Arabidopsis heterotrimeric G protein β subunit, AGB1, regulates brassinosteroid signalling independently of BZR1
Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University
, Harbin 150040,
Asian Natural Environmental Science Center (ANESC), The University of Tokyo
, 1-1-1 Midori-cho, Nishitokyo-shi,
Tokyo 188-0002, Japan
The Arabidopsis thaliana heterotrimeric G protein subunit, AGB1, is involved in both abscisic acid (ABA) signalling and brassinosteroid (BR) signalling, but it is unclear how AGB1 regulates these signalling pathways. Akey transcription factor downstream of BR, BZR1, and its gain-of-function mutant, bzr1-1, were overexpressed in an AGB1-null mutant, agb1-1, to examine their effects on the BR hyposensitivity and the ABA hypersensitivity of agb1-1, and to examine whether AGB1 regulates the functions of BZR1. Because the amino acid sequence of AGB1 contains 17 putative modification motifs of glycogen synthase kinase 3/SHAGGY-like protein kinases (GSKs), which are known components of BR signalling, the interaction between AGB1 and one of the Arabidopsis GSKs, BIN2, was examined. Expression of bzr1-1 alleviated the effects of a BR biosynthesis inhibitor, brassinazole, in both the wild type and agb11, and overexpression of BZR1 alleviated the effects of ABA in both the wild type and agb1-1. AGB1 did not affect the phosphorylation state of BZR1 in vivo. AGB1 interacted with BIN2 in vitro, but did not affect the phosphorylation state of BIN2. The results suggest that AGB1 interacts with BIN2, but regulates the BR signalling in a BZR1-independent manner.
Heterotrimeric GTP-binding proteins (G proteins, consisting
of subunits G, G, and G) are signalling molecules found
in a variety of eukaryotic organisms. They mediate
ligandbinding signals from G protein-coupled receptors (GPCRs)
to downstream pathways, and thus are involved in diverse
cellular processes. In contrast to humans, which have 21 G
genes, five G genes, and 12 G genes, Arabidopsis thaliana
has only one G gene (GPA1), one G gene (AGB1), and
three G genes (AGG1AGG3) (for a review, see Jones and
Assmann, 2004; Chakravorty etal., 2011). The physiological
functions of plant G proteins can be evaluated by using G
protein-deficient mutants. Such studies have suggested that
plant G proteins have roles in signal transduction of
various stimuli such as a stress-related phytohormone, abscisic
acid (ABA) (for a review, see Perfus-Barbeoch et al., 2004).
GPCRs on the plasma membrane in A.thaliana were shown
to bind ABA (Liu etal., 2007; Pandey etal., 2009),
supporting the importance of the G proteins in ABA signal
transduction, although the validity of these studies is still in dispute
(Gao etal., 2007; Jaff etal., 2012).
G proteins are thought to mediate signal transduction via
interacting with effector proteins and regulating their
activities (for a review, see Pierce et al., 2002). Many G protein
effectors have been identified in animals, but only some of
them exist in Arabidopsis or other plant species (for a review,
see Jones and Assmann, 2004), suggesting that plants have
plant-specific mechanisms for G protein signalling. In fact,
some putative plant-specific G protein effectors have been
identified. For example, THYLAKOID FORMATION
1 (THF1) physically interacts with GPA1 and is involved
in GPA1-mediated sugar signalling and chloroplast
development (Huang et al., 2006; Zhang et al., 2009). A cupin
domain-containing protein, AtPirin1, also physically
interacts with GPA1 and mediates ABA signalling (Lapik and
Kaufman, 2003). NDL1 (N-MYC
DOWNREGULATEDLIKE1) physically interacts with AGB1 and regulates auxin
distribution in plants (Mudgil etal., 2009). An acireductone
dioxygenase-like protein, ARD1, also physically interacts
with AGB1, and its enzyme activity is enhanced by G dimer
(Friedman etal., 2011). Overexpression of a Golgi-localized
hexose transporter, SGB1, partially suppresses the phenotype
of the AGB1-null mutant, agb1-2 (H.X. Wang etal., 2006).
Acomprehensive analysis of the G-protein interactome
suggested that G proteins interact with cell wall-related proteins
and thereby regulate the cell wall composition (Klopffleisch
etal., 2011). However, the molecular mechanisms underlying
G protein-mediated signalling remain to be elucidated.
G proteins are suggested to play roles in signal
transduction of a phytohormone, brassinosteroid (BR). For example,
G deficiency causes BR hyposensitivities in both Arabidopsis
(Ullah etal., 2001, 2002) and rice (L. Wang etal., 2006), and
G deficiency in Arabidopsis enhances the dwarf phenotypes
of bri1-5 and det2-1, which have defects in BR biosynthesis
and BR perception, respectively (Gao etal., 2008). An
AGB1null mutant, agb1-2, is hyposensitive to BR in seed
germination (Chen et al., 2004). In addition, agb1-2 has rounder
leaves, more highly branched root systems, shorter siliques
(Ullah etal., 2003), and higher sensitivities to ABA (Pandey
etal., 2006). All these phenotypes of agb1-2 are similar to the
phenotypes of mutants that have defects in BR biosynthesis
or BR signalling (for a review, see Clouse, 2011).
