The enzymes, volume 35

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CONTRIBUTORS
Hiroo Fukuda
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Tokyo, Japan
Yuki Hirakawa
WPI-Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya,
Japan
Takato Imaizumi
Department of Biology, University of Washington, Seattle, Washington, USA
Toshinori Kinoshita
Institute of Transformative Bio-Molecules (WPI-ITbM) Nagoya, Japan
Yuki Kondo
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Tokyo, Japan
Jiayang Li
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research
(Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing, China
Chentao Lin
Department of Molecular, Cell & Developmental Biology, University of California, Los
Angeles, California, USA
Yasunori Machida
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya, Japan
Makoto Matsuoka
Bioscience and Biotechnology Center, Nagoya University, Chikusa Nagoya, Japan
Paula Nguyen

Department of Molecular, Cell & Developmental Biology, University of California, Los
Angeles, California, USA
Michiko Sasabe
Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University,
Hirosaki, Japan
Ken-ichiro Shimazaki
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan
Kazuo Shinozaki
RIKEN Center for Sustainable Resource Science, Tsukuba, Japan

ix


x

Contributors

Fuminori Takahashi
RIKEN Center for Sustainable Resource Science, Tsukuba, Japan
Ken-ichiro Taoka
Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara
Institute of Science and Technology, Nara, Japan
Hiroyuki Tsuji
Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara
Institute of Science and Technology, Nara, Japan
Miyako Ueguchi-Tanaka
Bioscience and Biotechnology Center, Nagoya University, Chikusa Nagoya, Japan
Taishi Umezawa
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
Qin Wang

The Basic Forestry and Biotechnology Center, Fujian Agriculture and Forestry University,
Fuzhou, China, and Department of Molecular, Cell & Developmental Biology, University of
California, Los Angeles, California, USA
Xu Wang
The Basic Forestry and Biotechnology Center, Fujian Agriculture and Forestry University,
Fuzhou, China, and Department of Molecular, Cell & Developmental Biology, University of
California, Los Angeles, California, USA
Yin Wang
Institute for Advanced Research, Nagoya University, and Institute of Transformative BioMolecules (WPI-ITbM) Nagoya, Japan
Yonghong Wang
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research
(Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing, China
Guosheng Xiong
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research
(Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing, China
Hideki Yoshida
Bioscience and Biotechnology Center, Nagoya University, Chikusa Nagoya, Japan
Brian D. Zoltowski
Department of Chemistry, Southern Methodist University, Dallas, Texas, USA


PREFACE
Plant growth is regulated by physiologically active substances called plant
hormones and is further influenced by various environmental stimuli,
including light. Studies of such active substances can be traced back to observations and writings of Charles Darwin and his son Francis more than
100 years ago. They observed that light induces bending of the plant hypocotyl and stimulates the stomatal opening, and hypothesized the involvement
of effective substances in these phenomena. Research on the molecular
mechanisms behind such phenomena had to wait until molecular genetic

studies with model plants such as Arabidopsis thaliana were developed.
Although plants endogenously produce plant hormones, exogenously
supplied plant hormones can also trigger responsive reactions similar to those
of endogenously induced ones. Based on these characteristics, mutants of
model plants that exhibit abnormalities in response to specific plant hormones and environmental stimuli have been isolated, and causative genes
and corresponding proteins have been identified. Furthermore, molecular
studies using protein–protein interactions, plant–pathogen interactions,
and actions of growth inhibitors have also contributed to the identification
of key molecules, such as receptors and downstream controllers that transmit
signals generated by these stimuli. These molecular studies have accelerated
biochemical understanding of the intracellular signaling pathways responsible for plant responses to stimuli.
This book includes reviews on current understanding of signaling pathways that control physiologically critical processes in plants. Most of the key
molecules in these pathways were discovered within the past decade. In
2005, Matsuoka’s group reported on the F-box-containing receptor of
gibellin (GA), which was selected as a “Breakthrough of the Year 2005”
by Science. In the history of abscisic acid (ABA) research, although several
receptor candidates had been considered, one molecule was eventually proposed in 2009 to control activities of a specific protein phosphatase and
kinase downstream of the receptor. Strigolactone (SL), identified in 2008
as the 8th plant hormone, is the new functional substance that induces germination of a parasitic plant and controls the branching pattern. Although at
least two factors and a receptor candidate, including the F-box, have been
identified within the SL signaling pathway, some points still remain to be
elucidated.
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Preface

Discoveries of peptides that act as signaling molecules in plants and their

corresponding receptors contributed important knowledge to the field of
developmental biology. Genome analyses have predicted that thousands
of functional peptides exist in plants. Although florigen, which induces
flowering, had long been hypothesized, its molecular features were eventually revealed around 2000 in Arabidopsis and rice. Cytokinesis in plants is
distinctive from that occurring in other eukaryotes. Molecular studies with
plant cells demonstrated that the unidentified transcriptional signal specifically generated at the G2-M transition of the cell cycle activates the mitotic
kinesin-mediated MAP kinase cascade that is essential for the execution of
complex and integrated cytokinetic events.
Concerning the typical environmental stimulus light, research on a blue
light photoreceptor and its downstream signaling pathway has recently
shown remarkable progress. The plant cryptochrome (CRY) involved in
controlling photomorphogenesis and the circadian clock was first identified
in plants in 1993 as a blue light receptor that controls COP1-mediated protein degradation. Around 2000, two new blue light receptors, phototropin
controlling the stomatal opening and ZTL (ZEITLUPE) responsible for the
circadian clock, were also discovered in plants and together commonly
encode the LOV domain. In addition, the former also codes the protein
kinase domain, whereas the latter codes the F-box domain, which suggests
that they may function in light signaling pathways. Recent advancements in
these investigations are introduced in this book.
Interestingly, four out of nine signaling pathways (for GA, SL, CRY, and
ZTL) described in this book include protein degradation systems involving
an F-box protein family. Additionally, signaling pathways stimulated by
auxin and jasmonic acid, although not touched on here, also include the
F-box proteins. Thus, the ubiquitin–proteasome system of protein degradation is widely conserved as a central mechanism for the perception of various
signals in plants. Although protein kinase and phosphatase are responsible for
many plant signaling pathways, by connecting with characteristic interacting
factors, they are integrated uniquely into plant systems. This book introduces typical pathways mediated by such stimuli as ABA, peptide ligands,
cell cycle signaling, and phototropin.
Many factors remain unknown in the signaling pathways introduced
here. Identification of these as-yet unknown molecules should be critical

for our understanding of the overall frameworks of the pathways. Further
research advancements in this field will likely contribute to opening up
new research areas in basic plant biology as well as molecular breeding to


