Benefits of brassinosteroid crosstalk

Review

Benefits of brassinosteroid crosstalk Sikander Pal Choudhary1,2, Jing-Quan Yu1, Kazuko Yamaguchi-Shinozaki3, Kazuo Shinozaki4 and Lam-Son Phan Tran5 1

Department of Horticulture, Zhejiang University, Hangzhou 310058, China Department of Botany, University of Jammu, Jammu 180003, India 3 Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan 4 Gene Discovery Research Group, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan 5 Signaling Pathway Research Unit, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan 2

Brassinosteroids (BRs) are a group of phytohormones that regulate various biological processes in plants. Interactions and crosstalk between BRs and other plant hormones control a broad spectrum of physiological and developmental processes. In this review, we examine recent findings which indicate that BR signaling components mainly interact with the signaling elements of other hormones at the transcriptional level. Our major challenge is to understand how BR signaling independently, or in conjunction with other hormones, controls different BR-regulated activities. The application of a range of biotechnological strategies based on the modulation of BR content and its interplay with other plant growth regulators (PGRs) could provide a unique tool for the genetic improvement of crop productivity in a sustainable manner. BRs regulate plant growth and stress responses Over the past decade, extensive research efforts have characterized all components of the BR signal relay from the site of its perception to the ultimate point of the regulation of gene expression. Intensive genetic and biochemical strategies have been used to elucidate the complete core of the BR signaling cascade [1,2]. In the past few years, the BR metabolism and signaling pathway have become a major focus of plant biology research [1,3]. Recent studies have demonstrated the significance of BR homeostasis for maintaining normal plant growth [2,4]. Crosstalk and interactions between BRs and other PGRs occur through either the modification or intersection of their primary signaling cascades and function to regulate a large and diverse array of biological processes [5–10]. In turn, the execution of such a myriad of activities depends on BR signal integration with various transcription factors (TFs) that are implicated in the regulation of several developmental and physiological processes, including responses to abiotic and biotic stresses [6]. Synergistic BR and auxin (Aux) actions have been attributed to their signaling elements, such as BRI1 and ARF2 (see Glossary) [2,6]. Recently, BR signaling components have been shown to play a role in innate immunity and stomatal development in Arabidopsis thaliana [11–14]. BRs have also been demonstrated to regulate biotic and abiotic stress responses in Corresponding author: Tran, L S ([email protected]).

594

plants [14–21]. In recent years, there has been a major increase in the utilization of BRs in agricultural applications as a means to boost crop productivity and stress tolerance [15–18,22]. In this review, we highlight the most recent advances regarding our understanding of BR metabolism and BR signaling. In addition, we examine the roles played by BRs, either alone or in cooperation with other PGRs, such as abscisic acid (ABA), Aux, cytokinins (CKs), ethylene (ET), jasmonic acid (JA), salicylic acid (SA) and gibberellins (GAs), in plant growth and development and in abiotic and biotic stress responses. Furthermore, we will also briefly discuss the implications of this knowledge for plant biotechnology. BR metabolism, perception and the execution of BR signaling BR metabolism BR metabolism encompasses the synthesis of bioactive BRs to conversions, modifications or the addition of side chains resulting in the change of bioactive BRs to biologically inactive forms [1,23–25]. Various enzymatic steps in BR biosynthesis and catabolism, such as glycosylation, hydroxylation and sulfonation, are depicted in Figure 1. Perception and execution of BR signaling The signal transduction of BRs has been extensively studied. In plants, the BR signaling cascade involves the perception of the BR signal and further downstream relay of events leading to BR-induced gene expression [1–3,26–30] (Figure 2). The BR signal is perceived by BRI1, a leucinerich repeat (LRR) receptor-like kinase (RLK), which functions with its coreceptor BAK1/SERK3 in BR signaling. BRI1 has a large extracellular ligand-binding domain of 25 LRRs and a 70-amino acid island domain between LRR20 and LRR21, a transmembrane domain and a cytoplasmic domain with kinase activity [1,3]. In addition to its role in BR signaling, BAK1 is also involved in light signaling, cell death control and innate immunity [11,12,14,31]. The SERK1, SERK2 and SERK4 also participate in the early steps of BR signaling [32]. In the absence of BRs, BRI1 remains an inactive homodimer owing to an interaction between its cytoplasmic domain and the negative regulator BKI1, preventing the heterodimerization of BRI1 with BAK1 [1,2,33]. In the presence of BRs, BR binding at

1360-1385/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2012.05.012 Trends in Plant Science, October 2012, Vol. 17, No. 10

Review Glossary 14-3-3 proteins: conserved regulatory proteins found in all the eukaryotic cells. ABI1 (ABSCISIC ACID-INSENSITIVE 1): a gene encoding a protein phosphatase 2C involved in abscisic acid (ABA) signal transduction. ABI2 (ABA-INSENSITIVE 2): An ABI1 homologous gene encoding a protein phosphatase 2C involved in ABA signal transduction. ACC: 1-aminocyclopropane-1-carboxylic acid. ACC oxidase: catalyzes the formation of ethylene from ACC. ACS (ACC SYNTHASE): enzyme involved in ethylene synthesis. ARF (AUXIN RESPONSE FACTOR): transcription factor regulating auxinmediated transcriptional activation–repression. AtIWS (Arabidopsis thaliana INTERACT-WITH-SPT6): an evolutionarily conserved protein involved in RNA polymerase II (RNAPII) post-recruitment and transcriptional elongation processes. BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1): also known as SERK3, a member of the somatic embryogenesis receptor kinase (SERK) family, acting as a coreceptor of BRASSINOSTEROID INSENSITIVE 1 (BRI1). BES1 (BRI1-EMS SUPRESSOR 1): transcription factor responsible for BRregulated gene expression. bes1-D (BES1 dominant): mutant with enhanced BES1 signaling. BIK1 (BOTRYTIS-INDUCED KINASE1): the membrane-anchored BIK1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. BIM1 (BES1-INTERACTING MYC-LIKE 1): basic helix–loop–helix (bHLH) transcription factor involved in BR signaling and embryonic patterning in Arabidopsis. BIN2 (BRASSINOSTEROID INSENSITIVE 2): a glycogen synthase kinase with negative regulation in BR signaling. BKI1 (BRI1 KINASE INHIBITOR 1): inhibits heterodimerization of BRI1 with BAK1. BL (Brassinolide): a highly active form of BR. BR6OX (BRASSINOSTEROID-6-OXIDASE): such as CYP85A1 and CYP85A2 implicated in BR biosynthesis. BRI1 (BRASSINOSTEROID INSENSITIVE 1): a leucine-rich repeat receptor-like kinase that perceives the BR signal. BRX (BREVIS RADIX): root meristem growth regulator, an auxin-responsive gene. Brz (Brassinazole): a BR biosynthesis inhibitor. BSK1 (BR-SIGNALING KINASE 1): a member of the BSK family that activates BR signaling downstream of BRI1. BSU1 (BRI1 SUPPRESSOR 1): a phosphatase involved in BR signaling. BU1 (BRASSINOSTEROID UPREGULATED 1): encoding a bHLH protein that is involved in BR signaling and controls bending of the lamina joint in rice. BZR1 (BRASSINAZOLE RESISTANT 1): a master regulator controlling BRrelated gene expression. bzr1-D (BZR1 dominant): mutant with enhanced BZR1 signaling. CDG1 (CONSTITUTIVE DIFFERENTIAL GROWTH 1): a receptor-like cytoplasmic kinase implicated in the inactivation of BIN2 through dephosphorylation. CESTA: a bHLH transcription factor positively regulating BR biosynthesis. ChIP (chromatin immunoprecipitation): method used to investigate the interactions between proteins and DNA in the cell. CKX3 (CYTOKININ DEHYDROGENASE/OXIDASE 3): CKX3 catalyzes the degradation of cytokinins. COI1 (CORONATINE INSENSITIVE1): an F-box protein required for jasmonic acid responses. COR15A (COLD-REGULATED 15A): cold-responsive gene in Arabidopsis. CPD (CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM): a hydroxylase involved in BR biosynthesis. cps1-1: mutant of ent-copalyl diphosphate synthase, a gibberellin (GA) metabolic enzyme. d35: mutant of ent-kaurene oxidase enzyme involved in early step of GA biosynthesis. DELLA proteins: negative regulators of GA signaling, containing the DELLA motif of 17 amino acids in their N-terminal region. DET2 (DE-ETIOLATED 2): a key enzyme of the BR biosynthetic pathway. DWF1 (DWARF1): catalyzes the rate-determining step in BR biosynthesis. DWF4 (DWARF4): catalyzes the rate-determining step in BR biosynthesis. DWF5 (DWARF5): encodes a putative sterol delta-7 reductase involved in BR biosynthesis. EBR (24-epibrassinolide): a biologically active type of BR. Eix (Ethylene-inducing xylanase): Eix serves as receptor for the fungal elicitor. ELF6 (EARLY FLOWERING 6): involved in flowering time in Arabidopsis. EMS (ETHYL METHANESULFONATE): a compound that is often used in mutagenesis. ERD10 (EARLY RESPONSIVE TO DEHYDRATION 10): implicated in stress tolerance and induced by water stress, ABA and cold stress. EXP (EXPANSIN): cell wall-loosening protein. FCA (FLOWERING CONTROL LOCUS A): a flowering time control gene in Arabidopsis that encodes a protein containing RNA-binding domain.

