Download Brassinosteroid biosynthesis and signalling in Arabidopsis thaliana

Brassinosteroid biosynthesis and signalling in
Arabidopsis thaliana and Petunia hybrida
The research presented in this thesis was performed at the Department of Genetics, Institute
of Molecular and Cell Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV
Amsterdam. The research was funded by the Netherlands Organisation for Scientific
Research (NWO).
Cover design: Nathalie Verhoef & Sylvia Klop
Printing was done by Wöhrman print service, Zutphen
VRIJE UNIVERSITEIT
Brassinosteroid biosynthesis and signalling in
Arabidopsis thaliana and Petunia hybrida
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Aard- en Levenswetenschappen
op woensdag 6 april 2011 om 15.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Nathalie Verhoef
geboren te Sliedrecht
promotor:
prof.dr. R.E. Koes
copromotor: dr. E. Souer
Voor mijn ouders
Contents
Chapter 1
General introduction: Brassinosteroid biosynthesis
and signalling in plants
9
Chapter 2
Group II GSK3/SHAGGY-like kinases act redundantly
in the brassinosteroid signalling pathway
35
Chapter 3
Towards identification of group II ASKs containing
complexes in Arabidopsis
57
Chapter 4
Brassinosteroid biosynthesis and signalling in petunia
81
Summary
113
Samenvatting
117
Dankwoord
121
Chapter 1
Brassinosteroid biosynthesis and signalling in plants
General introduction
Nathalie Verhoef, Ronald Koes and Erik Souer
General introduction: Brassinosteroid biosynthesis and signalling in plants
Introduction
Early in the 1970’s a new plant hormone was described as a substance present in pollen of
Brassica napus that, when applied in minute concentrations, could induce growth of bean
internodes (Mitchell et al., 1970). It took till 1979 before the chemical nature of this
growth-promoting factor was elucidated as a steroid compound. This discovery was the
birth of a new class of plant hormones, since then known as brassinosteroids (BRs).
Pioneering work using feeding experiments in Catharanthus roseus and Arabidopsis
thaliana lead to insight in the biosynthesis pathway (Suzuki et al., 1993; Suzuki et al.,
1994). Subsequent analysis of mutants from Arabidopsis combined with detailed
biochemical analysis of identified proteins led to a near complete picture of BR
biosynthesis and signalling (Fujioka and Yokota, 2003; Gendron and Wang, 2007; Kim et
al., 2009). In this chapter, we will give an overview of the current knowledge on BR
biosynthesis and signalling in plants.
Biosynthesis of brassinosteroids
The first BR compound, brassinolide, was discovered in bee-collected pollen of Brassica
napus L. (Grove et al., 1979). To date more than 65 free BRs and 5 BR conjugates have
been isolated from plants with brassinolide as the most bioactive compound (Bajguz, 2007).
BRs are widely distributed among the plant kingdom and so far they have been found in
gymnosperms, monocots, dicots, a pteridophyte, a bryophyte and a chlorophyte (Bajguz
and Tretyn, 2003). They are found in almost every part of the plant, but are most abundant
in the reproductive organs (Bajguz and Tretyn, 2003).
The sterol biosynthesis pathway can be divided into three biosynthetic domains (Fig. 1). In
the first domain the mevalonate derivative squalene is converted into 24methylenelophenol. Next, the pathway diverges into two branches, one branch producing
the more abundant sterols that are used to produce membrane components, while the
second part produces brassinosteroids from the precursor campesterol. Detailed studies in
Arabidopsis thaliana and Catharanthus roseus revealed that synthesis of catasterone from
the precursor campesterol can be accomplished via two pathways, the early and late C-6
oxidation pathways (Choi et al., 1996; Nomura et al., 2001; Fujioka and Yokota, 2003).