Preface

xiii

generate useful plants. We hope that readers in many research areas will find
topics of interest in this book.
We thank the authors for their excellent contributions and Helene Kabes
and Mary Ann Zimmerman of Elsevier for their guidance and editing of the
chapters.
YASUNORI MACHINDA
CHENTAO LIN
FUYUHIKO TAMANOI
June 2014


CHAPTER ONE

Regulatory Networks Acted Upon
by the GID1–DELLA System After
Perceiving Gibberellin
Hideki Yoshida, Miyako Ueguchi-Tanaka, Makoto Matsuoka1
Bioscience and Biotechnology Center, Nagoya University, Chikusa Nagoya, Japan
1
Corresponding author: e-mail address:

Contents
1. Gibberellin Perception System in Higher Plants
2. Suppression of DNA-Binding Activity of TFs by DELLA (Trapping Function of DELLA)
2.1 Phytochrome-Interacting Factor Family of Proteins Involved in Hypocotyl
Elongation and Chlorophyll Biosynthesis
2.2 Alcatraz and Spatula Involved in Valve Margin Development and Cotyledon
Expansion, Respectively
2.3 Squamosa Promoter Binding-Like Proteins Involved in Floral Transition
2.4 Ethylene-Insensitive 3 and EIN3-Like 1 Involved in the GA–Ethylene Crosstalk
for Apical Hook Development
2.5 Brassinazole-Resistant 1 Involved in the GA–Brassinosteroid Crosstalk for
Hypocotyl Elongation
2.6 Jasmonate ZIM Domain and MYC2 Proteins Involved in the GA–Jasmonate
Acid Crosstalk Under Certain Conditions
3. Transcriptional Regulation of Downstream Genes Via the Interaction of DELLA with
Their Promoters (Direct Targeting Function of DELLA)
3.1 Backgrounds
3.2 ABA-Insensitive 3 and ABI5 Involved in GA–Abscisic Acid Crosstalk
3.3 Indeterminate Domain Proteins Involved in the Feedback Regulation of GA
Signaling
3.4 Botrytis-Susceptible Interactor and Its Related Proteins Involved in the
Transrepression Activity of DELLA
4. Other Functions of DELLA Besides Transcriptional Regulation
4.1 Prefoldin 3 and PFD5 Involved in Cortical Microtubule Arrangement
4.2 D14 Involved in GA–Strigolactone Crosstalk
5. Future Perspectives
References

The Enzymes, Volume 35
ISSN 1874-6047

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Abstract
Recent studies have revealed that DELLA proteins (DELLAs) interact with various kinds of
transcription factors (TFs) and other kinds of proteins to regulate GA signaling. Here, we
enumerate some of these DELLA interactors to show the multiple functions of DELLAs in
the GA signaling pathway. Through interaction with TFs, DELLAs regulate the expression
of many genes in an inhibitory or enhancing manner under various biological events
including the crosstalk between GA and other phytohormones, and the development
of organs and tissues. DELLA-interacting TFs can be categorized into two types in terms
of the effect of DELLA on their transacting activity. The first group includes those that are
inhibited by DELLAs in terms of their DNA-binding activity, which includes the phytochrome interacting factor family of proteins involved in hypocotyl elongation, chlorophyll biosynthesis, fruit patterning, and cotyledon expansion; squamosa promoter
binding-like proteins involved in floral transition; ethylene insensitive 3 and EIN3-like
1 proteins involved in GA–ethylene crosstalk; brassinazole-resistant 1 involved in GA–
brassinosteroid crosstalk; and jasmonate ZIM domain and MYC2 proteins involved in
GA–jasmonate crosstalk. The second group includes those TFs that affected in terms
of their transcriptional activity but not DNA-binding activity upon interaction with
DELLA, which includes the ABA-insensitive 3 and ABI5 involved in GA–abscisic acid
crosstalk, indeterminate domain involved in feedback regulation of GA signaling, and
Botrytis-susceptible interactor proteins involved in DELLAs transrepression activity.
We also mentioned that interaction of DELLAs with proteins besides TFs regulates
the crosstalk between GA and strigolactone, and tubulin folding. The interaction of
all of these various TFs and proteins with DELLAs strongly demonstrates that DELLA
functions as a hub protein linking GA signaling to a myriad of biological events.

1. GIBBERELLIN PERCEPTION SYSTEM IN HIGHER
PLANTS
Gibberellin (GA) is one of the plant hormones that regulate a wide
range of processes involved in plant growth, organ development, and environmental responses. These include seed germination, stem elongation, leaf
expansion, transition to flowering, and the development of flowers, fruits,

and seeds [1]. About 75 years ago, GA was first identified in the pathogenic
fungus, Gibberella fujikuroi, as the causal chemical for the rice “bakanae”
(foolish seedling) disease in which infected plants show excessive leaf elongation. Since its first discovery, more than 130 GAs have been identified in
plants, fungi, and bacteria, although only a few of them possess biological
activity [2].
During the past decade, most of the components important for GA perception or signaling have been identified through genetic studies on rice and