Trends in Plant Science October 2012, Vol. 17, No. 10

FER (FERONIA): receptor-like kinase mediating male–female interactions during pollen tube reception. FLC (FLOWERING LOCUS C): a MADS [MCM1, AGAMOUS, DEFICIENS and SRF (serum response factor)] domain transcription factor that acts as a repressor of flowering. flg22 (FLAGELLIN 22): a microbial-associated molecular pattern. FLS2 (FLG-SENSING 2): a binding receptor for flg22. GA20x (Gibberellin 20-oxidase): implicated in feedback regulation of GA biosynthesis. GASTs (Gibberellin-stimulated transcripts): genes whose expression is stimulated by GAs. GATA: GATA-binding transcription factors that contain either one or two highly conserved zinc-finger DNA-binding domains. GhGAI1 (Gossypium hirsutum gibberellic acid-insensitive 1): gene encoding a DELLA-type repressor of GA signaling in cotton. GUS: a b-glucuronidase-encoding gene that is usually used to make promoter:GUS fusion constructs for histochemical assays. IPT (ISOPENTYL TRANSFERASE): catalyzes the rate-limiting step of isoprenoid cytokinin biosynthesis. LD (LUMINIDEPENDENS): a gene involved in the control of flowering time in Arabidopsis. MAMP (MICROBIAL ASSOCIATED MOLECULAR PATTERNS): highly conserved molecular signatures that are recognized by cells of the innate immune system. MAPK (MITOGEN-ACTIVATED PROTEIN KINASE): implicated in the broad regulation of multiple physiological and defense responses in plants. NaJAR4 (Nicotiana attenuta JASMONATE RESISTANT4): enzyme catalyzing the joining of isoleucine (Ile) to JA to form JA-Ile. NaJAR6 (Nicotiana attenuta JASMONATE RESISTANT6): enzyme catalyzing the joining of isoleucine (Ile) to JA to form JA-Ile. NaTD (Nicotiana attenuta threonine deaminase): NaTD catalyzes the conversion of threonine to isoleucine in N. attenuata. NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1): a master regulatory protein of plant disease resistance. OsGSK1 (Oryza sativa GLYCOGEN SYNTHASE KINSASE1): a rice ortholog of Arabidopsis BIN2. OsGSR1 (Oryza sativa GAST family gene in rice 1): a member of GAST family in rice. OsNPR1 (Oryza sativa NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1): a master regulatory protein of plant disease resistance. OsSLR1 (Oryza sativa Slender Rice 1): a repressor of GA signaling in rice. OsWRKY45 (Orzya sativa WRKY): a WRKY transcription factor playing a crucial role in benzothiadiazole-induced blast resistance in rice. PCF: promoter-linked coupling element-binding factor. PIN (PIN-FORMED): a group of transmembrane proteins with a predicted function of secondary transporters and a rate-limiting role in the catalysis of auxin efflux from cells. PP2A (Protein Phosphatase 2A): a phosphatase involved in regulation of BR signaling. psc1 ( partially suppressing coi1): partial suppressor mutant of coil1. PYK10 (PHOSPHATE STARVATION-RESPONSE 3.1): encodes a hydrolase that hydrolyzes O-glycosyl compounds in Arabidopsis. RD22 (RESPONSIVE TO DESSICATION 22): gene implicated in stress tolerance and induced by water stress, ABA and salt stress. RD29A (RESPONSIVE TO DESSICATION 29A): gene implicated in stress tolerance and induced by water stress, ABA, cold and salt stresses. REF6 (RELATIVE OF EARLY FLOWERING 6): a repressor of FLC that is involved in histone demethylation and deacetylation. RNAi (ribonucleic acid interference): a technology used to repress the activity of given gene(s) in the cell. ROP (RHO OF PLANTS): Rho family GTPases implicated in auxin transport. ROT3 (ROTUNDIFOLIA 3): a BR biosynthetic pathway enzyme. sax1 (HYPERSENSITIVE TO ABSCISIC ACID AND AUXIN): a BR biosynthetic mutant with hypersensitivity to ABA and auxin. SBI1 (SUPPRESSOR OF BRI1): a leucine carboxyl methyltransferase involved in methylation of PP2A to deactivate BRI1. SDG (SET domain-containing group): SDG genes encode H3K36 methyltransferases, which regulate the methylation of histone lysine residues in plants. SERK family: somatic embryogenesis receptor kinase family to which the BAK1/SERK3 and its homologs (SERK1, SERK2 and SERK4) belong. SET [Su(var)3-9/E(z)/Trithorax]: SET domain is a conserved amino acid motif of proteins that are implicated in the epigenetic control of gene expression. SPCH (SPEECHLESS): a basic helix–loop–helix (bHLH) stomatal initiating factor. TCP1 [TEOSINTE BRANCHED 1, CYCLOIDEA and PCF1 (promoter-linked coupling element-binding factor)]: TCP1 is a TCP domain-containing transcription factor involved in the regulation of DWF4 expression. Waito-C (GA biosynthetic mutant): mutant of GA biosynthetic 3b-hydroxylase enzyme. YDA: also known as YODA, a MAPK Kinase Kinase (MAPKK) negatively regulating stomatal development.