On the other hand, an established model of the BR
signalling consists of specific types of protein kinases, protein
phosphatases, and transcription factors. When a receptor kinase,
BRI1 (BR INSENSITIVE1), binds BR, it phosphorylates
BSK (BR signalling kinase) family protein kinases, which
activate Kelch repeat-containing protein phosphatases, such
as BSU1 (BRI1 SUPPRESSOR1) and BSL1 (BSU1-LIKE1).
When activated, these protein phosphatases dephosphorylate
and deactivate GSKs (glycogen synthase kinase
3/SHAGGYlike protein kinases). As a result, two GSK-substrate
transcription factors, BZR1 (BRASSINAZOLE RESISTANT1,
also known as BES2: BRI1 EMS SUPPRESSOR2) and
BZR2 (also known as BES1) are dephosphorylated, and
regulate their target gene expression, which leads to BR responses
(Kim and Wang, 2010; for a review, see Sun etal., 2010). Type
2A protein phosphatases (PP2As) are involved in
dephosphorylating BZR1 and BZR2 (Tang etal., 2011). BZR1 and
BZR2 share 88% amino acid sequence identities, and
redundantly function in BR signalling (Kim and Wang, 2010; for
a review, see Sun etal., 2010). The phosphorylation states of
BZR1 and/or BZR2 and their target gene expression have
often been examined to characterize mutant and transgenic
plants that have different BR responses (He etal., 2002; Yin
etal., 2002; Vert and Chory, 2006; Peng etal., 2008; Kim etal.,
2009; Yan etal., 2009; Rozhon etal., 2010; Tang etal., 2011).
It is still unclear how G proteins participate in the BR
signalling, or whether the well-studied components of BR
signalling interact with G proteins. Here, to gain further insights
into the interaction between G proteins and BR signalling,
the functions of BZR1 in agb1-1 were examined. In addition,
because AGB1 has many putative GSK modification sites,
the interaction between AGB1 and one of the GSKs, BIN2,
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used
throughout the experiments. Seeds of gpa1-4 (CS6534), agb1-1 (CS3976),
and agb1-2 (CS6536) were obtained from the Arabidopsis
Biological Resource Center (ABRC, http://www.arabidopsis.org).
Surface-sterilized seeds were sown on 0.5 MS medium (0.8% w/v
agar, 0.5 MS salts, 1% w/v sucrose, 0.5 g l1 MES, pH 5.8) with or
without ABA (Wako, Japan), the brassinosteroid brassinolide (BR)
(Brassino Co., Ltd., Japan), brassinazole (BRZ) (Tokyo Kasei,
Japan), or bikinin (Calbiochem) (concentrations are shown in the
figures), and then incubated for 3 d at 4 C (stratification). After
stratification, plants were grown at 22C under a 16h light/8 h dark
photoperiod or in the dark. To generate transgenic plants expressing
BZR1green fluorescent protein (GFP), the genomic region
corresponding to the open reading frame (ORF) of BZR1 (AT1G75080)
was amplified by genomic PCR as previously described (Tsugama
et al., 2011) using the following primer pair: 5-GGGTCTAGAA
TGACTTCGGATGGAGCTACG-3 and 5-TCCTCTAGAACC
ACGAGCCTTCCCATTTCC-3 (XbaI sites are underlined). The
PCR products were digested by XbaI, and inserted into the SpeI
site of pBI121-35SMCS-GFP (Tsugama etal., 2012a), generating
pBI121-35S-BZR1-GFP. To generate transgenic plants expressing
bzr1-1GFP, PCR was performed using pBI121-35S-BZR1-GFP
as template and either of the following two primer pairs: (i) 5-GT
TTCATACCCTGGCTACTATACCTGAATGTGATG-3 and 5-T
CCTCTAGAACCACGAGCCTTCCCATTTCC-3; or (ii) 5-GG
GTCTAGAATGACTTCGGATGGAGCTACG-3 and 5-GGTA
TAGTAGCCAGGGTATGAAACTGGTGGCGATG-3. The two
kinds of PCR products obtained with (i) and (ii) were mixed and
used as template for PCR using the following primer pair: 5-GG
GTCTAGAATGACTTCGGATGGAGCTACG-3 and 5-TCC
TCTAGAACCACGAGCCTTCCCATTTCC-3 (XbaI sites are
underlined). The resultant PCR products, which correspond to the
bzr1-1 DNA fragment, were digested by XbaI, and inserted into
the SpeI site of pBI121-35SMCS-GFP, generating
pBI121-35Sbzr1-1-GFP. The wild type (WT) and agb1-1 were transformed
with either pBI121-35S-BZR1-GFP or pBI121-35S-bzr1-1-GFP by
the Agrobacterium-mediated floral dip method (Clough and Bent,
1998). GFP expression in T2 plants was checked by fluorescence
microscopy as previously described (Zhang etal., 2008), and only
GFP-positive plants were used for measuring hypocotyl lengths,
scoring green cotyledons, and western blotting.
Western blot analysis of BZR1 phosphorylationstates
Transgenic plants expressing BZR1GFP were grown in the
presence or absence of BR, BRZ, or bikinin as described above, and
sampled at the time points indicated in the figures. Cell extracts were
prepared as previously described (Tsugama et al., 2011), and used
for western blotting using anti-GFP antibody (MBL, Japan). Signals
were detected using SuperSignal West Pico Chemiluminescent
Substrate (Thermo Fisher Scientific) and an LAS-1000 plus image
analyzer (Fuji Film, Japan). Images were processed with Canvas X
software (ACD Systems).