DELLA-Mediated Gibberellin Signaling Pathways

3

Arabidopsis mutants. Among these components, DELLA proteins (DELLAs)
have been considered as key intracellular repressors of the GA response of
downstream genes [3–7]. DELLAs comprise a subfamily within a family
of plant-specific putative transcriptional regulators called GRAS, the name
of which was coined after its first three members: GA insensitive (GAI),
repressor of ga1-3 (RGA), and scarecrow (SCR). All proteins of the GRAS
family contain a GRAS domain consisting of five distinct motifs: leucinerich region I, VHIID, leucine-rich region II, PFYRE, and SAW [8].
On the other hand, DELLAs are distinguishable from other GRAS proteins by way of additional DELLA and TVHYNP motifs at their
N-terminus. The DELLA subfamily is highly conserved among angiosperms, gymnosperms, and ferns, but not in Physcomitrella patens, a model
organism for mosses (bryophytes) [9,10]. Arabidopsis thaliana, a model plant
for dicots, has five kinds of DELLAs, namely, GAI, RGA, RGA-like 1
(RGL1), RGL2, and RGL3 [3,4,11,12], whereas, rice, a model plant
for monocots, only has one DELLA, namely, slender rice1 (SLR1) [6].
In GA signaling, DELLAs become rapidly degraded in the presence of
GA, resulting in various GA responses.
Another important component for GA perception is the GA receptor,
GA-insensitive dwarf1 (GID1). In rice, it is encoded by only a single gene
and its loss of function results in the gid1 mutant [13]. In Arabidopsis, however,

three GID1 orthologs (GID1a, GID1b, and GID1c) exist, all of which exhibit
some overlapping and yet distinct functions in regulating different developmental processes [14–16]. Biochemical analyses revealed that GID1 proteins
bind specifically and strongly to bioactive GAs but not to inactive ones
[13,17]. GID1 is related to the α/β-hydrolase fold superfamily due to their
similarity in terms of primary structure [18,19]. However, although α/βhydrolases possess three conserved amino acids (serine, aspartic acid, and histidine) or catalytic triad important for their enzymatic activity, GID1 has other
amino acids in place of histidine, making it devoid of hydrolase activity [13].
In GA signaling, GID1 is known to interact with DELLA after initially binding with GA. Although GID1s are localized in both the cytoplasm and the
nucleus [13,16], their interaction with DELLAs is only confined to the
nucleus where DELLA is present. Such interaction is important for the rapid
degradation of DELLA [13]. Recently, it has been revealed that the receptor
for another phytohormone, strigolactone, is also a member of the α/βhydrolase family, as exemplified by the D14 of rice and Arabidopsis, and the
DAD2 of petunia [20–23]. Unlike GA receptor GID1, the strigolactone
receptors retain the conserved catalytic triad, and thus function as an enzyme


4

Hideki Yoshida et al.

to hydrolyze the enol–ether linkage of active SLs, the catabolism of which is
essential for strigolactone perception in plants [21–23].
Interaction between the GA-binding GID1 receptor and DELLA induces
a subsequent interaction between DELLA and an F-box protein (GID2 in
rice; sleepy1 (SLY1) and sneezy (SNZ) in Arabidopsis), the third important
component for GA perception [24–26]. F-box proteins exist widely throughout the eukaryote kingdom, ranging from yeast to humans, and they function
as substrate-recruiting subunits of the Skp1-cullin1-F-box-protein (SCF)
ubiquitin ligase. The SCFGID2/SLY1/SNZ promotes the polyubiquitination
of DELLA in the GID1–GA–DELLA complex and induces its degradation
via the 26S proteasome complex, thus, triggering the downstream GA
response (Fig. 1.1). Although the above molecular mechanism involving

GID1, DELLA, and F-box proteins satisfy the basic principle for GA perception, we are still far from unveiling the complete picture of how DELLAs
repress the broad range of GA responses. Recently, however, the identification of some of the DELLA-interacting factors (discussed here) helped reveal
the diverse functions of DELLAs for GA signaling. In this review, we provide
an overview of the role of DELLA in the GA signaling pathway, particularly in
terms of its dual transcriptional regulatory function (trapping function and
direct targeting function; discussed earlier) on downstream genes, and also
its crosstalk with other signaling pathways through its interaction with various
kinds of transcription factors (TFs) and proteins.

Figure 1.1 GA perception mechanism mediated by GID1 and DELLA.


DELLA-Mediated Gibberellin Signaling Pathways

5

2. SUPPRESSION OF DNA-BINDING ACTIVITY OF TFs
BY DELLA (TRAPPING FUNCTION OF DELLA)
In recent years, intensive studies on the transcriptional regulatory
activity of DELLAs in Arabidopsis have revealed the dual function of
DELLAs in terms of regulating downstream gene expression. The following sections enumerate the TFs that lose their binding ability to promoters
of downstream genes upon their interaction with DELLA.

2.1. Phytochrome-Interacting Factor Family of Proteins
Involved in Hypocotyl Elongation and Chlorophyll
Biosynthesis
Phytochrome-interacting factor (PIF) proteins, characterized by their
bHLH DNA-binding domain (DBD), belong to one subfamily of the
bHLH superfamily [27–29]. PIFs were initially identified as components
of the light-mediated developmental regulation in which light promotes

the degradation of PIFs upon their phosphorylation by phytochromes [30].
Using yeast two-hybrid (Y2H) assay, de Lucas et al. and Feng et al. independently discovered the interaction between DELLAs and PIFs, of which PIF3
and PIF4 were the first to be identified in Arabidopsis [31,32]. Furthermore,
they found that PIFs promote hypocotyl elongation in Arabidopsis in a manner similar to GA, whereas DELLAs inhibit such event, indicating the contrasting physiological functions of PIFs and DELLAs. They revealed that the
interaction of DELLA with the bHLH domain of PIF4 diminishes the ability
of the latter to bind to the promoters of its target genes. In their model
(Fig. 1.2A), GA positively regulates the expression of such PIF-targeted
downstream genes by inducing the degradation of DELLAs, thereby restoring the DNA-binding activity of PIFs. The model also explains the crosstalk
between GA and light signals to modulate cell elongation in the hypocotyl.
Further studies have revealed that other PIFs such as PIF1 and PIF-like 2
(PIL2) also bind to DELLAs [33]. In addition, PIFs are negative regulators
of the expression of chlorophyll and carotenoid biosynthetic genes such as
conditional chlorina (CHLH) and phytoene synthase (PSY). In this case, DELLAs
derepress their expression by interfering with the DNA-binding ability of
PIFs, thereby modulating the levels of chlorophyll and carotenoids
(Fig. 1.2B) [34].