595

Review

Trends in Plant Science October 2012, Vol. 17, No. 10

BR homeostasis

BR biosynthesis

BR biosynthesis Aux

TCP1

BZR1

Active 24epiBR

CESTA

26

Sulfate addition

tio

AtST1

yla

x

BRX

o dr

Hy

Inactive BL and CS or 24-epiBR

Active BL and CS

C-

BEN1, C YP72C1 Competition to and CS prec BL ursors

n

AtST4a

at

n ti o yla os 3 2 yc Gl at C C5 3 T7 C6 UC T73 UG

Active BL and CS

1

4A

3 P7

CY

Inactive BL and CS

Inactive BL and CS

BR homeostasis BR homeostasis

OH

OH

DE T2

OH

22-HCR

ROT3 CYP90D1 T3 1 RO 90D OH P Y OH C

OH

DE

OH

T2

OH O

22-d-dTE

H

OH

DWF 4

O

ROT3 CYP90D

1

H

22-H-ergostan3-one

OH

HO

H

OH

H

O

HO

dCT

H

dTE

H

OH

OH

OH

OH

OH

OH

OH

CPD ROT3

HO

dDT

HO

dTY

H

HO

H

DE

T2

CN

CY CY P85 P8 A1 5A 2

3-epi-6dCT

H OH

DWF4 HO

BR biosynthesis

22-dHCR

CYP85A1 CYP85A2

DWF4 HO

dCS

OH

CYP85A1 CYP85A2 HO

CYP85A1 CYP85A2

CR OH

HO

HO HO

6-oxo-CN

HO

H O

CT

OH

OH H

TE

H

OH

DT

P8

BL

2

CY

OH HO

HO O

OH

OH

OH O

O

HO

O

5A

OH

CPD ROT3

OH

HO

H

HO O

TY

HO

CS

TRENDS in Plant Science

Figure 1. Proposed model for brassinosteroid (BR) biosynthesis and BR homeostasis in Arabidopsis. BR biosynthesis: DET2 (DE-ETIOLATED 2) catalyzes the conversion of campesterol (CR, precursor of BRs) to campestanol (CN); DWF4 (DWARF4) catalyzes the conversion of CN to 6-oxocampestanol (6-Oxo-CN); DWF4 brings conversion of CN and 6-Oxo-CN to 6-deoxocathasterone (dCT) and cathasterone (CT); CPD (CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM) and ROT3 (ROTUNDIFOLIA 3) catalyze the formation of 6-deoxoteasterone (dTE) and teasterone (TE) from dCT and CT, respectively; CYP85A1 and CYP85A2 catalyze the formation of 3-dehydro-6deoxoteasterone (dDT), 3-dehydroteasterone (DT), 6-deoxotyphasterol (dTY), typhasterol (TY), 6-deoxocastasterone (dCS) and castasterone (CS) in three independent steps; CYP85A2 catalyzes the formation of brassinolide (BL, an active BR) from CS; DWF4 also catalyzes the conversion of CR to 22-hydroxycampesterol (22-HCR); ROT3 and CYP90D1 bring the conversion of 22-HCR to 22,23-dihydroxycampesterol (22-dHCR); DET2 catalyzes the formation of (22S, 24R)-22-hydroxy-ergostan-3-one (22-H-ergostan3-one) and 3-dehydro-6-deoxoteasterone (22-d-dTE) from 22-HCR and 22-dHCR; 22-H-ergostan-3-one isomerizes to 3-epi-6-deoxocathasterone (3-epi-6dCT); ROT3 and CYP90D1 catalyze the formation of dTY from 3-epi-6dCT; CYP85A1 and CYP85A2 catalyze the formation of dDT from 22-d-dTE. In BR homeostasis, the strong repression of CPD and DWF4 genes encoding key BR biosynthetic enzymes by BZR1 (BRASSINAZOLE RESISTANT 1) represents an intrinsic BR homeostatic control. DWF4 is under positive regulation of the TCP1 (TEOSINTE BRANCHED 1, CYCLOIDEA and PCF1). Auxin (Aux)-induced transport of BRX (BREVIS RADIX) to the nucleus also promotes CPD and DWF4 expression in Arabidopsis roots. CESTA positively regulates BR biosynthesis by stimulating the expression of CPD. BR homeostasis is also maintained through inactivation of active brassinolide (BL) and CS by hydroxylation at the C-26 position by CYP734A1. Glycosylation at C-23 by uridine diphosphate (UDP)-glycosyltransferases (UGTs), namely UGT73C5 and UGT73C6, also renders BRs inactive. CYP72C1 binding to BL precursors inactivates the BL precursors to maintain BR homeostasis. The bri1-5 ENHANCED 1 (BEN1) encoding a protein homologous to dihydroflavonol 4-reductase and anthocyanidin reductase regulates BR homeostasis by reducing the synthesis of active BRs by competing with BL and CS precursors for the active site on BR biosynthesis enzymes. Sulfonation of BL, CS and 24-epibrassinosteroids (24-epiBR) by Arabidopsis sulfotransferases (AtST), namely AtST4a and AtST1, also renders BRs nonfunctional.

LRR21 induces partial BRI1 kinase activity that phosphorylates BKI1 on Tyr211, resulting in its dissociation from the membrane. The removal of BKI1 initiates the association and sequential transphosphorylation of BRI1with BAK1 [34]. Moreover, detached BKI1 can enhance BR signaling by antagonizing 14-3-3 proteins. The 14-3-3 proteins are involved in the binding and cytoplasmic retention of BZR1 and BES1 (also named as BZR2), the two master TFs of BR signaling [33,35–38]. Activated BRI1 phosphorylates BR signaling kinases (BSKs) at Ser230 and the CDG1 at Ser234, which 596

subsequently activate the BSU1 phosphatase, which in turn inactivates BIN2 through dephosphorylation [1,39,40]. Dephosphorylation relieves BIN2 suppression on BZR1 and BES1. This results in the accumulation of dephosphorylated BZR1 and BES1, which move to the nucleus to regulate BR-related gene expression directly or via an interaction with other TFs, such as AtIWS, BIM and GATA-binding TFs [6,41–44]. A recent study has indicated that SBI1 and PP2A are also involved in BR signaling. Specifically, methylated PP2A dephosphorylates BRI1, whereas BR-induced SBI1 methylates PP2A

Review

Trends in Plant Science October 2012, Vol. 17, No. 10

BRI1

BRI1

– BRs

+ BRs

BRI1:BAK1 heterodimer

BAK1

BR BR-binding site

BAK1

SBI1

SBI1

BSK1 CDG1 B

CDG1 CD

BSK1 CH 3

BKI1 1 PP2A

P

P P P

P

P

PP2A

BSU1 P B BKI1

PB BIN2

BSU1 1 P BRI1 endocytosis P BIN2 B

BIN2 14 -3 -3

Proteasomal degradation

P

P

BZR1/BES1 B P P P P

PP2A

P B BIN2

BZR1/BES1 B Other TFs

P P BZR1/BES1 S1

P P

BZR1/BES1 BZR1/BES1

Nucleus

BR-target genes

Cytosol BR-induced regulation of target genes

TRENDS in Plant Science

Figure 2. Current model of brassinosteroid (BR) signaling in the presence and absence of BRs in Arabidopsis. In the absence of BRs, BRI1 (BRASSINOSTEROID INSENSITIVE 1), a BR receptor kinase, remains inactive and does not heterodimerize with BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1), its coreceptor. BIN2 (BRASSINOSTEROID INSENSITIVE 2), a negative regulator of BR signaling, constitutively phosphorylates BZR1 (BRASSINAZOLE RESISTANT 1) and BES1 (BRI1-EMS SUPRESSOR 1), the two master transcription factors (TFs) of BR-induced responses. Phosphorylation of BZR1 and BES1 by BIN2 attracts the binding of 14-3-3 proteins. This in turn promotes the cytoplasmic retention of the TFs, rendering them unable to enter the nucleus and terminates the BR-induced response. In the presence of BRs, the binding of BRs to BRI1 initiates autophosphorylation to induce partial BRI1 kinase activity. This results in the dissociation of BRI1 kinase inhibitor BKI1 (attached at the BRI1 kinase domain), followed by the heterodimerization of BRI1 with BAK1 and transphosphorylation to impart full BRI1 kinase activity. Fully activated BRI1 then phosphorylates BR-SIGNALING KINASES (BSKs) and CDG1 (CONSTITUTIVE DIFFERENTIAL GROWTH 1). Subsequently, activated BSKs and CDG1 phosphorylate BSU1 (BRI1 SUPPRESSOR 1), which finally dephosphorylates BIN2. This in turn relieves the suppression of BIN2 on BZR1 and BES1, leading to the accumulation of dephosphorylated BZR1 and BES1. Dephosphorylated BZR1 and BES1 enter the nucleus and function to regulate the expression of BR target genes, either through direct interaction or via an interaction with other TFs. PP2A (Protein Phosphatase 2A) also positively regulates BR signaling by dephosphorylating BZR1 and BES1, whereas the SBI1 (SUPPRESSOR OF BRI1) is involved in the deactivation of BRI1 through methylation of PP2A.

and controls its membrane-associated subcellular localization [45,46]. BR homeostasis and plant growth As indicated by stunted root and shoot growth of BR biosynthetic mutants (dwf7-1, det2, cpd, br6ox, rot3) or abnormal shoot and root growth patterns of BR signaling mutants (bri1, bak1, bzr1-D, bes1-D), BR homeostasis at endogenous BR levels and at BR signaling levels is vital for normal biological processes [1,2,25,47]. For instance, analyses of loss-of-function bri1-116 and gain-of-function bes1D mutants indicated that a balance in BR signaling is required to maintain optimal meristem size and overall root growth. The bri1-116 mutant has reduced root meristem size, which is linked with decreased mitotic activity and low activity in the quiescent center. Interestingly, in BR-treated plants and in the bes1-D mutant, which has enhanced BR signaling, reduced root meristem sizes were

attributed to the premature cell cycle exit, which resulted in the early differentiation of meristematic cells [4]. Reduced BR titers (det2-1 mutation) or perturbed BR signaling (bri1-301 mutation) also have adverse impacts on cellulose biosynthesis, which contributes to cell wall formation during cell expansion and elongation [48]. BR homeostasis is primarily achieved through tight feedback regulation of BR metabolic genes by BR signaling elements, with strong repression of transcription following BR application and significant upregulation in response to inhibition of BR biosynthesis. Furthermore, the inactivation of bioactive BRs through hydroxylation, glycosylation and sulfonation constitutes vital components that control BR homeostasis [49–51] (Figure 1). BR signaling in plant growth and immunity Over the past several years, intensive research efforts have elucidated the roles of BR signaling elements regarding 597