Although both pathways are found throughout the plant kingdom, it seems that for species
such as tobacco and tomato the late C-6 oxidation pathway is more prevalent (Fujioka and
Yokota, 2003). In Arabidopsis and tomato, these two pathways are linked to each other via
the cytochrome P450 enzyme CYP85A1/A2, implying that they are not totally autonomous
11
Chapter 1
(Shimada et al., 2001). Fujioka et al. (2002) revealed upstream of the C-6 oxidation
pathways an early C-22 oxidation pathway.
Epoxidase
SMT1
Lupeol synthase
Squalene-2,3epooxide
Squalene
Mevalonate
24-Methylenecycloartenol
Cycloartenol
CYP51G1
CYP710A
Stigmasterol
DIM
Sitosterol
DWF5
DWF7
5-Dehydroavenasterol
Isofucosterol
Cycloeucalenol
δ 7-Avenasterol
Cyclopropyl isomerase
Obutusifoliol
24-Ethylidenelophenol
membranes
CYP51G1
δ8,14-Sterol
SMT2
FK
24-Methylenecholestrol
DWF5
5-Dehydroepisterol
DWF7
24-Methylene
lophenol
Episterol
HYD1
4α-Methylfecosterol
DIM
Campesterol
Campest-4-en3β-one
DWF4
22α-Hydroxy-5−
campesterol
Campest-4en-3-one
DWF4
SAX1
22α-Hydroxycampest-4-en-3-one
DET2 5α-Campestan-
6-Oxocampestenol
DWF4
DWF4
DET2 22α-Hydroxy-5α-
DWF4
6-Deoxocathasterone
campestan-3-one
CYP90
C1/D1
CYP90C1/D1
6α-Hydroxy
campestenol
Campestenol
3-one
Cathasterone
CYP90C1/D1
6-Deoxoteasterone
CYP90C1/D1
CYP90C1/D1
CYP85A1/A2
Teasterone
3-Epi-6-deoxocathasterone
22,23-Dihydroxy
campesterol
22,23-Hydroxycampest-4-en-3-one
3-Dehydro-6-deoxoteasterone
CYP85A1/A2
3-Dehydroteasterone
CYP85A2
7-Oxateasterone
CYP90C1/D1
6-Deoxotyphasterol
6-Deoxycatasterone
CYP85A1/A2
6α-Hydroxycatasterone
CYP85A1/A2
Typhasterol
CYP85A2
7-Oxatyphasterol
Catasterone
CYP85A2
Brassinolide
Figure 1. The proposed sterol/brassinosteroid biosynthesis pathway in Arabidopsis thaliana. The metabolic
products are shown in boxes and the enzymes involved in the pathway are depicted in red. Mevalonate is
converted via multiple steps into squalene (black dashed arrow). The early C-22 oxidation pathway is represented
by light green arrows. The late C-6 oxidation pathway is visualized by red arrows and the early C-6 oxidation
pathway by dark green arrows.
Sterol/BR biosynthesis genes have been cloned from a number of species such as
Arabidopsis, rice and tomato (Choe et al., 1998; Choe et al., 1999a; Choe et al., 1999b;
Koka et al., 2000; Shimada et al., 2001; Tanabe et al., 2005). Most of the identified BR
biosynthesis genes encode for cytochrome P450 monooxygenases. BR biosynthesis mutants
and sterol mutants with defects in late steps of the sterol pathway such as dwarf7 (dwf7),
dim (diminuto) and dwarf5 (dwf5) have a dwarfish appearance and can be rescued by
exogenous application of BRs. Adult sterol mutants with defects in genes functioning in the
early steps of the sterol biosynthesis pathway, such as fackel and hydra1 (hyd1), have
12
General introduction: Brassinosteroid biosynthesis and signalling in plants
phenotypes similar to BR-biosynthesis mutants. However, mostly these mutants are
arrested early in development as during embryogenesis they show distinctive defects
(except for sterol methyl transferase 2; smt2) that cannot be rescued by exogenous
application of BR. This suggests that these early sterols or their metabolites are involved in
developmental processes during embryogenesis that are not dependent on BRs. There are
multiple indications for a role of these sterols in correct auxin signalling. For instance, hyd1
and fackel mutants were shown to be defective in auxin and ethylene signalling (Lindsey et
al., 2003). Schrick et al. (2004) showed that sterols are also important for cellulose
synthesis and play a crucial role in cell elongation and cell wall expansion during the
building of the primary cell wall.
