Gibberellin signalling pathway

489
Gibberellin signalling pathway
Kenji Gomi and Makoto Matsuoka
Recent molecular biological and genetical studies have identified
several positive and negative regulators of gibberellin (GA)
signalling pathways in higher plants. The DELLA protein
functions as a negative regulator of GA signalling; its degradation
through the ubiquitin/proteasome pathway is a key event in the
regulation of GA-stimulated processes.
Addresses
BioScience Centre, Nagoya University, Nagoya 464-8601, Japan
e-mail: [email protected]
Current Opinion in Plant Biology 2003, 6:489–493
This review comes from a themed issue on
Cell signalling and gene regulation
Edited by Kazuo Shinozaki and Elizabeth Dennis
1369-5266/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/S1369-5266(03)00079-7
Abbreviations
d1
dwarf1
GA
gibberellin
gai
gibberellic-acid insensitive
GAMYB GA-regulated MYB transcription factor
GFP
green fluorescent protein
gid1
GA-insensitive dwarf1
KGM
KINASE ASSOCIATED WITH GAMYB
PHOR1
PHOTOPERIOD-RESPONSIVE1
rga
repressor of ga1–3
Rht
Reduced height
RSG
REPRESSION OF SHOOT GROWTH
SCF
Skp1–cullin–F-box
Skp1
Suppressor of kinetochore protein1
sln1
slender1
slr1
slender rice1
SLY1
SLEEPY1
spy
spindly
an almost complete cascade of the GA biosynthetic
process [1]. In contrast to the GA-biosynthesis cascade,
the mechanisms of GA signal transduction are still poorly
understood. Recent studies have revealed that a negative
regulator functions as a molecular switch in GA signalling
[6–10,11]. In this review, we summarise recent findings
on the GA signalling pathway, focusing on studies carried
out in the past two years.
Positive regulation of GA signalling
GAMYB is a GA-regulated MYB transcription factor that
was first identified as an activator of a-amylase expression
in barley aleurone cells [12–14]. A recent study demonstrated that the role of GAMYB is not restricted to
aleurone cells but is also involved in anther development
in barley [15]. Furthermore, GAMYB interacts with
KGM (KINASE-ASSOCIATED WITH GAMYB), which
was isolated by yeast two-hybrid screening using GAMYB
as bait. KGM is a member of an emerging subgroup of
protein kinases and it represses GAMYB function in
barley aleurone [16]. Although the phosphorylation of
GAMYB by KGM has not been demonstrated and the
detailed function of KGM is not yet clear, the characterisation of KGM function will provide new insights into
GA signalling pathways.
Introduction
The rice dwarf1 (d1) mutant has a GA-insensitive dwarf
phenotype. The D1 gene encodes an a-subunit of heterotrimeric G proteins [17,18]. Phenotypic analysis revealed
that increased expression of the GA 20-oxidase gene led to
the accumulation of high concentrations of bioactive GA in
the stunted internodes of d1 mutants. Furthermore, analysis of a d1 and slender rice1 (slr1) double mutant revealed
that SLR1 is epistatic to D1 [19]. These results indicate
that D1 functions as a positive regulator of GA signalling.
Some recent studies have demonstrated, however, that the
D1 protein also functions in auxin signalling and disease
resistance [20,21]. More precise analysis will be necessary
to identify the overall function of the D1 protein.
Gibberellins (GAs) are a large family of tetracyclic diterpenoid plant growth regulators. To date, 126 GAs have
been identified in higher plants, fungi and bacteria [1].
They are associated with several plant growth and development processes, such as seed germination, stem elongation, flowering, fruit development and the regulation
of gene expression in the cereal aleurone layer [2–4].
Many GA-related mutants have been isolated from various plant species [3,4,5], and these mutants have
helped to determine the physiological role of GA and
to elucidate its biosynthetic pathways. Genes that
encode GA-catalytic enzymes have been identified using
GA-deficient mutants, enabling researchers to build
REPRESSION OF SHOOT GROWTH (RSG) is
thought to be a positive regulator of the GA-biosynthetic
pathway in tobacco. Transgenic tobacco plants expressing
a dominant-negative form of RSG had a dwarf phenotype
and a reduced concentration of the active GA, GA1. RSG,
which contains a basic leucine-zipper (bZIP) domain,
transactivated the expression of the ent-kaurene oxidase
gene through interaction with its promoter sequence [22].
Recently, it has been demonstrated that 14-3-3 proteins
bind RSG and control its subcellular localisation, thus
regulating its efficiency as a transcriptional effector of
GA-synthesis genes in the nucleus [23].
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Current Opinion in Plant Biology 2003, 6:489–493
490 Cell signalling and gene regulation
PHOTOPERIOD-RESPONSIVE1 (PHOR1) encodes the
armadillo-repeat (arm-repeat) protein, which is upregulated in potato leaves under conditions that induce
tuberisation. PHOR1-antisense plants have a semi-dwarf
phenotype similar to that of GA-deficient mutants and
exhibit reduced GA responsiveness. A PHOR1::green
fluorescent protein (GFP) construct was transported from
the cytosol into the nucleus in response to GA treatment
[24], suggesting that PHOR1 acts as a positive regulator in
GA signalling.