Review overall plant growth, development and immunity. Here, we discuss the most recent findings that have emerged from this research area. Vegetative growth Previous expression studies have demonstrated that BRs regulate the expression of cell expansion genes. Expansins (EXPs) induce cell wall extension for the growth of plant cells. A recent study demonstrated that BRs are involved in the regulation of EXP levels in Arabidopsis. When treated with exogenous BRs, the expression of an AtEXP gene (AtEXPA5) was downregulated in det-2 and bri1-301 mutants but upregulated in the dominant bzr1-D mutant and in wild-type (WT) plants. Furthermore, the bzr1-1 DXexpA5-1 double mutant showed reduced growth compared with the bzr1-D single mutant. Taken together, these data provide evidence for the regulation of AtEXPA5 by BR signaling downstream of BZR1 [52]. Methylation of histone lysine residues is implicated in epigenetic regulation of gene expression in plants. In rice (Oryza sativa), it was recently reported that a H3K36 methyltransferase encoded by SDG725 is crucial in the methylation of histone lysine residues. Downregulation of SDG725 was associated with dwarfism, shortened internodes, erect leaves and small seeds; features that are typical of BR mutants. Deletion of SDG725 resulted in more than a twofold downregulation of several key BRrelated genes, such as OsDWARF11, BRI1 and BU1. In addition, ChIP (chromatin immunoprecipitation) data showed a reduced level of H3K36me2 and H3K36me3 in chromatin at several regions of the examined 30 -coding regions of these BR-related genes in SDG725 knockdown plants. Moreover, the SDG725 protein was able to bind directly to the BR target genes. These results demonstrate the role of SDG725-mediated H3K36 methylation in modulating the expression of BR target genes, which is crucial for regulating the growth and development of rice [53]. Reproductive growth Floral development is an important transition from the vegetative phase and is regulated by several endogenous and environmental signals [54,55]. BR signaling promotes flowering by enhancing the expression level of LD and FCA autonomous pathway genes, which in turn suppress the FLC gene [54,56]. BR induction of the floral pathway independent of LD and FCA has also been suggested in Arabidopsis. Studies conducted in bri1 mutants exhibiting a weak late flower phenotype and partial sensitivity to vernalization did not show altered expression of LD and FCA genes. These data support the existence of a LD- and FCA-independent pathway of floral induction that is mediated by BR-induced suppression of FLC [56,57]. In addition, BES1 was shown to regulate the expression of ELF6 and REF6 genes, which are involved in the control of flowering time. Specifically, ELF6 functions in the photoperiodic flowering pathway and REF6 acts as a repressor of FLC [58]. Stomatal development Although BRs inhibit chloroplast development in Arabidopsis [59], the direct functional role of BRs in stomatal 598

Trends in Plant Science October 2012, Vol. 17, No. 10

development is not entirely understood. Recently, BRs have been shown to play a crucial regulatory role in stomatal development by activating the MAPK kinase kinase (MAPKKK) YDA [13], which has been implicated as a negative regulator of stomatal development in Arabidopsis [60,61]. In the absence of BRs, BIN2 represses YDA function through phosphorylation, leading to a reduction in MAPK pathway activity. This in turn leads to the derepression of SPCH (SPEECHLESS), allowing SPCH to initiate stomatal development. In the presence of BRs, BR signaling activates the MAPK pathway by inactivation of BIN2 through BRI1, BSK1 and BSU1, resulting in the inhibition of stomatal production [13]. Contrary to the aforementioned report [13], a recent study in Arabidopsis shows positive implication of BRs in stomatal development by inhibition of BIN2-mediated phosphorylation of SPCH [62]. In the absence of BRs, in addition to MAPK, BIN2 also phosphorylates residues overlapping the MAPK target domain and four residues located in the amino-terminal region outside of the MAPK target domain of SPCH. These phosphorylation events of SPCH by BIN2 inhibit SPCH activity and limit epidermal cell proliferation. Conversely, the presence of BRs inhibits BIN2 activity, thereby stabilizing SPCH that initiates excessive stomatal and non-stomatal cell formation [62]. Innate immunity The triggering of innate immunity is a costly tradeoff between growth and immunity in plants and animals [14,63–65]. Although it is known that BRs induce disease resistance in rice and tobacco (Nicotiana tabacum) [66], the molecular mechanisms underlying this functional role have remained elusive. Intensive studies have deciphered complex positive and negative roles of BRs and BR signalings in innate immunity that involve crucial functions of BRI1 and BAK1 [14,31,67,68]. Among the pathogen- or microbial-associated molecular patterns (PAMP or MAMPs), flagellin 22 (flg22) and chitin are well characterized. The binding of flg22 to its receptor flg-sensing 2 (FLS2) initiates metabolic activities to arrest pathogen proliferation [67–69]. Similar to the BR-induced BRI1 signaling, the binding of flg22 to FLS2 triggers an association and transphosphorylation with BAK1, thereby activating FLS2. The activated FLS2 subsequently phosphorylates BIK1 to transduce the target response [67,70,71]. The function of BAK1 as a coreceptor of BR-induced BRI1 signaling and flg22-induced FLS2 signaling indicates the potential tradeoff between BR and FLS2 signalings through the mediation of BAK1. However, a recent study has suggested the existence of BAK1-independent immune signaling. In plants treated with both BRs and flg22, BRs were found to significantly decrease flg22-induced MAMP-triggered immunity (MTI) responses. By contrast, treatment with flg22 had no effect on BR-induced responses. When BRs and flg22 were applied to plants separately, they induced distinct gene expression profiles and biochemical responses. Collectively, these data suggest a unidirectional inhibition of FLS2mediated immune signaling by BR perception that occurs independently of complex formation with BAK1 and associated downstream phosphorylation [11]. Consistent with

Review

Trends in Plant Science October 2012, Vol. 17, No. 10

(a)

ing

flg22

nd

Bi

BRI1

FLS2

FLS2

BSK CDG

BAK1

Inactive BSU BSUs

BAK1 Active BSUs

BIN2 ism

Unknown mechan

ism

Unknown mechan

MAPK

Immunity

BZR1 BES1

MAPK

BIN2 BZR1 BES1

anism

n mech

BR-induced responses

Reduced immunity

Abiotic or biotic stress tolerance

(b) Abiotic stress tolerance

anism

n mech

Unknow

Unknow

No BR-induced responses

Cytosol

BR

BSK CDG

BAK1

BIK1

BAK1

BIK1

g

din

Bin

BRI1

+ BRs

BAK1 Innate immunity

Association and Transphosphorylation

Association

– BRs

flg22

ing

nd Bi

BRs

BRs Active BR signaling

NO Abiotic stress

H2O2

ABA

NADPH oxidase BZR1 and BES1

Abiotic stress response

ROS Damage to nucleic acids, proteins and membrane

Upregulated an tioxidant system , PCs, GSH, AS A, proline etc.

Nucleus

BR-regulated gene expression Cytosol

Reduced cell viability

Improved cell viability TRENDS in Plant Science

Figure 3. Current model of brassinosteroids (BRs) implicated in innate immunity and abiotic stress responses in Arabidopsis. (a) In the absence of BRs, flg22 (FLAGELLIN 22) binds to FLS2 (FLG-SENSING 2), a receptor of flg22, and initiates the association and transphosphorylation between BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1) and FLS2. Activated FLS2 phosphorylates the downstream receptor-like cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1) to trigger innate immunity via a MAPK (MITOGEN-ACTIVATED PROTEIN KINASE) pathway. In the presence of BRs, the FLS2-mediated signaling pathway is suppressed through BRI1 (BRASSINOSTEROID INSENSITIVE 1) in a BAK1-dependent or -independent manner, depending upon endogenous BR and BRI1 levels. Other BR signaling components, such as BIN2 (BRASSINOSTEROID INSENSITIVE 2), BZR1 (BRASSINAZOLE RESISTANT 1) and BES1 (BRI1-EMS SUPRESSOR 1), might also be involved in the suppression of FLS2 signaling downstream of BIK1, perhaps independently of BAK1, by an unknown mechanism. (b) Abiotic stress leads to the production of reactive oxygen species (ROS), which damage nucleic acids, proteins and cell membranes, resulting in reduced cell viability. Active BR signaling regulates the expression of BR target genes that are involved in the induction of antioxidant systems and metabolites that render protection to nucleic acids, proteins and cell membranes. As a result of this induced antioxidant activity and cell viability, the overall stress tolerance is improved through this beneficial activity imparted by BR function. Active BR signaling can induce gene expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to regulate H2O2 production to confer abiotic and biotic stress tolerance. Active BR signaling can also upregulate nitric oxide (NO) production, which promotes abscisic acid (ABA) biosynthesis, resulting in stress tolerance. Abbreviations: ASA, ascorbic acid; GSH, glutathione; PCs phytochelatins.

