolethat was co-opted by systemin during evolution in theSolaneae subtribe of the Solanaceae family [38]. Althoughsystemin is only found in a subtribe of Solanaceae plants,various peptides that induce similar cellular responseshave been found in other plants [38], and proteolyticprocessing of peptides has been implicated in BR signalingin Arabidopsis [25]. It is possible that BRI1 in Arabidopsisand other plants also perceives peptide signals and has arole in defense.

Whereas it might be wise for Drosophila to use onereceptor efficiently for two responses, it is surprising thatthe dual functions of tBRI1 have not separated after suchdramatic expansion of this receptor gene family in plants.There is possibly a benefit for coupling BR and woundingsignals with one receptor, such as coordinating cell expan-sion with cell wall synthesis in order to avoid cell rupture.Signals generated by cell damage can regulate either cellwall synthesis or defense responses, perhaps depending onthe developmental stage of the tissue. Systemin mighthave evolved recently from a local wounding signal thatbecame jasmonic acid inducible or from a jasmonic acid-induced peptide that acquired tBRI1 binding activity.

Further studies of BR and systemin signaling in tomatowill shed light on the specificities of LRR-RLKs at thelevels of both ligand binding and downstream signaling.Like the Toll pathway, some of the downstream com-ponents might also be shared between BR and systeminresponses, and it will be interesting to determine whetherBAK1, BIN2, BZR1 and BES1 play roles in systeminsignaling. Conversely, analysis of BR responses of theother systemin-related tomato mutants and identificationof systemin signaling components will provide a betterunderstanding of how the specificity of downstreamsignaling is achieved. The questions about the mechanismof dual function remain to be answered by further studiesof the BRI1 receptor complex. Perhaps a more importantquestion is whether the dual function has been evolution-arily conserved. The wound and BR responses are two ofthe best-studied plant signaling pathways, and theirmerging promises to bring more excitement in the future.

AcknowledgementsWe thank David Ehrhardt, Yu Sun and Zhiping Deng for critical reading ofthe manuscript. Research on BR signal transduction in Z-Y.W.’s laboratoryis supported by Carnegie Institution and the National Institute of Health.

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