The GA-insensitive dwarf1 (gid1) rice mutant has a GAinsensitive dwarf phenotype [25]. The GID1 gene
encodes a member of the serine hydrolase family, which
includes esterases, lipases, and proteases [25,26]. Although the enzymatic function of GID1 has not yet been
identified, analysis of a gid1 and slr1 double mutant has
revealed that SLR1 is epistatic to GID1. Consistent with
the idea that GID1 acts upstream of SLR1, SLR1 was not
degraded in the gid1 mutant when treated with GA,
whereas GA treatment causes rapid degradation of SLR1
in wildtype plants [26]. Recent studies have indicated
that the GID1 protein may be directly involved in the
degradation of the SLR1 protein (M Ueguchi-Tanaka,
M Matsuoka, unpublished data).
Negative regulation of GA signalling
GA-insensitive mutants that are defective in the DELLA
genes have been identified in screens of various plant
species, such as Arabidopsis (repressor of ga1–3 [rga] and
gibberellic-acid insensitive [gai]), barley (slender1 [sln1]),
maize (Dwarf8 [D8]), wheat (Reduced height [Rht]), and
rice (slr1) [6–10,11,27]. These mutants fall into two
classes: those caused by semi-dominant gain-of-function
mutations in Arabidopsis, maize, barley, and wheat, which
lead to GA-insensitive dwarfism; and those caused by
recessive loss-of-function mutations in barley and rice,
which lead to increased growth. The wheat Rht allele was
used to produce the wheat varieties that enabled the
‘green revolution’ [8].
The DELLA proteins are members of the GRAS family,
which also includes SCARECROW and SHORT ROOT
[28]. In addition to the GRAS family consensus motifs,
GA-signal-related DELLA proteins also contain unique
motifs in their amino-terminal region called DELLA
domains. These domains are absent from other GRAS
proteins. The sequence of the Arabidopsis gai allele
demonstrated that in-frame deletion mutations in the
DELLA domain induced the GA-insensitive dwarf phenotype of gai mutants [6]. Similarly, wheat Rht-B1/Rht-D1
and maize D8 alleles also have an in-frame deletion in the
DELLA or TVHYNP domain, respectively [8]. Furthermore, transgenic rice plants that overproduced an SLR1
protein that had a truncated DELLA domain showed a
dominant GA-insensitive dwarf phenotype [10,11]. On
the other hand, null alleles for these proteins, such as
Current Opinion in Plant Biology 2003, 6:489–493
those of rice slr1 and barley sln1 mutants, induced a
constitutive GA-responding phenotype [10,11]. These
results demonstrate that DELLA proteins function as
negative regulators of GA signalling, and that the
DELLA and TVHYNP domains are essential for this
function. Further domain analysis using transgenic rice
plants that overproduced different kinds of truncated
SLR1 proteins revealed that the SLR1 protein can be
divided into four parts: a GA signal perception domain at
the amino terminus (DELLA and TVHYNP), a regulatory domain that controls the protein’s repression activity
(S/T-rich domain), a dimer-formation domain (leucine
zipper), and a repression domain at the carboxyl terminus
[11]. DELLA proteins are localised in the nuclei where
they suppress the downstream GA action and are rapidly
degraded in response to GA signals [29–31]. It has been
suggested recently that GA-dependent degradation of
DELLA proteins is a key event for GA-signalling.
Arabidopsis spindly (spy) was first identified as a mutant
that is resistant to the GA-biosynthesis inhibitor paclobutrazol (PAC); spy germinates in the presence of PAC,
which blocks the germination of wildtype Arabidopsis
[32]. The SPY gene encodes a protein that is homologous
to O-GlcNAc transferase. Recent studies on the overexpression of SPY have indicated that SPY functions not
only as a negative regulator of GA signalling but also in
other signalling pathways. Filardo and Swain [33] discussed the possibilities of the negative regulation of
PHOR1 function by SPY and of crosstalk between the
GA response and the circadian clock.
Involvement of the ubiquitin/proteasome
pathway in GA signalling
Very recently, we isolated a new GA-insensitive dwarf rice
mutant, gid2, which has a severe dwarf phenotype without
any GA-responses [34]. The GID2 gene encodes a putative F-box protein, which interacted with a rice Skp1
(Suppressor of kinetochore protein1) homologue in a yeast
two-hybrid assay. GID2 is therefore expected to form a
Skp1–cullin–F-box (SCF) complex and to function as an
E3 ubiquitin ligase. In gid2 mutants, SLR1 accumulates in
a phosphorylated form even though these mutants also
accumulate high levels of active GA. In contrast, SLR1 is
rapidly degraded by GA through ubiquitination in the
wildtype [34]. The Arabidopsis SLEEPY 1 (SLY1) gene
has been isolated by positional cloning and found to
encode a putative F-box protein with structural similarity
to rice GID2. In the sly1 mutant, the DELLA protein
RGA accumulates to high concentrations even after GA
treatment [35]. Furthermore, a recent phenotypical analysis of barley sln1 revealed that the degradation of SLN1
protein is inhibited by proteasome and kinase inhibitors
[36]. These findings indicate that GA induces the degradation of DELLA-repressor proteins through the ubiquitin/proteasome pathway, mediated by the SCF complex.