Reverse transcriptionPCR (RTPCR)
Plants were grown with 0.5M ABA for 20 d or without ABA for
10 d, and sampled. Total RNA was prepared as previously described
(Chomczynski and Sacchi, 1987), and cDNA was synthesized from
2 g of the total RNA with PrimeScript Reverse Transcriptase
(TakaraBio, Japan) using an oligo(dT) primer. The reaction
mixtures were diluted 25 times with distilled water and used as templates
for PCR. GoTaq Green Master Mix (Promega) was used for
semiquantitative RTPCR, and GoTaq qPCR Master Mix (Promega)
for quantitative RTPCR. Primers used for RTPCR are given
in Supplementary Table S1 available at JXB online. In
quantitative RTPCR, the PCR was run using a StepOne Real-Time PCR
System (Applied Biosystems), and relative expression levels were
calculated by the comparative CT method using UBQ5 as an
internal control gene.
Prediction of three-dimensional structure and GSK modification
The three-dimensional structure of AGB1 (AT4G34460.1) was
predicted using SWISS-MODEL (http://swissmodel.expasy.org). The
output pdb file was read by RasMol (http://www.openrasmol.org)
to visualize the structure. GSK modification sites in AGB1 were
predicted by the Eukaryotic Linear Motif database (ELM, http://elm.
eu.org). The predicted 17 GSK modification sites in AGB1 (amino
acid positions 4653, 6774, 93100, 109116, 132139, 142149,
146153, 173180, 185192, 206213, 208215, 221228, 231238,
264271, 296303, 347354, and 351358) were highlighted using
In vitro GST pull-downassay
Hexahistidine-tagged AGB1 (His-AGB1) was expressed in
Escherichia coli and purified as previously described (Tsugama
etal., 2012b). Expression of His-AGB1 in E.coli was confirmed by
western blotting using HisProbe-HRP (Thermo Fisher Scientific).
To express glutathionine S-transferase (GST)-fused BIN2 (GST
BIN2), the ORF of BIN2 (AT4G18710) was amplified by RTPCR
using the following primer pair: 5-GAGGATCCATGGCTGATGA
TAAGGAGATGCC-3 and 5-CCCACTAGTTCCAGATTGATT
GATTCAAGAAGC-3 (BamHI site is underlined). The PCR
products were digested by BamHI and inserted into the BamHISmaI
site of pGEX-6P-3 (GE Healthcare). This construct was
transformed into the E.coli strain, BL21 (DE3). Transformed cells were
cultured at 37 C in LB medium until the OD600 reached 0.5, and
was then incubated at 28C for 2 h after addition of isopropyl--d
thiogalactopyranoside (IPTG) to a final concentration of 0.2mM
to express GSTBIN2. Expression of GSTBIN2 was confirmed
by western blotting using an anti-GST antibody (GE Healthcare).
Crude E. coli extracts were prepared as previously described
(Tsugama et al., 2012b). GSTBIN2 in the E. coli extracts was
bound to glutathioneSepharose 4 Fast Flow (GE Healthcare)
following the manufacturers instructions, and washed four times with
1 Tris-buffered saline (TBS). A solution containing purified
HisAGB1 was added to the GSTBIN2-bound resin, and the mixture
was incubated at room temperature for 30 min with gentle shaking.
The resin was then washed four times by TBS, resuspended in 20 mM
reduced glutathione in 50 mM TRIS-HCl, pH 8.0, and incubated at
room temperature for 10 min to elute GSTBIN2. His-AGB1 in the
elutant was analysed by western blotting using HisProbe-HRP. For
negative controls, 250 mM imidazole was used instead of the
solution containing His-AGB1, and GST alone instead of GSTBIN2.
After detecting His-AGB1, the blot was washed three times by
Tweenphosphate-buffered saline (PBS)EDTA, which was made
by adding 0.5 M EDTA, pH 8.0, to TweenPBS (0.1% v/v
Tween20 in PBS) to 10 mM final concentration of EDTA, for deprobing
the HisProbe-HRP. The blot was then washed twice by TweenTBS
and used for a western blot analysis of phosphoproteins using
Phostag Biotin BTL-104 (Wako, Japan) (Kinoshita etal., 2006). Signal
detection and image processing were performed as described above.