Figure 1.2 Suppression of DNA-binding activity of TFs by DELLA (trapping function of
DELLA) (A and B) DELLA–PIF interaction is involved in cell elongation (A) and biosynthesis
of chlorophyll and carotenoid (B) under the GA–light crosstalk. Pf, an inactive form of phytochrome; Pfr, an active form of phytochrome. (C) DELLA–ALC interaction is involved in cell
differentiation in the valve margin development. (D) DELLA–SPT relationship involved in
cotyledon expansion. (E) DELLA–SPL interaction is involved in the phase transition.
(F) DELLA–EIN3/EIL1 interaction is involved in the crosstalk between GA and ethylene signaling to regulate apical hook formation. EIN2, an ER-located protein whose C-terminal can
directly stabilize EIN3. (G) DELLA–BZR1–PIF module is involved in crosstalk among GA, BR,
and light signaling to regulate cell elongation in hypocotyl. BIN2, a glycogen synthase
kinase 3-like kinase which can phosphorylate and inactivate BZR1. (H and I) DELLA–JAZ–
MYC1 module is involved in crosstalk between GA and JA signaling to regulate root length,
photogene defense, and sesquiterpene synthesis. COI1, a JA receptor; LOX2, lipoxygenase
2, one of the jasmonate synthesis genes; TATs, tyrosine aminotransferases.

DELLA-Mediated Gibberellin Signaling Pathways

7

2.2. Alcatraz and Spatula Involved in Valve Margin
Development and Cotyledon Expansion, Respectively
Alcatraz (ALC) and spatula (SPT) are also members of the PIF subfamily [30],
and thus, also have the ability to interact with DELLAs [33,35]. Arabidopsis
fruit development has been studied as a model system for tissue patterning in
plants, and for understanding the genetic control of seed dispersal, which has
a key role in crop domestication and improvement. The valve margin is a
specific tissue comprised of two specific types of cells (lignification layer
and separation layer) that help to fuse valves of siliques [36] and is also
involved in fruit opening and in efficient seed dispersal [37,38]. Arnaud
et al. found that overproduction of a GA-catabolic enzyme, GA
2-oxidase (GA2ox), causes a defect in valve margin development and fruit
opening. At that time, ALC, another type of PIF, was already reported to be
positively involved in the differentiation of valve margin. Thus, they used
the alc mutant to compare its valve margin cell patterning with that of the
GA-deficient ga4-1 mutant, and found that their phenotypes were quite
similar to each other. They also demonstrated that ALC and DELLAs such
as GAI, RGA, and RGL2, can physically interact, and that mutations in both
GAI and RGA can rescue the defective valve margin development in ga4-1.
Taking such observations together, they discussed that GA promotes valve
margin development through the degradation of DELLA that binds and prevents ALC from activating its target genes (Fig. 1.2C) [35].
On the other hand, Josse et al. studied the physiological relationship
between DELLAs and SPT [39]. According to their study, unlike some PIFs
that positively regulate hypocotyl elongation, loss of function of SPT promotes cotyledon expansion, which is also observed in quadruple della mutant

plants. As the penta mutant plant carrying spt and quadruple della mutations
developed larger cotyledon than the spt and quadruple della mutants, they
discussed that SPT and DELLA are quite similar in terms of physiological
function. Furthermore, they found that DELLA and SPT act independently
to regulate common downstream genes. The protein level of RGA was
unchanged in the spt mutant and in the SPT overexpressor, whereas the level
of SPT was reduced in gai-1 (gain-of-function mutant of DELLA) but
increased by GA treatment. Based on these observations, they presented a
model for the SPT–DELLA compensatory circuit for maintaining cotyledon
size (Fig. 1.2D). That is, both SPT and DELLAs act as suppressors of cotyledon expansion by regulating both distinct and common gene subsets; but
at the same time, SPT level is being negatively regulated by DELLAs [39].


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Hideki Yoshida et al.

They emphasized that this cross-regulation generates a compensatory action
where SPT responds reciprocally to changes in the levels of DELLAs.
Although SPT also interacts with DELLA [33], the physiological function
of SPT seems to be different from that of other PIFs. Given the results mentioned in Section 2.1, various combinations between DELLAs and PIFs may
guarantee the extensive plasticity of various plant growth responses [39].

2.3. Squamosa Promoter Binding-Like Proteins Involved in
Floral Transition
In Arabidopsis, GA is known to promote floral initiation and the development of floral organs, seeds, and siliques [1] as evidenced by plants carrying
a defect in a GA-biosynthetic gene, copalyl diphosphate synthase that fail to
undergo flower formation [40]. Later on, Griffiths et al. revealed that gid1, a
GA receptor mutant, showed flower formation under long days but was
much delayed as compared to wild-type (WT) plants [14]. In this context,

the ability of GA to accelerate flowering is known to be linked with the degradation of DELLA, although the underlying mechanism remains poorly
understood even up to now. The pathway mediated by microRNA156
(miR156) targeting a group of transcription factors called squamosa promoter binding-like (SPLs) is known to be crucial for establishing flower formation [41–43]. Recently, Yu et al. showed that DELLAs directly bind to
some miR156-targeted SPLs, such as SPL2, SPL3, SPL9, SPL10, and
SPL11, in Arabidopsis (Fig. 1.2E) [44]. By interacting with DELLAs, the
transcriptional activity of these SPLs become inhibited, consequently
delaying the floral transition due to the inactivation of its targets miR172
in leaves and MADS box genes in the shoot apex (both positive regulators
of floral induction) aside from the suppressive action of miR156 on floral
transition. These results demonstrate a complicated regulatory mechanism
for floral transition, that is, under conditions where GA is absent, the function of SPLs is doubly downregulated by miR156 and DELLAs under the
juvenile stage, whereas at the adult stage, DELLA becomes the sole suppressor of SPL function. Based on this double downregulation system, floral
induction consequently occurs only under the absence of miR156 (reproductive stage) and presence of GA (absence of DELLAs) (see Fig. 1.2E).