The degradation mechanisms are probably not uniform
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Gibberellin signalling Gomi and Matsuoka 491
for all DELLA proteins, however, because Arabidopsis
GAI is not degraded as a result of GA3 treatment [37].
Further biochemical studies are needed to solve the
overall function of the DELLA proteins in the GA
signalling pathway.
degradation of target proteins [41]. Recent studies have
demonstrated that U-box proteins in mammals possess
ubiquitin ligase activity [42,43]. In this context, PHOR1
may act as an E3 ubiquitin ligase that degrades DELLA
proteins in the GA signalling pathway.
PHOR1, which contains an U-box domain at its amino
terminus, may also be involved in the degradation of
DELLA proteins. The U-box motif was first identified
in yeast UFD2, an E4 multiubiquitin chain assembly
factor, and was later recognised as a conserved domain
that is shared with proteins in various kinds of organisms
[38]. In Arabidopsis, 37 predicted U-box proteins have
been identified in database searches [39], although their
biological function has not yet been characterised. The
predicted three-dimensional structure of the U-box
domain is similar to that of RING fingers [40], some of
which have E3 ubiquitin ligase activity in triggering the
Conclusions
The processing of the GA signal in the nucleus depends
directly on the presence or absence of DELLA proteins,
which are therefore presently considered to be a ‘molecular switch’ for GA signalling (Figure 1). An SCF complex is essential for the degradation of DELLA proteins,
which results in the transduction of the GA signal. Rice
GID2 and Arabidopsis SLY1 are F-box proteins, which are
typically components of SCF complexes. Many F-box
proteins contain a protein–protein interaction domain,
such as a leucine-rich repeat (LRR) or a WD-40 repeat
sequence, which allows their interaction with target
Figure 1
GA
GA
D1
GA signal
PHOR1
Kinase
SPY
GID1
GID2/SLY1
SLR1/RGA
14-3-3 protein
PHOR1
KGM
RSG
RSG
GAMYB
Nucleus
GA-response genes
GA-mediated action
Cytoplasm
Current Opinion in Plant Biology
Possible roles of recently identified factors in the GA signalling pathway. In the absence of GA, DELLA protein (SLR1/RGA) directly or indirectly inhibits
the expression of GA-induced genes, including GAMYB. KGM inhibits GAMYB activity by phosphorylation. SPY activates the negative regulator and
inhibits the activity of the positive regulator by O-GlcNAc modification. GA binds to an unidentified GA receptor(s) and activates G proteins (D1) that
enhance the GA signal. PHOR1 is translocated into the nucleus, where it acts as a positive regulator by GA signalling. The GA signal also activates
protein kinase and GID1 to trigger GID2/SLY1-mediated degradation of SLR1/RGA. 14-3-3 proteins regulate the subcellular localisation of RSG,
which controls the expression of the ent-kaurene oxidase gene.
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Current Opinion in Plant Biology 2003, 6:489–493
492 Cell signalling and gene regulation
proteins [44–46]. GID2 and SLY1 do not contain such
interactive sequences, however, suggesting that they may
not interact directly with SLR1 and RGA. Additional
protein(s) may be required as mediators of the interaction
between GID2/SLY1 and their target proteins, SLR1/
RGA, respectively.
Phosphorylation of the DELLA protein (SLR1) triggers
its ubiquitin/proteasome-mediated degradation through
interaction with SCFGID2. In barley, a tyrosine kinase
inhibitor blocked the GA-induced degradation of SLN1
[36], indicating that GA-dependent phosphorylation of
DELLA protein forms part of the GA signalling pathway.
Thus, the next steps in unravelling the GA signalling
pathway are to identify DELLA-protein-specific kinases
and the phosphorylation site of DELLA proteins.
Furthermore, as DELLA proteins may not contain
DNA-binding domain, even though they seem to function as trans-acting factors, other interacting factors that
contain DNA-binding motifs should exist. At present,
there is little information on the relationship between
GAMYB and DELLA proteins. GA-induced expression
of GAMYB was inhibited in the barley dominant Sln1
mutant, indicating that SLN1 functions upstream of the
transcription of GAMYB in barley aleurone cells [31,47].
Whether GAMYB and DELLA proteins interact directly
is unclear; other protein(s) may be necessary to transduce
the signal from a DELLA protein to GAMYB. Finally, the
nature of the primary GA receptor is still obscure, despite
great efforts to identify it. At present, favoured hypotheses assume that there may be two types of GA receptor in
plant cells; one a plasma-membrane-bound receptor and
the other a cytoplasm-type receptor. The isolation and
characterisation of the GA receptor is one of the most
important targets in this field.
Acknowledgements
Our research on GA is supported by a Grant-in-Aid for Centres of Excellence,
a Grant-in-Aid from the program for the Promotion of Basic Research
Activities for Innovative Bioscience, and by the Ministry of Agriculture,
Fisheries and Food (MAFF) Rice Genome Project.
References and recommended reading
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