Yeast three-hybrid (Y3H)assays
pGBK-AGB1 (Tsugama et al., 2012b) was digested by HpaI and
SalI, and the resultant DNA fragment containing the full-length
ORF of AGB1 and a partial coding sequence (CDS) of the GAL4
DNA-binding domain (GAL4BD) was inserted into pBridge
(Clontech), generating pBridge-AGB1. The ORF fragment of
AGG1 (AT3G63420) was obtained by PCR using pGAD-AGG1
(Tsugama et al., 2012b) as template and the following primer
pair: 5-GAGAGATCTATGCGAGAGGAAACTGTGGT-3 and
5-CCTAGATCTAAGTATTAAGCATCTGCAGCC-3 (BglII sites
are underlined). The PCR products were digested by BglII,
and inserted into the BglII site of pBridge-AGB1,
generating pBridge-AGB1-AGG1. The ORF of AGG1 was obtained
by digesting pGAD-AGG1 by NdeI and XhoI, and inserted
into the NdeISalI site of pGBKT7 (Clontech), generating
pGBK-AGG1. The ORF of AGG1 was obtained by PCR using
pGAD-AGG1 as template and the following primer pair: 5-G
AGGAATTCATGCGAGAGGAAACTGTGGT-3 and 5-CC
TAGATCTAAGTATTAAGCATCTGCAGCC-3 (EcoRI and BglII
sites are underlined). The PCR products were digested by EcoRI
and BglII, and inserted into the EcoRIBamHI site of pBridge,
generating pBridge-AGG1. The ORF of AGB1 was obtained by PCR
using pGBK-AGB1 as template and the following primer pair: 5-G
AGGGATCCATGTCTGTCTCCGAGCTCAAAG-3 and 5-C
CCGGATCCTCAAATCACTCTCCTGGTCC-3 (BamHI sites
are underlined). The PCR products were digested by BamHI, and
inserted into the BglII site of either pBridge or pBridge-AGG1,
generating pBridge-2-AGB1 or pBridge-AGG1-AGB1, respectively. The
ORFs of BIN2 were obtained by digesting pGEX-6P-BIN2 by NcoI
and SpeI, and were inserted into the NcoIXbaI site of
pGADT7Rec (Clontech), generating pGAD-BIN2. The ORF fragments of
BIN2 were obtained again by digesting pGAD-BIN2 by NcoI and
BamHI, and were inserted into the NcoIBamHI site of pGBKT7
(Clontech), generating pGBK-BIN2. pGBK-BIN2 was digested
by HpaI and PstI, and the resultant fragment containing the
fulllength ORF of BIN2 and a partial CDS of GAL4BD was inserted
into the HpaIPstI site of pBridge-2-AGB1, generating
pBridgeBIN2-AGB1. The ORF of BZR1 was amplified by PCR using a
cDNA clone of BZR1 (RAFL04-20-E20), which was obtained from
RIKEN BRC Experimental Plant Division (Seki et al., 2002), as
template and the following primer pair: 5-GAGGAATTCATGAC
TTCGGATGGAGCTACG-3 and 5-TCCTCTAGAACCACGAG
CCTTCCCATTTCC-3 (EcoRI and XbaI sites are underlined). The
PCR products were digested by EcoRI and XbaI, and inserted into
the EcoRIXbaI site of pGADT7-Rec, generating pGAD-BZR1.
The Saccharomyces cerevisiae strain AH109 was transformed with
combinations of pGAD and pBridge constructs. After
transformation, at least four colonies grown on synthetic dextrose (SD) medium
lacking leucine and tryptophan (SD/Leu/Trp), were streaked on
SD/Leu/Trp and SD/Leu/Trp lacking histidine or lacking both
histidine and adenine. Reporter gene activation was quantified by a
-galactosidase assay as described in the Yeast Protocols Handbook
Bimolecular fluorescence complementation(BiFC)
The ORF of BIN2 was amplified using pGBK-BIN2 as
template and the following primer pair: 5-GAGTCTAGAATGGC
TGATGATAAGGAGATGCC-3 and 5-CCCACTAGTTCCAGA
TTGATTGATTCAAGAAGC-3 (XbaI and SpeI sites are
underlined). The PCR products were digested by XbaI and SpeI, and
inserted into the SpeI site of pBS-35SMCS-cYFP (Tsugama etal.,
2012b), generating pBS-35S-BIN2-cYFP. The ORF of AGG1 was
amplified by PCR using pGBK-AGG1 as template and the
following primer pair: 5-GGGACTAGTATGCGAGAGGAAACTGT
GG-3 and 5-CCACTAGTAAGTATTAAGCATCTGCAGCC-3
(SpeI sites are underlined). The PCR products were digested by SpeI
and inserted into the SpeI site of pBS-35SMCS-cYFP, generating
pBS-35S-AGG1-cYFP. These cYFP (C-terminal yellow fluorescent
protein) constructs and pBS-35S-nYFP-AGB1 (Tsugama et al.,
2012b) was used to co-express cYFP-fused protein and nYFP-fused
AGB1 in Arabidopsis mesophyll protoplasts. Arabidopsis mesophyll
protoplasts were prepared and transformed as previously described
(Yoo etal., 2007; Wu etal., 2009). Recovered YFP fluorescence was
observed by fluorescence microscopy 12h after transformation.
ABA enhances the BR hyposensitive phenotype of
The leaves of agb1-1 and agb1-2 are rounder and their
petioles are shorter than those of the WT. It was discovered
that in the presence of ABA, leaves of agb1-1 and
agb12 become even rounder and their petioles become even
shorter. These ABA-induced phenotypes of agb1-1 and
agb1-2 were similar to the phenotypes of mutants which
have severe defects in BR biosynthesis or BR signalling (for
a review, see Clouse, 2011). To examine whether the agb1
phenotypes are due to impaired BR signalling, a BR
biosynthesis inhibitor, BRZ, was tested for its effects on the
phenotypes of agb1-1 and agb1-2. BRZ made leaves rounder
and petioles shorter in all the genotypes including the WT
and gpa1-4. However, the leaf morphologies of agb1-1 and
agb1-2 appeared to be more strongly affected by BRZ than
those of the WT and gpa1-4 (Fig.1A; Supplementary Fig.