2.4. Ethylene-Insensitive 3 and EIN3-Like 1 Involved in the
GA–Ethylene Crosstalk for Apical Hook Development
Dark-grown Arabidopsis seedlings germinated in soil develops an apical
hook, which protects the cotyledons and apical meristem when breaking


DELLA-Mediated Gibberellin Signaling Pathways

9

through the soil. It has been known that development of apical hook is
under the control of multiple hormonal signaling pathways [45]. Ethylene
induces hook formation in dark-grown seedlings via ethylene insensitive
3 (EIN3) and EIN3-like 1 (EIL1) (key transcription factors in ethylene signaling) [46], and hookless1 (HLS1), an N-acetyltransferase-like protein
[47,48]. Hook formation is repressed by GA deficiency but restored by
mutations in DELLA [49]. This indicates a crosstalk between GA and ethylene signals for the control of apical hook formation. An et al. proposed that

such crosstalk requires the physical interaction between DELLAs and EIN3/
EIL1 (Fig. 1.2F) [50]. They actually demonstrated that overexpression of
EIN3 partially rescues the impaired hook formation of dark-grown GA deficient or paclobutrazol (PAC; GA biosynthesis inhibitor)-treated seedlings.
Furthermore, WT seedlings treated with both ethylene and GA were found
to exhibit exaggerated hook bending, which was mimicked by the overexpression of EIN3 in the presence of exogenous GA. In contrast, loss of
function of EIN3–EIL1 remarkably suppressed the constitutive hookbending phenotype in the della mutant. Based on these observations, they
proposed a coordinated regulation of apical hook development by GA
and ethylene in dark-grown Arabidopsis seedlings. They also demonstrated
that HLS1 is a direct target gene of EIN3 and that GA-induced HLS1
expression is dependent on EIN3/EIL1. Finally, they revealed that DELLAs
physically interact with EIN3, EIL1, and EIL2, through their DNA-binding
domains and that accumulation of DELLAs leads to the inhibition of EIN3/
EIL1 transactivation activity for HLS1 expression, without changing EIN3
content (Fig. 1.2F) [50].

2.5. Brassinazole-Resistant 1 Involved in the GA–
Brassinosteroid Crosstalk for Hypocotyl Elongation
GA and brassinosteroid (BR) are known to cause many similar developmental responses in plants, such as cell elongation and seed germination [51],
although their relationship still remains unclear. Bai et al. showed that GA
and BR act interdependently via the direct interaction between DELLAs
and brassinazole-resistant 1 (BZR1) [52]. BZR1, a TF with an atypical
bHLH motif, plays a key role in BR signaling and its transcriptional activity
is regulated via its phosphorylation/dephosphorylation under the influence
of BR. In the absence of BR, BRZ1 is phosphorylated and rapidly degraded,
whereas, in the presence of BR, BZR1 is maintained in a dephosphorylated
state and functions as a TF [53,54]. Bai et al. found that GA cannot increase
hypocotyl length in BR-deficient or BR-insensitive mutants and discussed


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Hideki Yoshida et al.

that DELLA degradation by GA is not sufficient for inducing hypocotyl
elongation in the absence of BR. In contrast, the accumulation of active
BZR1 due to brassinolide (BL; bioactive BR) treatment or introduction
of a dominant gain-of-function mutation, bzr1-1D, partially rescued the
defective hypocotyl elongation of a GA-deficient mutant, ga1-3. Furthermore, GA-deficient and -insensitive mutants, and PAC-treated WT plants
were found to respond to BL, resulting in induced hypocotyl elongation.
They also explained that DELLAs inhibit the DNA-binding activity of
BZR1 via the physical interaction between BZR1 and DELLAs. Interestingly, BZR1 also interacts with PIF4, both of which share common target
downstream genes including expansins and paclobutrazol-resistants (PREs)
involved in hypocotyl elongation, suggesting that BZR1 and PIF4 may form
a functional complex to regulate a large number of genes (Fig. 1.2G) [52,55].
Further comparative studies on transcriptomic data of BZR1-, PIF4-, and
GA-regulated genes revealed a complex regulatory system consisting of
DELLAs, BZR1, and PIF4, all of which have the ability to interact with
each other, namely, DELLAs can potentially block the transcriptional activity of BZR1 and PIF4 by independently interacting with them, thereby regulating the expression of their unique targets, and/or by blocking the
BZR1–PIF4 heterodimer to modulate their common targets. (Fig. 1.2G)
[51,52,55]. This regulatory model also demonstrates that GA, BR, and light
signals interact with the DELLA–BZR1–PIF module to control cell elongation [51,52].

2.6. Jasmonate ZIM Domain and MYC2 Proteins Involved in the
GA–Jasmonate Acid Crosstalk Under Certain Conditions
DELLAs are known to be involved in the GA–JA crosstalk by physical interaction with the jasmonate ZIM domain (JAZ) family of proteins (Fig. 1.2H)
[56–58]. JAZs function as repressors of the jasmonate acid (JA) signaling
pathway by binding to downstream TFs such as MYC2, MYC3, and
MYC4 in the absence of JA to diminish their transcriptional activity.
MYC2, MYC3, and MYC4 are TFs containing a bHLH motif and function
as master regulators of most aspects of JA signaling in Arabidopsis [59,60]. In

the presence of JA, JAZs are degraded, thereby allowing these transcription
factors to freely interact with the promoter of JA-inducible genes and promote their expression [61–63]. Interaction between DELLAs and JAZs
attenuates the activity of each other, striking a balance between plant growth
and defense responses in accordance with developmental cues and environment changes (Fig. 1.2G) [56–58].