all of the aforementioned observations, another independent study has provided evidence that implicates BRs in MTI responses in both BAK1-dependent and -independent pathways. The association of BRs with MTI responses was dependent upon endogenous BR and BRI1 levels [12] (Figure 3a). Interactions and crosstalk between BRs and other hormones in plant growth and stress responses BRs and ABA ABA regulates both stress- and nonstress-related plant responses by acting as a signal molecule [72,73]. Interactions between BRs and ABA regulate the expression of

many genes that govern several biological processes, such as seed germination, stomatal closure and responses to environmental stresses [74–77]. Analyses of the BR-related mutants (det2-1 and bri1-1) have demonstrated that they show increased sensitivity to the inhibitory effects of ABA during seed germination in comparison with WT [74]. However, primary root and hypocotyl elongation assays in the sax1 mutant revealed hypersensitive responses to ABA, as well as Aux and ET [78]. Both BRs and ABA exert a promoting effect on stomatal closure which is most likely mediated via nitric oxide (NO) functioning as a mediator of ABA-induced stomatal closure in plants. This hypothesis is supported by the observation that NO was shown to 599

Review mediate both ABA-induced stomatal closure and BR-induced ABA biosynthesis [75,79,80]. In addition, the coapplication of BRs and ABA has been reported to result in a synergistic increase in drought-protective effects that were greater than those observed with either ABA or BR treatments alone [81]. Furthermore, Arabidopsis and Brassica napus seedlings treated with BRs showed enhanced tolerance to drought and cold stresses, which was accompanied by the induction of ABA- and multiple stress-inducible RD29A, ERD10 and RD22 genes [76]. Moreover, microarray analyses have shown that a large proportion of BR-responsive genes are also regulated by ABA [82]. An additional independent study provided further evidence demonstrating that the exogenous application of BRs enhances water stress tolerance by increasing ABA biosynthesis via the mediation of BR treatment-induced NO production [80]. Recently, it was proposed that the signaling cascades of ABA and BRs crosstalk just after the perception of BRs and before their transcriptional activation [5]. Analysis of the BR signaling mutant bri1-116 and subcellular localization of BKI1 revealed that the BR–receptor complex was not required by ABA to act on BR signaling outputs. However, ABA action in BR signaling was dependent on BIN2 because application of ABA was unable to inhibit BR signaling outputs when BIN2 was inhibited by application of LiCl, a BIN2 inhibitor. Furthermore, studies on ABA insensitive abi1 and abi2 mutants indicated that ABA was unable to act on BR signaling outputs in these mutants, suggesting that early ABA signaling components (ABI1 and ABI2) are implicated in ABA regulation of BR signaling outputs [5]. BRs and Aux The synergistic interactions of BRs and Aux play prominent roles in several aspects of plant growth and development [30]. The direct interaction between BIN2 and ARF2 represents an example of the synergistic effects of BRs and Aux in photomorphogenesis. Phosphorylation of ARF2 by BIN2 results in a loss of the DNA-binding repression activities of ARF2, indicating that BIN2 is capable of regulating the expression of Aux-induced genes through direct inactivation of ARF repressors, leading to the synergistic effect of BRs and Aux [83]. In addition, the application of BRs enhances polar Aux transport and endogenous Aux distribution by upregulating the expression of PIN and ROP genes, which are functionally involved in the distribution of Aux [84]. Furthermore, high levels of BRI1 expression in the epidermis results in an enlarged root meristem, whereas reduced BRI1 expression accounts for the small root meristem size in the bri1 mutant. The activity of BRs in the meristem was later shown to be accompanied by transcriptional modulation and post-transcriptional regulation of PIN2 and PIN4 genes [8,85]. The influence of BRs on PIN Aux efflux carriers, which control mitotic activity and cell differentiation, suggests a possible mechanism that contributes to BR-mediated root growth through regulation of Aux distribution. The specific signals that initiate BR biosynthesis are largely unknown. In a recent study, Aux was shown to function as a biosynthesis signal for BRs in Arabidopsis [9]. 600

Trends in Plant Science October 2012, Vol. 17, No. 10

Exogenous applications of Aux to DWF4pro:GUS plants were shown to enhance DWF4 expression and concomitantly increase endogenous BR levels. Analysis of the DWF4pro:GUS construct in BR- and Aux-mutant backgrounds indicated that Aux-induced expression of DWF4 occurs independently of BRs and only requires the Auxsignaling pathway. These data suggest that Aux exerts a direct control on BR biosynthesis in plants. In addition, Aux was shown to directly control BR signaling by inhibiting the binding of the BZR1 repressor to the DWF4 promoter. However, disruption in BR biosynthesis has been shown to have an adverse impact on Aux-responsive genes, which is consistent with previous findings [83]. BRs and ET ET biosynthesis is regulated by a large number of extrinsic and intrinsic cues [86]. The ACSs are rate-limiting factors of ET biosynthesis [87]. BRs positively influence ET biosynthesis through regulation of ACS and ACC oxidase activities [88]. Recently, BRs have been shown to be involved in ET-induced growth responses through FER. The FER gene encodes a RLK that is implicated in pollen tube perception and cell elongation and is required for a normal ET response. The etiolated fer-2 mutant exhibited hypersensitivity to ET but partial insensitivity to BRs. Moreover, BRs were unable to antagonize the ET effects on hypocotyl growth in the fer-2 mutant, indicating FER-dependent BR effects on ET-induced growth responses [89,90]. There is also an increasing amount of evidence suggesting interactions between BRs and ET in defense responses. Eix proteins are strong elicitors of plant defense responses in several cultivars. For instance, two Eix RLK receptors of tomato (Solanum lycopersicum LeEix1 and LeEix2) both bind to Eix. However, only LeEix2 is actually involved in mediating defense responses, whereas LeEix1 heterodimerizes with LeEix2 and attenuates the signaling of LeEix2. Furthermore, the attenuating function of LeEix1 requires the binding of LeEix1 to BAK1, providing evidence for the interaction between BRs and ET in responses to Eix [7]. BRs and CKs The roles of CKs in plant development and responses to various stresses have been widely characterized [91–96]. Recently, several lines of evidence have suggested the existence of interactions and crosstalk between BRs and CKs in various biological processes. Overexpression of the CKX3 gene with the root-specific promoter PYK10 resulted in a reduction of CK levels in Arabidopsis roots, leading to enhanced root growth and slightly reduced leaf growth [92]. Overexpression of BRI1 in PYK10:CKX3 plants using its own promoter further enhanced root growth and resulted in leaf growth similar to WT levels. These findings are in agreement with those observed when PYK10:CKX3 plants were treated with BRs, suggesting that crosstalk between BRs and CKs is involved in the regulation of plant growth [10]. In addition, overexpression of an IPT gene, which results in an increase in CK content, also provides possible links between CK and BR signaling pathways [97]. An increase in CK content just before the onset of senescence by the conditional overexpression of IPT in rice

Review results in enhanced tolerance to drought stress. The observed increase of CKs coincided with the upregulation of several BR-related genes, such as BAK1, BSK1, SERK1 and DWF5, suggesting that crosstalk between BRs and CKs may contribute to the modification of source–sink relations, leading to increased drought tolerance [97]. BRs and JA The interaction of BRs and JA plays crucial roles in plant development and stress responses [98–100]. The COI1 is an F-box protein that is required for the execution of JA signaling and JA responses in Arabidopsis [101]. The psc1 mutant with partial suppression of coi1insensitivity to JAinduced inhibition of root growth is a leaky mutation of DWF4 [98]. Application of BRs was able to eliminate partial restoration of JA sensitivity of the psc1 mutant in coi1-2 and JA hypersensitivity of psc1 in WT. Furthermore, expression analysis indicated that DWF4 expression was repressed by JA in a COI1-dependent manner. These results demonstrated that BRs negatively regulate JAinduced inhibition of root growth. Secondly, these data confirm that JA-induced downregulation of DWF4 occurs downstream of COI1 in the JA signaling pathway [98]. Recently, BAK1 was shown to confer resistance to Nicotiana attenuata against its specialist herbivore, Manduca sexta [100]. Altered expression of NaBAK1 was observed in plants exposed to larval oral secretions (LOS) of M. sexta. NaBAK1-silenced plants, when wounded or applied with LOS, exhibited attenuated JA and JA–isoleucine bursts independently of compromised MAPK activity or elevated SA levels. JA application to the NaBAK1-silenced plants induced higher levels of defensive trypsin proteinase inhibitors. In addition, transcription of NaTD was reduced, whereas NaJAR4 and NaJAR6 expression was increased in the NaBAK1-silenced plants. These data demonstrate that BAK1 plays an essential role in mediating the resistance of N. attenuata to M. sexta by modulating herbivoryinduced JA accumulation and controlling the activity of defensive secondary metabolites [100]. BRs and SA Increasing evidence suggests that crosstalk between BRs and SA plays an important role in plant response to biotic and abiotic stresses. As a challenge to the prevailing view that BRs positively regulate innate immunity [66], a recent study showed that BR-induced modulation of immunity responses, partly through the negative crosstalk between BRs with SA, could enhance the susceptibility of rice to the root oomycete Pythium graminicola [102]. Rice seedlings treated with BRs showed increased susceptibility towards P. graminicola. Application of Brz, a BR biosynthesis inhibitor, to rice plants, consistently revealed reduced susceptibility against P. graminicola. This was shown to be the result of the repression of SA defense responses by BRs downstream of SA biosynthesis but upstream of key defense regulators, namely OsNPR1 and OsWRKY45. These data support the hypothesis that P. graminicola exploits the BR pathway as a decoy to counteract the effective SA-induced defense responses in rice. This report clearly highlights the implication of BRs as a negative regulator of immunity against pathogens and also