S1 at JXB online). BRZ also inhibited hypocotyl
elongation in the dark more severely in agb1-1 and agb1-2 than
in the WT and gpa1-4 (Fig. 1B; Supplementary Fig. S2),
suggesting that agb1 is hypersensitive to BRZ. Exogenously
added BR induced hypocotyl elongation in agb1-1 and
agb1-2, but the elongation was less than that in the WT
(Supplementary Fig. S3). Together, these results suggest
that agb1-1 and agb1-2 are hyposensitive to BR and that
their BR-hyposensitive phenotypes are enhanced by ABA.
The BR hyposensitivities of agb1 in seed germination were
previously reported (Chen etal., 2004), but AGB1 was not
well characterized as a regulator of BR signalling. In
previous studies, BR signalling mutants showed ABA
hypersensitivities (for a review, see Clouse, 2011), supporting the
idea that the ABA hypersensitivities of agb1 are at least
partly dependent on its BR hyposensitivity. Based on these
results, it was hypothesized that AGB1 regulates BR
signalling, and the interactions between AGB1 and BR signalling
were further examined.
Expression of bzr1-1 alleviates effects of BRZ in both
the wild type and agb1
BZR1 is a transcription factor downstream of BR signalling
(for a review, see Kim and Wang, 2010). To examine BZR1
functions in agb1, BZR1 and its point-mutated (PL at
amino acid position 234) version, bzr1-1, were expressed as
GFP-fused proteins (BZR1GFP and bzr1-1GFP) in agb1-1
as well as in the WT (Table1). Expression of BZR1-GFP and
bzr1-1-GFP in transgenic plants was confirmed by RTPCR
(Supplementary Fig. S4 at JXB online). Expression of bzr1-1
is known to alleviate BRZ-induced inhibition of hypocotyl
elongation in the dark (Wang etal., 2002; Ryu etal., 2007).
In the presence of BRZ, the hypocotyl length of bzr1-1/a was
greater than that of agb1-1 but smaller than the hypocotyl
lengths of the WT and bzr1-1/WT. In the absence of BRZ,
the hypocotyl length of bzr1-1/a was comparable with that of
agb1-1 and slightly smaller than the hypocotyl lengths of the
WT and bzr1-1/WT (Fig.2A; Supplementary Fig. S5). These
results suggest that expression of bzr1-1 partially suppresses
the BRZ hypersensitivity of agb1 but does not fully suppress
the agb1 phenotypes.
BR is known to induce dephosphorylation and activation
of BZR1 (He et al., 2002). However, neither BR nor BRZ
Fig.1. BRZ hypersensitivity of agb1. (A) ABA- and
BRZhypersensitive phenotypes of agb1. Plants were grown under a
16 h light/8 h dark photoperiod in the presence of either 0.5M
ABA or 5M BRZ for 25 d (+ ABA or + BRZ, respectively), or in
the absence of ABA or BRZ for 10 d (Control). Arepresentative
plant is shown for each genotype. Scale bars=3 mm. (B) BRZ
hypersensitivity of agb1 in hypocotyl elongation in the dark. Plants
were grown for 5 d in the dark in the presence of 0, 0.5, or 1M
BRZ. Relative hypocotyl lengths are shown (for absolute lengths,
see Supplementary Fig. S2 at JXB online). Values are means SE
(n=1118). *P<0.05 vs. the WT by Students t-test.
Table1. Transgenic plants used in this study
Fig.2. Functions of BZR1 in agb1. (A) Expression of bzr1-1
partially suppresses BRZ hypersensitivity of agb1-1. Plants were
grown in the dark in the presence of 0, 0.5, or 1M BRZ for 4 d,
and their hypocotyl lengths were measured. Relative hypocotyl
lengths are shown (for absolute lengths, see Supplementary Fig.
S5 at JXB online). Values are means SE (n=1524). *P<0.05 vs.
non-transgenic lines by Students t-test. (B) AGB1 is not involved
in BR-dependent regulation of BZR1 phosphorylation states.
BZR1-GFPox/WT #9 and BZR1-GFPox/agb1-1 #9 (only genetic
backgrounds, WT and agb1-1, are shown) were grown in the
presence of 20 nM BR for 15 d (+ BR), in the presence of 2.5M
BRZ for 25 d (+ BRZ), or in the absence of BR and BRZ (Control),
and used for western blotting using an anti-GFP antibody (WB:
GFP). Experiments were performed in triplicate, and representative
results are shown.
caused differences in BZR1GFP phosphorylation states
between BZR1/WT and BZR1/a (Fig. 2B), suggesting that
AGB1 is not involved in BR-dependent changes of BZR1
Overexpression of BZR1 suppresses effects of ABA in
both the wild type and agb1
BZR1/WT, bzr1-1/WT, BZR1/a, and bzr1-1/a were grown
in the presence of ABA. Interestingly, BZR1/WT but not
bzr1-1/WT was larger in size than the WT. Similarly, BZR1/a
but not bzr1-1/a was larger in size than agb1-1. The leaf
morphology of BZR1/a was similar to that of agb1-1 rather than
that of the WT (Fig.3A). The expresson level of bzr1-1-GFP
in bzr1-1/a #6 was higher than the expression level of
BZR1GFP in BZR1/a #9 (Supplementary Fig. S6 at JXB online).