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11

Interestingly, DELLAs directly interact not only with JAZs but also
MYC2 (Fig. 1.2I) [64]. Hong et al. found that Arabidopsis MYC2 activates
the expression of the sesquiterpene synthase genes, terpene synthase 21
(TPS21) and TPS11 both involved in the synthesis of volatile terpenes
and important for plant–insect interactions by directly interacting with their
promoters. Such MYC2-mediated expression is not only induced by JA but
also by GA. In this context, DELLAs were found to negatively regulate sesquiterpene biosynthesis by physically interacting with MYC2 thereby
suppressing the expression of TPS genes (Fig. 1.2I) [64]. However, further
studies should be needed to completely understand DELLA–JAZ and
DELLA–MYC2 interactions. In the present model (Fig. 1.2H and I),
DELLAs positively regulate JA signaling by relieving the suppressive function of JAZ by directly binding with it. On the other hand, DELLAs negatively regulate JA signaling by extinguishing the transactivation activity of
MYC2 upon their interaction. It is possible that DELLAs selectively bind
with JAZs or MYC2, depending on existing physiological and environmental conditions thus providing a broad spectrum of gene expression [64].

3. TRANSCRIPTIONAL REGULATION OF DOWNSTREAM
GENES VIA THE INTERACTION OF DELLA WITH
THEIR PROMOTERS (DIRECT TARGETING FUNCTION
OF DELLA)
3.1. Backgrounds
In contrast to the above trapping function of DELLA to suppress the DNAbinding function of TFs, there are alternative observations to support that

DELLAs can directly regulate the expression of downstream genes by binding to their promoters in conjunction with TFs acting as scaffolds.
Ogawa et al. first observed that DELLAs possess a strong transactivation
activity in yeast [5]. Later on, Zentella et al. and Gallego-Bartolome´ et al.
tried to identify the genes that are directly upregulated by DELLAs by performing a transcriptome analysis using transgenic Arabidopsis plants
expressing gain-of-function versions of DELLAs, namely, GAI and RGA,
which serve as model systems for DELLA function under GA-deficiency
[65,66]. Their results included the GA-biosynthetic genes, GA 3-oxidase1
(GA3ox1) and GA 20-oxidase2 (GA20ox2); the GA receptor genes, GID1a
and GID1b; and a positive regulator in GA signaling, SCL3, among others.
This indicated that DELLAs may promote gene expression, at least in the
context of feedback regulation of GA signaling. In this context, Sarnowski


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Hideki Yoshida et al.

et al. reported that such enhancement of gene expression due to DELLAs
might be related to the interaction between DELLAs and SWI3C, one of
the core subunits of the chromatin remodeling complex switch/sucrose
nonfermenting (SWI/SNF) [67]. SWI/SNF consists of a central Snf2-type
ATPase and several core subunits, all of which are evolutionary conserved in
eukaryotes including plants [68]. Sarnowski et al. also found that mutation of
the Snf2-type ATPase modified the expression of genes under the control of
several signaling pathways including the GA signaling pathway. Mutant
swi3c plants exhibit several GA-related phenotypes such as delayed flowering
time under short day conditions, and reduction of hypocotyl and root
length, leaf blade size, lateral root number, and size of seed coat epidermal
cells. The swi3c mutants also showed reduced expression of genes that are
directly targeted and upregulated by DELLA such as GA3ox1 as mentioned

earlier, resulting in a lower GA4 content compared with WT plants. Since
SWI3C physically interacts with DELLAs as confirmed by bimolecular fluorescence complementation and pull down assays, Sarnowski et al. concluded
that the SWI/SNF complex enhances the transactivation activity of DELLA
proteins through protein–protein interaction [67].
Hirano et al. demonstrated the negative correlation between the transactivation activity of a rice DELLA, SLR1, and the plant height of rice [69].
They first clarified that the transactivation activity of SLR1 largely depends
on its DELLA and TVHYNP motifs located at its N-terminus. To do this,
they produced various truncated versions of SLR1, measured their transactivation activity in yeast cells, and overexpressed them in the rice plant.
After evaluating the plants, they found that the transactivation activity of
modified SLR1s positively corresponds with different levels of dwarfism
in rice plants, indicating that SLR1 suppresses plant growth through its
transactivation activity, although the mechanism behind this is still unclear.
They also investigated the function of the GRAS domain of SLR1 from the
viewpoint of its inhibitory function on plant growth. They produced a chimeric version of SLR1 (ΔDELLA-SLR1-VP16) that has its GID1-binding
DELLA motif replaced by the activation domain of the herpes simplex virus
protein VP16 [70,71]. After overproducing it in a loss-of-function mutant,
slr1-1, they found that the plants exhibited extremely severe dwarf phenotype. They also transformed slr1-1 with mutant versions of ΔDELLASLR1-VP16 containing various mutations in the conserved motifs of
the GRAS domain to determine the motifs important for its repressive
function. Almost all of the mutations in the LHRI, PFYRE, and SAW
motifs completely prevented the dwarf phenotype elicited by the intact


DELLA-Mediated Gibberellin Signaling Pathways

13

ΔDELLA-SLR1-VP16. Based on these observations, they discussed that
mutations in the LHRI, PFYRE, and SAW motifs alter the repressive effects
of SLR1 without affecting its transactivation activity [69], thus, suggesting
that the LHRI, PFYRE, and SAW motifs might be involved in direct association with gene promoters. However, since DELLAs are believed to be

devoid of DBD, other TFs carrying DBD may serve as a transcriptional scaffold between the GRAS domain of DELLAs and the promoter sequence of
their downstream genes [69].