Trends in Plant Science October 2012, Vol. 17, No. 10

uncovers the pathogen-mediated modulation of steroid homeostasis as a central virulence strategy [102]. Several lines of evidence have also shown that BR crosstalk with SA regulates plant responses to abiotic stress. Exogenous application of BRs was unable to confer salt stress tolerance in the SA-insensitive npr1-1 mutant in comparison with WT [16]. This result indicated that BRinduced salt tolerance in Arabidopsis partially depends on NPR1, a master regulator of the SA-mediated defense signaling pathway [16]. Recently, exogenous applications of BRs and SA have been shown to increase salinity stress tolerance in Brassica juncea. The combined application of BRs and SA was most effective in alleviating the salt stress when compared with their individual treatments [103]. BRs and GAs Recent research has supported the existence of extensive crosstalk between BRs and GAs in a wide range of biological processes, including plant development and responses to environmental stimuli [102,104]. The GAST family is critically involved in GA signaling. OsGSR1, a member of the GAST family in rice, is induced by GA and suppressed by BRs [104]. RNAi plants with reduced OsGSR1 expression exhibited reduced sensitivity to GAs, enhanced level of GAs, reduced levels of endogenous BRs and a dwarf phenotype that could be rescued by exogenous BR application. OsGSR1 was shown to activate BR biosynthesis through direct interaction with DWF1, suggesting that OsGSR1 is a probable crosstalk point in GA and BR signaling pathways. Another piece of evidence showing the negative crosstalk of BRs with GAs is the enhanced stabilization of OsSLR1 by exogenous BR treatment. OsSLR1 is the only DELLA GA-signaling repressor in rice, which functions as a key negative regulator in the resistance to P. graminicola. Additionally, expression of OsSLR1 is upregulated in response to both pathogen infection and exogenous BR treatment. These data suggest that BRs may antagonize the GA-induced defense responses in rice through interference with GA signaling [102]. In contrast to the antagonistic crosstalk of BRs and GAs, the impact of BRs on cotton (Gossypium hirsutum) DELLA genes in fiber cell initiation and elongation was shown to be positive. Exogenous BR treatment triggers the downregulation of four DELLA genes in cotton fiber cells, including GhGAI1, which is engaged in fiber cell initiation [105]. Taken together, these reports demonstrate that the crosstalk between BRs and GAs is complex and warrants future investigation. Biotechnological manipulation of BR actions to improve stress tolerance Exogenous applications of BRs are widely used to improve stress tolerance in plants [77] (Figure 3b) and the application of BRs alone or in conjunction with other PGRs has become a routine stress management protocol [15,103,106]. Treatment of cucumber (Cucumis sativus) with BRs has been reported to enhance tolerance to photo-oxidative and cold stresses and is accompanied by H2O2 accumulation and systemic induction of genes associated with stress responses, such as MAPK1 and MAPK3 [17,18]. Similarly, application of BRs to radish (Raphanus sativus) improves 601

Review

Trends in Plant Science October 2012, Vol. 17, No. 10

Table 1. BR metabolic and BR signaling genes involved in stress responses Gene ID AtBRI1 AtBAK1

Description BR receptor kinase Coreceptor of BRI1

Regulatory function in stress response Negative Positive

AtBKK1

BKK1/SERK4, a homolog of BAK1 BR transcription factor BR biosynthetic gene Ortholog of BAK1 in rice Ortholog of BIN2 in rice

AtBZR1 AtDWF4 OsSERK1 OsGSK1

Refs [12,114] [11,113]

Positive

Type of stresses Immunity response, cold Immunity response, TCV infection, cell death and chlorosis TCV infection, cell death and chlorosis

Positive Positive Positive Negative

Cold Cold Disease resistance Cold, heat, salt, and drought

[120] [110] [112] [111]

[113]

Abbreviations: At, Arabidopsis thaliana; BAK1, BRI1-ASSOCIATED RECEPTOR KINASE 1; BIN2, Brassinosteroid insensitive 2; BKK1/SERK4, BAK1-like kinase1; BRI1, BRASSINOSTEROID INSENSITIVE 1; BZR1, BRASSINAZOLE RESISTANT 1; DWF4, DWARF4; GSK1, glycogen synthase 3-like protein kinase; Os, Oryza sativa; SERK, somatic embryogenesis receptor kinase; TCV, Turnip crinkle virus.

tolerance to Cu and Cr stress owing to the upregulation of a protective antioxidant system and the modulation of endogenous ABA, Aux and polyamine profiles. These results highlight the existence of crosstalk between BRs and ABA or Aux in stress responses [107,108]. Furthermore, the positive effect of BRs in Cr detoxification can be significantly improved with the addition of the polyamine spermidine, indicating that the advantageous interplay between BRs and polyamines may serve as a promising approach to enhance the mitigation of abiotic stresses in plants [109]. The influence of BRs on stress responses has led to several biotechnological strategies to enhance plant tolerance to abiotic and biotic stresses (Table 1). Seed-specific overexpression of AtDWF4 in Arabidopsis enhanced cold tolerance, which was attributed to the upregulation of the cold-responsive gene COR15A [110]. Loss-of-function of OsGSK1, a BIN2 homolog of rice, improved tolerance to cold, heat, salt and drought stresses when compared with the WT [111]. These data suggest that BR signaling components may also serve as potential targets for genetic engineering of abiotic stress tolerance. The induction of selective BR signaling components by biotechnological approaches can also enhance disease resistance. For instance, constitutive overexpression of OsSERK1, a BAK1 homolog of rice, led to an increase in the resistance of transgenic rice plants to blast fungus [112], while loss-offunction of BAK1 in Arabidopsis enhanced susceptibility to Turnip crinkle virus (TCV) infection [113]. The effects of BRs on stress tolerance depend on the concentration of the BRs that were applied under the test conditions. Excessive use of BRs may exert detrimental consequences on the adaptive responses of plants because appropriate levels of BRs are required for optimal BR signaling [12,14,31,114]; a phenomenon that has also been observed with CKs [96]. Concluding remarks Although recent research has elucidated the BR signaling pathway from the receptor to BZR1 and BES1, the key regulators of BR-induced responses in plants, the mechanisms behind the pleiotropic action of BRs and the execution of BR-induced responses still remains poorly understood at this time. The versatile role of BRs may be attributed to multilayer interactions with other PGRs affecting the post-transcriptional fate of the target response. Taken together, forward genetic approaches 602

combined with genome-wide transcriptional analyses have identified several hundreds of BR targets, shedding additional light on the complexity of the diverse activity of BRs in plants. Concerted and focused efforts by the research community are required to further elucidate the mechanisms of BRs in innate immunity, stomatal development and crosstalk with other PGRs to acquire or confer abiotic and biotic stress tolerance. Furthermore, the high redundancy of BR signaling components demands additional attention to clarify the functional roles of the mechanisms involved with BR signaling in plants. An important challenge in the coming years will be unraveling the exact mechanism of the BR-regulated gene network in a systems biology-based manner [115,116]. Challenges ahead will also include uncovering the crosstalk behavior of BR signaling components with other PGRs under various stresses. Given the huge potential and value of BRs in stress management, targeted genetic engineering of BR biosynthesis and/or BR signaling is likely to become a popular tool in the coming years to improve stress tolerance, and perhaps biomass and yield of agricultural crops [117–119]. As a result, it is hoped that advances in this area of BR signaling could bring us one step closer to meeting the demands for increased food production to feed the growing world population. Acknowledgments We apologize to those colleagues who have contributed to this field but were not cited because of space limitations. This work was supported by a grant (No. AP24-1-0076) from the RIKEN Strategic Research Program for R & D to L-SPT. SPC and J-QY are grateful to the support from the National Basic Research Program of China (2009CB119000).