These results suggest that BZR1 rather than bzr1-1
alleviates the ABA responses. ABA responses of BZR1/WT and
BZR1/a were further evaluated by scoring their green
cotyledons in the presence of ABA. The cotyledon greening rate
in the presence of ABA was in the order of BZR1/WT > WT
> BZR1/a > agb1-1 (Fig.3B), suggesting that overexpression
of BZR1 alleviates ABA-induced growth retardation in both
the WT and agb1, and that overexpression of BZR1 cannot
fully suppress the ABA hypersensitivity of agb1. Among
BZR1GFP-overexpressing lines, BZR1 expression levels
and cotyledon greening rates in the presence of ABA were
not correlated (Supplementary Fig. S7), which suggests that
BZR1-dependent responses are saturated in the transgenic
The expression levels of ABA-responsive genes, RAB18
and RD29A (Umezawa etal., 2006), were lower in BZR1/WT
and BZR1/a than in the WT and agb1-1 in the presence of
ABA (Fig.3C, upper panels), which is in agreement with the
finding that overexpression of BZR1 alleviates the effects of
ABA on cotyledon greening and subsequent growth (Fig.3A,
B). Under control conditions, the expression of BZR1 target
(thereby BR-responsive) genes, CPD and DWF4 (He et al.,
2005), was higher in agb1-1 than in the WT, which is
consistent with the finding that agb1 is hyposensitive to BR. The
CPD expresson level was lower in BZR1/a than in agb1-1, but
no significant difference was observed in the DWF4
expression levels between agb1-1 and BZR1/a (Fig.3C, lower
panels). ABA increased the DWF4 expression levels in both the
WT and agb1-1, but both BZR1/WT and BZR1/a showed
lower expression levels of DWF4 than either the WT or
agb11 in the presence of ABA (Fig.3C, lower right panel), again
supporting the idea that BZR1 overexpression alleviates the
effects of ABA. ABA significantly decreased the CPD
expression levels and, in the presence of ABA, no difference was
observed in the CPD expression levels among all the
genotypes studied (Fig.3C, lower left panel).
AGB1 interacts withBIN2
A motif-scanning program (ELM, http://elm.eu.org)
identified 17 possible GSK modification sites in the amino acid
sequence of AGB1. GSKs are known to regulate BR signalling
negatively in Arabidopsis (He etal., 2002; Rozhon etal., 2010).
Some of the putative GSK modification sites are located on the
surface of the predicted three-dimensional structure of AGB1
(Fig.4A), raising the possibility that AGB1 interacts with GSKs
and is phosphorylated by them. To test this idea, the
interaction between AGB1 and BIN2, the best-characterized plant
GSK, was examined by an in vitro GST pull-down assay using
His-AGB1 and GSTBIN2. His-AGB1 and GSTBIN2 were
expressed in E. coli (Supplementary Fig. S8 at JXB online).
GSTBIN2 was bound to resin and mixed with purified
HisAGB1. After incubation, GSTBIN2 was eluted from the resin
and His-AGB1 in the elutant was analysed by western blotting.
His-AGB1 was detected only when both His-AGB1 and GST
BIN2 were present in the reaction mixture (Supplementary
Fig. S9), suggesting that AGB1 and BIN2 interact in vitro.
Neither ATP, which is required for phosphorylation by protein
kinases, nor a GSK inhibitor, bikinin (De Rybel etal., 2009),
affected the level of His-AGB1 detected in the pull-down assay
(Fig. 4B, upper panel), suggesting that the kinase activity of
BIN2 does not affect the interaction between AGB1 and BIN2.
After the pull-down assay, phosphorylated proteins on the
same blot were detected using a phosphoprotein probe,
Phostag (Kinoshita etal., 2006). However, no clear difference was
observed in the banding patterns of phosphoproteins in the
presence or absence of His-AGB1 (Fig.4B, lower panel),
suggesting that AGB1 is not phosphorylated by BIN2, and that
AGB1 does not affect BIN2 autophosphorylation.
The AGB1BIN2 interaction was further examined by a
yeast three-hybrid (Y3H) assay. In the Y3H system, three
kinds of protein of interest were co-expressed as GAL4
activation domain (AD)-fused, GAL4 DNA-binding domain
(BD)-fused, and haemagglutinin (HA)-tagged forms,
respectively, in yeast cells, and subsequent reporter gene activation
was checked by culturing the transformed cells on a selection
medium. Yeast cells could grow on the selection medium when
transformed with AD-fused BIN2 (AD-BIN2), BD-fused
AGG1 (BD-AGG1), and HA-tagged AGB1 (HA-AGB1),
whereas yeast cells could not grow when one of AD-BIN2,
BD-AGG1, or HA-AGB1 was not expressed (Fig.4C, upper
four panels), suggesting that these three proteins can form
a complex in yeast cells. Yeast cells also did not grow when
the combination AD-BIN2, BD-AGB1, and HA-AGG1 was
used (i.e. AGB1 and AGG1 for BD/HA fusions were swapped)
(Fig.4C, bottom). In this case, the BD may have interrupted
the interaction between BIN2, AGB1, andAGG1.