3.2. ABA-Insensitive 3 and ABI5 Involved in GA–Abscisic Acid
Crosstalk
The germination of Arabidopsis seeds is inhibited by high temperature and
induced by light. The hormones abscisic acid (ABA) and GA critically control the regulation of high-temperature-induced signals for seed germination
[72–77]. High temperature increases ABA levels in Arabidopsis seeds during
imbibition by activating and repressing the expression of ABA biosynthetic
and catabolic genes, respectively. At the same time, high temperature also
decreases GA levels by repressing the expression of GA biosynthesis
genes [76]. Consistent with this, ABA-deficient and -insensitive mutants,
and also GA-hypersensitive mutants, such as spindly and rgl2, can germinate
at higher rates under high temperature than WT plants [74,76]. Somnus
(SOM), which encodes a CCCH-type zinc finger protein [78] and negatively regulates seed germination at high temperature, possibly serves as a
crucial intersection for GA and ABA signaling (Fig. 1.3A) [79]. The expression of SOM is under the control of GA and ABA through the physical
interaction between DELLAs and the transcription factors ABA-insensitive
3 (ABI3) and ABI5 that both positively regulate ABA signaling by directly
interacting with its promoter. Aside from interacting with DELLAs, ABI3,
and ABI5 also interact with each other, additively promoting the expression
of SOM as observed in transient expression assay. Based on these results, they
concluded that DELLAs use ABI3 and ABI5 as transcriptional scaffolds to
bind to the SOM promoter to activate SOM expression at high temperature,
resulting in the inhibition of seed germination (Fig. 1.3A) [79].

3.3. Indeterminate Domain Proteins Involved in the
Feedback Regulation of GA Signaling
The expression of GA-biosynthetic genes is feedback-regulated based on the
levels of bioactive GAs. Such feedback mechanism is known to be governed
by the GA–GID1–DELLA system, and concerns not only biosynthetic

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Hideki Yoshida et al.

Figure 1.3 Models for DELLA-dependent transcriptional activity of TFs through
protein–protein interaction. (A) DELLA–BI3–ABI5 module is involved in SOM expression
to inhibit seed germination under high-temperature conditions. (B) DELLA–IDD–SCL3
module is involved in feedback regulation of GA signaling. (C) DELLA–BOI interaction
is involved in seed germination, phase transition, and chlorophyll accumulation.

genes but also GA signaling genes [80]. As mentioned earlier, DELLAs promote the expression of genes that positively regulate GA signaling. Yoshida
et al. also found the transcriptional scaffolds that allow DELLAs to bind with
the promoter of downstream genes for such upregulation [81]. These
include some members of the indeterminate domain (IDD) family of proteins that carry a C2H2-type zinc finger motif as DBD [82,83]. By doing
yeast one-hybrid (Y1H) and Y2H assays using a cDNA library of Arabidopsis


DELLA-Mediated Gibberellin Signaling Pathways

15

TFs [84] as prey, they found five IDDs, namely, AtIDD3, AtIDD4,
AtIDD5, AtIDD9, and AtIDD10, that bind with the GRAS domain of
RGA. DELLA and IDD were also found to interact in the nucleus by using
bimolecular fluorescence complementation test, and such interaction was
through IDDs C-terminal region which has no DNA-binding ability [85].
The five IDDs also bound to the promoter sequence of SCL3 (mentioned in
Section 3.1), resulting in the positive regulation of the latter [86]. Such

upregulation of SCL3 was also reported by Zhang et al. to be induced by
the interaction of RGA with SCL3’s promoter by chromatin immunoprecipitation assay [87]. Based on these observations, they hypothesized that
these IDDs function as transcriptional scaffolds between RGA and the
SCL3 promoter (Fig. 1.3B). To verify such hypothesis, they conducted a
transient reporter assay and confirmed that RGA and IDDs synergistically
promote the expression of SCL3. Next, they generated plants overproducing AtIDD3 fused with SRDX, a plant-specific repression domain,
to mimic mutants with defects in multiple IDDs. These plants exhibited typical GA-related phenotypic defects such as dwarfed leaves, shorter roots, delayed flowering, and reduced expression of the GA upregulated genes
EXPANSINs and PREs. Based on these results, they concluded that IDDs
function as transcriptional scaffolds to link DELLAs to the promoter
sequences of downstream genes in the DELLA–IDD network to promote
their expression in GA feedback regulation. They also found SCL3, another
GRAS protein, to interact with IDDs and such SCL3–IDD interaction
competes with DELLA–IDD interaction, and consequently the transactivation activity of DELLA–IDD complex is diminished by SCL3 in planta
(Fig. 1.3B). These results indicated that SCL3 functions to repress its own
expression by interacting with IDD. Based on these, they proposed the
coactivator/corepressor exchange system consisting of DELLAs, SCL3,
and IDDs in GA signaling regulation (Fig. 1.3B). In the model, the increase
in the levels of SCL3 and SCL3–IDD complexes causes a decrease in the
expression level of their downstream genes. As a result, SCL3 protein is
decreased and DELLA–IDD complex increases again (Fig. 1.3B) [81]. This
DELLA/SCL3-mediated feedback loop explains the homeostatic regulation
of downstream gene protein levels including the positive regulator SCL3,
resulting in homeostatic GA signaling.