References 1 Clouse, S.D. (2011) Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development. Plant Cell 23, 1219–1230 2 Gudesblat, G.E. and Russinova, E. (2011) Plants grow on brassinosteroids. Curr. Opin. Plant Biol. 14, 530–537 3 Kim, T.W. and Wang, Z.Y. (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu. Rev. Plant Biol. 61, 681–704 4 Gonzalez-Garcia, M.P. et al. (2011) Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots. Development 138, 849–859 5 Zhang, S. et al. (2009) The primary signaling outputs of brassinosteroids are regulated by abscisic acid signaling. Proc. Natl. Acad. Sci. U.S.A. 106, 4543–4548

Review 6 Li, J. (2010) Regulation of the nuclear activities of brassinosteroid signaling. Curr. Opin. Plant Biol. 13, 540–547 7 Bar, M. et al. (2010) BAK1 is required for the attenuation of ethyleneinducing xylanase (Eix)-induced defense responses by the decoy receptor LeEix1. Plant J. 63, 791–800 8 Hacham, Y. et al. (2011) Brassinosteroid perception in the epidermis controls root meristem size. Development 138, 839–848 9 Chung, Y. et al. (2011) Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis. Plant J. 66, 564–578 10 Vercruyssen, L. et al. (2011) Combining enhanced root and shoot growth reveals cross talk between pathways that control plant organ size in Arabidopsis. Plant Physiol. 155, 1339–1352 11 Albrecht, C. et al. (2012) Brassinosteroids inhibit pathogenassociated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. Proc. Natl. Acad. Sci. U.S.A. 109, 303–308 12 Belkhadir, Y. et al. (2012) Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc. Natl. Acad. Sci. U.S.A. 109, 297–302 13 Kim, T.W. et al. (2012) Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature 482, 419–422 14 Wang, Z.Y. (2012) Brassinosteroids modulate plant immunity at multiple levels. Proc. Natl. Acad. Sci. U.S.A. 109, 7–8 15 Divi, U.K. and Krishna, P. (2009) Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance. New Biotechnol. 26, 131–136 16 Divi, U.K. et al. (2010) Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways. BMC Plant Biol. 10, 151 17 Xia, X.J. et al. (2009) Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber. Plant Physiol. 150, 801–814 18 Xia, X.J. et al. (2011) Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus. New Phytol. 191, 706–720 19 Zhang, A. et al. (2010) ZmMPK5 is required for the NADPH oxidasemediated self-propagation of apoplastic H2O2 in brassinosteroidinduced antioxidant defence in leaves of maize. J. Exp. Bot. 61, 4399–4411 20 Miller, G. et al. (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 33, 453–467 21 Cui, J.X. et al. (2011) Role of nitric oxide in hydrogen peroxidedependent induction of abiotic stress tolerance by brassinosteroids in cucumber. Plant Cell Environ. 34, 347–358 22 Vriet, C. et al. (2012) Boosting crop yields with plant steroids. Plant Cell 24, 842–857 23 Santner, A. and Estelle, M. (2009) Recent advances and emerging trends in plant hormone signalling. Nature 459, 1071–1078 24 Bajguz, A. (2007) Metabolism of brassinosteroids in plants. Plant Physiol. Biochem. 45, 95–107 25 Ye, H. et al. (2011) Recent advances in the regulation of brassinosteroid signaling and biosynthesis pathways. J. Integr. Plant Biol. 53, 455–468 26 Kim, T.W. et al. (2009) Brassinosteroid signal transduction from cellsurface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 11, 1254–1260 27 Jaillais, Y. and Chory, J. (2010) Unraveling the paradoxes of plant hormone signaling integration. Nat. Struct. Mol. Biol. 17, 642–645 28 Gendron, J.M. and Wang, Z.Y. (2007) Multiple mechanisms modulate brassinosteroid signaling. Curr. Opin. Plant Biol. 10, 436–441 29 Tang, W. et al. (2010) Proteomics shed light on the brassinosteroid signaling mechanisms. Curr. Opin. Plant Biol. 13, 27–33 30 Vert, G. and Chory, J. (2011) Crosstalk in cellular signaling: background noise or the real thing? Dev. Cell 21, 985–991 31 Chinchilla, D. et al. (2009) One for all: the receptor-associated kinase BAK1. Trends Plant Sci. 14, 535–541 32 Gou, X. et al. (2012) Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling. PLoS Genet. 8, e1002452 33 Jaillais, Y. et al. (2011) Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–237

Trends in Plant Science October 2012, Vol. 17, No. 10

34 Wang, X. and Chory, J. (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313, 1118–1122 35 Yin, Y. et al. (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109, 181–191 36 He, J.X. et al. (2005) BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307, 1634–1638 37 Li, L. and Deng, X.W. (2005) It runs in the family: regulation of brassinosteroid signaling by the BZR1-BES1 class of transcription factors. Trends Plant Sci. 10, 266–268 38 Ryu, H. et al. (2010) Phosphorylation dependent nucleocytoplasmic shuttling of BES1 is a key regulatory event in brassinosteroid signaling. Mol. Cells 29, 283–290 39 Tang, W. et al. (2008) BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321, 557–560 40 Kim, T.W. et al. (2011) The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2. Mol. Cell 43, 561–571 41 Li, L. et al. (2010) Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression. Proc. Natl. Acad. Sci. U.S.A. 107, 3918– 3923 42 Luo, X.M. et al. (2010) Integration of light- and brassinosteroidsignaling pathways by a GATA transcription factor in Arabidopsis. Dev. Cell 19, 872–883 43 Yan, Z. et al. (2009) BIN2 functions redundantly with other Arabidopsis GSK3-like kinases to regulate brassinosteroid signaling. Plant Physiol. 150, 710–721 44 Saidi, Y. et al. (2012) Function and evolution of ‘green’ GSK3/Shaggylike kinases. Trends Plant Sci. 17, 39–46 45 Wu, G. et al. (2011) Methylation of a phosphatase specifies dephosphorylation and degradation of activated brassinosteroid receptors. Sci. Signal. 4, ra29 46 Tang, W. et al. (2011) PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat. Cell Biol. 13, 124–131 47 Cheon, J. et al. (2010) Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction. BMC Plant Biol. 10, 270 48 Xie, L.Q. et al. (2011) Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis. J. Exp. Bot. 62, 4495–4506 49 Hategan, L. et al. (2011) Regulation of brassinosteroid metabolism. In Brassinosteroids: A Class of Plant Hormone (Hayat, S. and Ahmad, A., eds), pp. 775–781, Springer 50 Yoshimitsu, Y. et al. (2011) Transcription of DWARF4 plays a crucial role in auxin-regulated root elongation in addition to brassinosteroid homeostasis in Arabidopsis thaliana. PLoS ONE 6, e23851 51 Poppenberger, B. et al. (2011) CESTA, a positive regulator of brassinosteroid biosynthesis. EMBO J. 30, 1149–1161 52 Park, C.H. et al. (2010) Brassinosteroids control AtEXPA5 gene expression in Arabidopsis thaliana. Phytochemistry 71, 380–387 53 Sui, P. et al. (2012) H3K36 methylation is critical for brassinosteroidregulated plant growth and development in rice. Plant J. 70, 340–347 54 Domagalska, M.A. et al. (2010) Genetic analyses of interactions among gibberellin, abscisic acid, and brassinosteroids in the control of flowering time in Arabidopsis thaliana. PLoS ONE 5, 14012 55 Koo, S.C. et al. (2010) Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6. Plant J. 62, 807–816 56 Li, J. et al. (2010) Involvement of brassinosteroid signals in the floralinduction network of Arabidopsis. J. Exp. Bot. 61, 4221–4230 57 Domagalska, M.A. et al. (2007) Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering. Development 134, 2841–2850 58 Yu, X. et al. (2008) Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 105, 7618–7623 59 Komatsu, T. et al. (2010) The chloroplast protein BPG2 functions in brassinosteroid-mediated post-transcriptional accumulation of chloroplast rRNA. Plant J. 61, 409–422 603