A BiFC assay was also performed. The ORF of AGB1 was
fused downstream of the ORF of nYFP and the ORF of BIN2
was fused upstream of the ORF of cYFP. When nYFP-fused
Effects of AGB1 on the interaction between BIN2
BZR1 is a substrate of GSKs (He etal., 2002; Rozhon etal.,
2010). The Y3H system was used to evaluate the effect of
HA-AGB1 on the reporter gene activations, which are
dependent on co-expressed AD-BZR1 and BD-BIN2. In either the
presence or absence of HA-AGB1, yeast cells transformed
with AD-BZR1 and BD-BIN2 could grow on
high-stringency selection medium, but the activity of -galactosidase,
one of the reporter gene products, was lower in the presence
of HA-AGB1 than in its absence (Supplementary Fig. S11 at
JXB online), suggesting that HA-AGB1 can interfere to some
extent with the interaction between BZR1 and BIN2 inyeast.
To examine the effects of AGB1 on the functions of GSKs
in vivo, BZR1/WT, bzr1-1/WT, BZR1/a, and bzr1-1/a were
grown in the presence of BR or bikinin, and their phenotypes
were compared. BR- or bikinin-induced hypocotyl
elongation was greater in BZR1/WT and bzr1-1/WT than in the
WT. Similarly, BR- or bikinin-induced hypocotyl
elongation was greater in BZR1/a and bzr1-1/a than in agb1-1. The
effect of bzr1-1 overexpression on the BR- or bikinin-induced
hypocotyl elongation was similar to the effect of BZR1
overexpression (Fig.5A). In a previous study, bzr1-1
overexpression caused greater BR-induced hypocotyl elongation than
BZR1 overexpression (Ryu etal., 2007), which conflicts with
the present result. Although the expression level of
bzr1-1GFP in bzr1-1/a #6 was higher than the expression level of
BZR1-GFP in BZR1/a #9 (Supplementary Fig. S6 at JXB
online), the expression levels of bzr1-1-GFP still might have
been insufficient to cause the greater hypocotyl elongation. In
the presence of bikinin, BZR1GFP was detected as a single,
lower sized band in both BZR1/WT and BZR1/a (Fig.5B).
In the presence of BR, BRZ, or bikinin, no clear difference
was observed in BZR1GFP localization between the WT
and agb1-1 (Supplementary Fig. S12). These results suggest
that AGB1 is not involved in GSK-dependent BZR1
phosphorylation in vivo.
This study has shown that the ABA hypersensitivity of agb1
is partially suppressed by overexpressing BZR1 (Fig.3),
suggesting that the ABA hypersensitivity of agb1 is at least partly
dependent on the BR hyposensitivity of agb1. Although
AGB1 is not phosphorylated by BIN2 (Fig.4B) and does not
affect the phosphorylation states of BZR1 or BIN2 (Figs 2,
4B, 5B), AGB1 has putative GSK modification sites on its
surface (Fig.4A) and physically interacts with BIN2 (Fig.4B,
C), supporting the idea that AGB1 regulates BR responses
via interaction with GSKs. Amodel proposed by this study is
shown in Fig.5C.
Overexpression of bzr1-1 suppressed BRZ-hypersensitive
phenotypes of agb1-1, but the hypocotyl lengths of
bzr11/a were still lower than those of the WT and bzr1-1/WT
(Fig. 2A). Similarly, BR- or bikinin-induced hypocotyl
Fig.5. AGB1 is not involved in GSK-dependent regulation of
BZR1 functions. (A) Overexpression of BZR1 and bzr1-1 partially
suppresses the BR hyposensitivity of agb1-1. Plants were
grown under a 16 h light/8 h dark photoperiod for 15 d in the
presence of either 20 nM BR (+ BR) or 40M bikinin (+ Bikinin),
or in the absence of BR or bikinin (Control), and their hypocotyl
lengths were measured. Values are means SE (n=1522).
*P<0.05 vs. non-transgenic lines by Students t-test. (B) AGB1
is not involved in GSK-dependent phosphorylation of BZR1.
BZR1-GFPox/WT #9 and BZR1-GFPox/agb1-1 #9 (only genetic
backgrounds, WT and agb1-1, are shown) were grown for 15
d in the presence of 0 (Control) or 40M bikinin (+ Bikinin),
and used for western blotting using an anti-GFP antibody
(WB: GFP). Experiments were performed in triplicate and a
representative result is shown. (C) Amodel proposed by this
elongation was not as great in BZR1/a and bzr1-1/a as it
was in the WT, BZR1/WT, and bzr1-1/WT (Fig.5A). These
results indicate that BZR1 can enhance BR responses even
in agb1, although AGB1 is required to activate BR responses
fully. Because AGB1 is not necessary for regulating either
BZR1 phosphorylation states or the subcellular
localization of BZR1 (Figs 2B, 5B; Supplementary Fig. S10 at JXB
online), AGB1 does not seem to be involved in regulating the
functions of BZR1. BZR2 is another key transcription factor
regulating the BR responses. It is also unlikely that AGB1
regulates the functions of BZR2 because BZR2 is similar
to BZR1 in several respects, including amino acid sequence
(88% identity), physiological function (Wang etal., 2002; Yin
etal., 2002; for a review, see Kim and Wang, 2010), BR- and
GSK-dependent phosphorylation states (He etal., 2002; Yin
etal., 2002; Vert and Chory, 2006; Yan etal., 2009; Rozhon
etal., 2010), and target genes (He etal., 2005; Vert and Chory,
2006; Sun etal., 2010).