3.4. Botrytis-Susceptible Interactor and Its Related Proteins
Involved in the Transrepression Activity of DELLA
In contrast, Park et al., proposed that DELLAs act as a transcriptional repressor for GA-responsive genes by interacting with some of the RING finger


16

Hideki Yoshida et al.

proteins, namely, Botrytis-susceptible interactor (BOI), BOI-related gene1
(BRG1), BRG2, and BRG3 [88]. They actually found DELLA–BOI interaction by Y2H screening using RGA as bait. Plants overexpressing BOI1
and quadruple mutants for boi and brg showed contrasting abnormal phenotypes in terms of seed germination, phase transition from juvenile to adult,
and also phase transition from vegetative to reproductive, which are all due
to reduced GA signaling and enhanced GA signaling, respectively. Based on
these, they discussed that the BOI family of proteins negatively regulate GA
signaling in a manner similar to DELLAs in terms of physiological events.
RING domain has been considered to participate in protein–protein interaction and protein ubiquitination, and many RING finger proteins work as
E3 ligase, a component of the 26S proteasome, which is used for the degradation of DELLAs [89–91]. However, the boi quadruple mutants did not
show altered RGA levels or PIF3 interaction. Further, both RGA and
BOI bind to the promoter of some GA-responsive genes such as expansin
8 and PRE1 and PRE8. Based on these observations, they concluded that
DELLA–BOI complexes act as transcriptional repressors for the expression
of some GA-responsive genes, resulting in the repression of GA signaling
under certain physiological events (Fig. 1.3C).

4. OTHER FUNCTIONS OF DELLA BESIDES
TRANSCRIPTIONAL REGULATION
4.1. Prefoldin 3 and PFD5 Involved in Cortical Microtubule
Arrangement
Plant morphogenesis relies on specific patterns of cell division and expansion, which are influenced by the cortical microtubule arrangement
[92,93]. In this context, GA is well known to be involved in orientating
the cortical microtubule array such that they are perpendicular to the growth
axis [92,94]. Recent works demonstrated that GAs regulate microtubule
orientation through the physical interaction between nuclear-localized
DELLAs and prefoldin 3 (PFD3) and PFD5 constituting the prefoldin complex, one of cochaperones required for tubulin folding (Fig. 1.4A) [95]. In
the presence of GA, DELLAs are rapidly degraded, and the prefoldin complex remains in the cytoplasm in a functional form. In the absence of GA, the

prefoldin complex moves into the nucleus, which severely compromises
α/β-tubulin heterodimer availability thus affecting microtubule organization. Locascio et al. also indicated that the daily rhythm of plant growth is


DELLA-Mediated Gibberellin Signaling Pathways

17

Figure 1.4 Models for the diverse DELLA functions showing DELLAs’ interaction with
proteins besides TFs. (A) In the absence of GA, DELLAs interact with PFD in nucleus.
In the presence of GA, due to DELLA degradation, PFD is released from the nucleus
to the cytosol and arranges the microtube orientation. (B) DELLA-D14 binding to hydrolyze SL is involved in the crosstalk between GA and SL signaling.

accompanied by coordinated oscillation in DELLA levels, prefoldin subcellular localization, and cortical microtubule reorientation. This is a good
example that DELLAs are directly involved in cytological events without
interacting with any TFs.

4.2. D14 Involved in GA–Strigolactone Crosstalk
Strigolactone (SL) is a plant hormone that controls shoot branching [96,97].
Recently, the SL receptor is identified as D14, a member of the α/βhydrolase family of proteins [98,99]. Unlike the GA receptor GID1, D14
functions as a cleavage enzyme for SLs, and the cleavage reaction induces
the interaction of D14 with SLR1, after which, the SL-induced D14–
SLR1 complex modulates downstream signaling (Fig. 1.4B) [23]. The
Arabidopsis GA-biosynthetic mutant ga1-3 exhibits enhanced shoot
branching while the overexpression of GA2ox genes in rice promotes tillering, suggesting the crosstalk between GA and SL, at least in part [100]. In this
context, it is possible that the D14–DELLA complex is an intersection for
such GA–SL crosstalk.


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Hideki Yoshida et al.

5. FUTURE PERSPECTIVES
The large number of DELLA-interacting proteins mentioned earlier
implies that DELLAs work as a hub protein in the GA signaling pathway.
There seems to be no apparent trend in terms of the TF families interacting
with DELLAs, indicating that the latter could be involved in various biological events. Actually, comprehensive studies by microarray analyses demonstrated that there is no significant overlapping in terms of gene expression
pattern among light-grown seedlings expressing rga-Δ17, flowers overexpressing RGA, and etiolated seedlings expressing the gain-of-function
version of DELLA, gai-1 [65,66,101], indicating that the partner TFs of
DELLAs may change depending on the demands of a particular biological
event. In the case of the GA–JA crosstalk, DELLAs interact with JAZ to
enhance JA signaling in the context of hypocotyl and root elongation and
plant defense against pathogens, whereas, DELLAs interact with MYC2
to diminish JA signaling in the context of the sesquiterpene biosynthesis
(Fig. 1.2H and I) [56–58,64]. This shows us that DELLAs function both
in a positive and negative manner to regulate gene expression under different
tissue and environmental conditions. Therefore, it should be more meaningful to discuss the biological function of DELLA on a case-to-case basis rather
than making general statements about DELLA’s function.
As mentioned earlier, the DELLA family is a subfamily of the GRAS
superfamily, which contains a total of 37 proteins in Arabidopsis. The
interaction between DELLAs and TFs completely depends on the GRAS
domain, at least in existing literature, indicating that some TFs interacting
with DELLAs might also be interacting with other GRAS proteins. Actually, some of the IDDs, which function as transcriptional scaffolds
between DELLAs and their target DNA sequences also physically interact
with other GRAS proteins such as SCL3, SCR, and SHR [81,102,103],
and such IDD–GRAS interaction regulates cell differentiation in root
ground tissue [81,87,102,104]. In this context, DELLAs and other GRAS
proteins may interact competitively with TFs, at least in the case of IDDs,
thereby creating a complicated transcriptional regulatory network. In

order to decipher the molecular interactions within this network, it
should be important to reveal the structures of the DELLA proteins
and their complex with the various TFs, in addition to the previous structural analyses done on GID1–GA and GID1–GA–DELLA peptide complexes [18,19]. Recently, Sato et al. reported a sophisticated method to


DELLA-Mediated Gibberellin Signaling Pathways

19

purify a large amount of GRAS domain [105]. This could be an important milestone toward fully understanding the properties of DELLAs and
other GRAS proteins.

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