Review 60 Wang, H. et al. (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19, 63–73 61 Lampard, G.R. et al. (2008) Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322, 1113–1116 62 Gudesblat, G.E. et al. (2012) SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat. Cell Biol. 14, 548–554 63 Tena, G. et al. (2011) Protein kinase signaling networks in plant innate immunity. Curr. Opin. Plant Biol. 14, 519–529 64 McCann, H.C. et al. (2012) Identification of innate immunity elicitors using molecular signatures of natural selection. Proc. Natl. Acad. Sci. U.S.A. 109, 4215–4220 65 Spoel, S.H. and Dong, X. (2012) How do plants achieve immunity? Defence without specialized immune cells. Nat. Rev. Immunol. 12, 89– 100 66 Nakashita, H. et al. (2003) Brassinosteroid functions in a broad range of disease resistance in tobacco and rice. Plant J. 33, 887–898 67 Chinchilla, D. et al. (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500 68 Heese, A. et al. (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. U.S.A. 104, 12217–12222 69 Segonzac, C. and Zipfel, C. (2011) Activation of plant patternrecognition receptors by bacteria. Curr. Opin. Microbiol. 14, 54–61 70 Lu, D. et al. (2010) A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl. Acad. Sci. U.S.A. 107, 496–501 71 Zhang, J. et al. (2010) Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7, 290–301 72 Umezawa, T. et al. (2010) Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol. 51, 1821–1839 73 Hadiarto, T. and Tran, L.S. (2011) Progress studies of droughtresponsive genes in rice. Plant Cell Rep. 30, 297–310 74 Steber, C.M. and McCourt, P. (2001) A role for brassinosteroids in germination in Arabidopsis. Plant Physiol. 125, 763–769 75 Haubrick, L.L. et al. (2006) Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts. Physiol. Plant. 128, 134– 143 76 Kagale, S. et al. (2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225, 353–364 77 Acharya, B.R. and Assmann, S.M. (2009) Hormone interactions in stomatal function. Plant Mol. Biol. 69, 451–462 78 Ephritikhine, G. et al. (1999) The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid. Plant J. 18, 303–314 79 Ribeiro, D.M. et al. (2009) Differential requirement for NO during ABA-induced stomatal closure in turgid and wilted leaves. Plant Cell Environ. 32, 46–57 80 Zhang, A. et al. (2011) Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves. Plant Cell Physiol. 52, 181–192 81 Xu, H.L. (2007) Effects of exogenous epibrassinolide and abscisic acid on grain yield of sorghum plants growing under soil water deficit. In Dryland Crop Production: Technology Breakthroughs and Study Cases (Xu, H.L., ed.), pp. 195–202, Research Signpost 82 Goda, H. et al. (2008) The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access. Plant J. 55, 526–542 83 Vert, G. et al. (2008) Integration of auxin and brassinosteroid pathways by auxin response factor 2. Proc. Natl. Acad. Sci. U.S.A. 105, 9829–9834 84 Li, L. et al. (2005) Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis. Plant Cell 17, 2738–2753

604

Trends in Plant Science October 2012, Vol. 17, No. 10

85 Hacham, Y. et al. (2012) BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers. Plant Signal. Behav. 7, 68–70 86 Wang, K.L. et al. (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14 (Suppl.), S131–S151 87 Tsuchisaka, A. et al. (2009) A combinatorial interplay among the 1aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics 183, 979–1003 88 Hansen, M. et al. (2009) Regulation of ACS protein stability by cytokinin and brassinosteroid. Plant J. 57, 606–614 89 Deslauriers, S.D. and Larsen, P.B. (2010) FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls. Mol. Plant 3, 626–640 90 Cheung, A.Y. and Wu, H.M. (2011) THESEUS 1, FERONIA and relatives: a family of cell wall-sensing receptor kinases? Curr. Opin .Plant Biol. 14, 632–641 91 Werner, T. and Schmulling, T. (2009) Cytokinin action in plant development. Curr. Opin. Plant Biol. 12, 527–538 92 Werner, T. et al. (2010) Root-specific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. Plant Cell 22, 3905–3920 93 Choi, J. et al. (2011) Cytokinins and plant immunity: old foes or new friends? Trends Plant Sci. 16, 388–394 94 Nishiyama, R. et al. (2011) Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis. Plant Cell 23, 2169–2183 95 Nishiyama, R. et al. (2012) Transcriptome analyses of a salt-tolerant cytokinin-deficient mutant reveal differential regulation of salt stress response by cytokinin deficiency. PLoS ONE 7, e32124 96 Ha, S. et al. (2012) Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Sci. 17, 172–179 97 Peleg, Z. et al. (2011) Cytokinin-mediated source/sink modifications improve drought tolerance and increase grain yield in rice under water-stress. Plant Biotechnol. J. 9, 747–758 98 Ren, C. et al. (2009) A leaky mutation in DWARF4 reveals an antagonistic role of brassinosteroid in the inhibition of root growth by jasmonate in Arabidopsis. Plant Physiol. 151, 1412–1420 99 Campos, M.L. et al. (2009) Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato. J. Exp. Bot. 60, 4347–4361 100 Yang, D.H. et al. (2011) BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata’s responses to herbivory. J. Exp. Bot. 62, 641–652 101 Xie, D.X. et al. (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280, 1091–1094 102 De Vleesschauwer, D. et al. (2012) Brassinosteroids antagonize gibberellin- and salicylate-mediated root immunity in rice. Plant Physiol. 158, 1833–1846 103 Hayat, S. et al. (2012) Comparative effect of 28 homobrassinolide and salicylic acid in the amelioration of NaCl stress in Brassica juncea L. Plant Physiol. Biochem. 53, 61–68 104 Wang, L. et al. (2009) OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice. Plant J. 57, 498–510 105 Hu, M.Y. et al. (2011) Brassinosteroids and auxin down-regulate DELLA genes in fiber initiation and elongation of cotton. Agr. Sci. China 10, 1168–1176 106 Peleg, Z. and Blumwald, E. (2011) Hormone balance and abiotic stress tolerance in crop plants. Curr. Opin. Plant. Biol. 14, 290–295 107 Choudhary, S.P. et al. (2010) Epibrassinolide induces changes in indole-3-acetic acid, abscisic acid and polyamine concentrations and enhances antioxidant potential of radish seedlings under copper stress. Physiol. Plant. 140, 280–296 108 Choudhary, S.P. et al. (2011) Epibrassinolide ameliorates Cr (VI) stress via influencing the levels of indole-3-acetic acid, abscisic acid, polyamines and antioxidant system of radish seedlings. Chemosphere 84, 592–600 109 Choudhary, S.P. et al. (2012) Chromium stress mitigation by polyamine-brassinosteroid application involves phytohormonal and physiological strategies in Raphanus sativus L. PLoS ONE 7, e33210

Review 110 Divi, U.K. and Krishna, P. (2010) Overexpression of the brassinosteroid biosynthetic gene AtDWF4 in Arabidopsis seeds overcomes abscisic acid-induced inhibition of germination and increases cold tolerance in transgenic seedlings. J. Plant Growth Regul. 29, 385–393 111 Koh, S. et al. (2007) T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses. Plant Mol. Biol. 65, 453–466 112 Hu, H. et al. (2005) Rice SERK1 gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection. Planta 222, 107–117 113 Yang, H. et al. (2010) BAK1 and BKK1 in Arabidopsis thaliana confer reduced susceptibility to turnip crinkle virus. Eur. J. Plant Pathol. 127, 149–156 114 Kim, S.Y. et al. (2010) Constitutive activation of stress-inducible genes in a brassinosteroid-insensitive 1 (bri1) mutant results in higher tolerance to cold. Physiol. Plant. 138, 191–204

Trends in Plant Science October 2012, Vol. 17, No. 10

115 Sun, Y. et al. (2010) Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev. Cell 19, 765–777 116 Yu, X. et al. (2011) A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 65, 634–646 117 Morinaka, Y. et al. (2006) Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice. Plant Physiol. 141, 924–931 118 Wu, C.Y. et al. (2008) Brassinosteroids regulate grain filling in rice. Plant Cell 20, 2130–2145 119 Li, D. et al. (2009) Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield. Plant Biotechnol. J. 7, 791–806 120 Qu, T. et al. (2011) Brassinosteroids regulate pectin methylesterase activity and AtPME41 expression in Arabidopsis under chilling stress. Cryobiology 63, 111–117

605