AGB1 was originally identified as ELK4
(ERECTALIKE4), a gene responsible for the erecta (er) phenotype.
agb1/elk4 shows several er phenotypes (e.g. rounder leaves
with shorter petioles, shorter stems, more highly clustered
flower buds, and shorter and wider siliques) (Lease et al.,
2001). The well-studied BR-related mutants have these
phenotypes as well (for a review, see Clouse, 2011), although
the phenotypes of the BR-related mutants seem much more
severe than those of er and agb1. ER encodes a receptor-like
kinase (RLK) which has 20 extracellular leucine-rich repeats
(LRRs), a single transmembrane domain, and an
intracellular serine/threonine protein kinase domain (Lease et al.,
2001). Interestingly, a BR receptor, BRI1, and its co-receptor,
BAK1, are both LRR-RLKs. BR-mediated
heterodimerization of BRI1 and BAK1 is thought to promote BR
signalling (for a review, see Kim and Wang, 2010). Further studies
are required to determine whether AGB1 and ER interact or
whether ER is involved in BR signalling.
Overexpression of BZR1 alleviated the effects of ABA on
cotyledon greening and subsequent growth in both the WT
and agb1-1 (Fig.3). This is consistent with previous studies
suggesting that mutants which have impaired BR responses
are hypersensitive to ABA (for a review, see Clouse, 2011).
BZR1-GFPox/agb1-1 showed lower cotyledon greening
rates in the presence of ABA than the WT (Fig. 3B). It is
unclear how BR signalling decreases the ABA responses, but
BZR1-mediated BR responses are thought to be insufficient
to suppress the ABA hypersensitivity of agb1 fully. It is also
interesting that BZR1 overexpression rather than bzr1-1
overexpression alleviated the ABA effects (Fig. 3A). BZR1
and bzr1-1 have been suggested to differ in their protein
stabilities (He etal., 2002; Wang etal., 2002; Tang etal., 2011),
phosphorylation states (He et al., 2002; Tang et al., 2011),
and subcellular localizations (Ryu et al., 2007; this study,
Supplementary Fig. S12 at JXB online). In addition, bzr1-1
has a higher affinity for a subunit of PP2A than does BZR1
(Tang etal., 2011). BZR1 and bzr1-1 may have different
affinities for other interaction partners that affect the expression
of BZR1 target genes. These differences might lead to the
different effects of BZR1 and bzr1-1 on the gene expression
involved in the ABA responses. Further studies are required
to elucidate the molecular mechanisms underlying
BZR1mediated ABA signalling.
AGB1 was shown to interact with BIN2 in vitro (Fig.4B).
However, AGB1 did not affect the kinase activity of BIN2
(Figs 4B, 5B) and was not phoshorylated by BIN2 (Fig.4B).
Thus the physiological relevance of the interaction between
AGB1 and BIN2 is unclear. One possibility is that AGB1
regulates the phosphorylation of BIN2 substrates other than
BZR1 and BZR2. ARF2 and YODA are two examples of
BIN2 substrates other than BZR1. ARF2 is a transcription
factor regulating auxin-responsive gene expression (Vert
et al., 2008). YODA is a mitogen-activated protein kinase
kinase kinase (MAPKKK) regulating stomatal development,
and, interestingly, YODA has been suggested to be under the
control of ER signalling as well as BIN2-dependent
signalling (Kim etal., 2012). AGB1 is also involved in auxin
signalling in Arabidopsis (Ullah etal., 2003), and Pisum sativum G
interacts with a MAPK (Bhardwaj etal., 2011). Therefore, it
will be interesting to examine whether AGB1 regulates the
BIN2-dependent phosphorylation of ARF2 and YODA.
Supplementary data are available at JXB online.
Figure S1. Phenotypes of agb1 grown in different
Figure S2. Absolute hypocotyl lengths of agb1 grown in
the presence ofBRZ.
Figure S3. BR-induced hypocotyl elongation in agb1.
Figure S4. Semi-quantitative RTPCR analysis of
BZR1GFP and bzr1-1-GFP expression.
Figure S5. Absolute hypocotyl lengths of
bzr1-1GFPoverexpressing plants grown in the presence ofBRZ.
Figure S6. Expression levels of BZR1-GFP and
bzr1-1GFP in transgenic plants.
Figure S7. BZR1 expression levels in
BZR1-GFPoverexpressing lines and their responses toABA.
Figure S8. Expression of His-AGB1 and GSTBIN2 in
Figure S9. In vitro GST pull-downassay.
Figure S10. BiFC between AGB1 andBIN2.
Figure S11. Effects of AGB1 on the interaction between
BIN2 and BZR1 in a Y3H system.
Figure S12. Subcellular localizations of BZR1GFP in agb1.
Table S1. Primer pairs used for RTPCR analyses.
This work was supported by a Grant-in-aid for Scientific
Research (21380002) to TT and (222144) to DT. We are
grateful to the ABRC for providing the Arabidopsis mutant
seeds. An Arabidopsis full-length cDNA clone
(RAFL0420-E20) was developed by the plant genome project of
RIKEN Genomic Sciences Center, and provided by RIKEN
BRC which is participating in the National Bio-Resource
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