Although the BR biosynthesis pathway has been intensively studied still some questions
remain. For some of the enzymes it is not completely clear in which BR conversion they are
involved. For example, the BR biosynthesis enzyme CPD (CONSTITUTIVE
PHOTOMORPHOGENIC DWARF, CYP90A1,) was suggested to act as a C-23
hydroxylase in Arabidopsis (Szekeres et al., 1996), but recently Ohnishi et al. (2006)
proposed that CPD rather acts in the early C-22 oxidation pathway and performs a function
different from C-23 hydroxylation.
BRs are structurally similar to mammalian steroids. Two of the BR biosynthetic enzymes,
the cytochrome P450 monooxygenase CPD and the 5α-reductase DE-ETIOLATED2
(DET2), are very similar to enzymes involved in animal steroid biosynthesis. Arabidopsis
DET2 protein was able to catalyse the 5α reduction of several animal steroid substrates in
human embryonic kidney cells (Li et al., 1997). Moreover, expression of the human steroid
5α-reductase types 1 or 2 both complemented the Arabidopsis det2 mutant.
In health sciences there are multiple claims on the beneficial action of plant sterols (often
refered to as phytosterols) as anti-cancer and cholesterol-lowering substances. However,
many conflicting reports have been published. Nevertheless, plant sterols as well as BRs
have been shown to inhibit the growth of several cancer lines (Malikova et al., 2008;
Woyengo et al., 2009) and were also shown to reduce the levels of cholesterol and possess
anti-inflammatory properties (Bouic, 2002; Woyengo et al., 2009).
Brassinosteroid signalling
In Arabidopsis, BRs are perceived by the membrane-localized leucine-rich repeat (LLR)
receptor-like kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1, Li and Chory, 1997).
BRI1 has an extracellular domain containing 25 LRRs, a transmembrane domain and a
cytoplasmic serine/threonine kinase domain (Li and Chory, 1997). The non-repetitive
13
Chapter 1
island found between the 21st en 22nd LRR in conjunction with LRR22 is of crucial
importance for binding to BR (Kinoshita et al., 2005). BRI1 orthologs have been identified
in a number of plant species such as tomato (CU-3), pea (LKA), rice (OsBRI1), barley
(HvBRI1), cotton (GhBRI1) and petunia (PhBRI1) (Yamamuro et al., 2000; Montoya et al.,
2002; Chono et al., 2003; Nomura et al., 2003; Sun et al., 2004) suggesting that plants
perceive BRs on a similar manner.
- BR
+ BR
BRI1
BRI1
BAK1
BAK1
BSK1
BSK1
BSK1
p
p
BKI1
p
p
p
BIN2
BSU1
BSU1
p
14-3-3
p
BKI1
BZR1/BES1
BZR1/BES1
BIN2
proteasome
BSU1
BZR1/BES1 p
p
p
BZR1/BES1
BIN2
BIN2
BSU1
BZR1
BES1
BR taget gene
BR taget gene
Figure 2. Predicted BR signalling pathway. In the absence of BR (left), BKI1 suppresses BRI1 and BSK1;
BAK1 and BSU1 are inactive. BIN2 is active and phosphorylates the transcription factors BZR1 and BES1,
resulting in inhibition of their activity through proteasome mediated degradation, cytoplasmic retention by 14-3-3
proteins and inhibition of their DNA-binding and transcriptional activities. In the presence of BR (right), BR binds
to the extracellular domain of BRI1 to activate its kinase, which in turn leads to disassociation of BKI1 from BRI1
and dimerization with and activation of BAK1. Subsequently, BRI1 phosphorylates BSK1, which in turn activates
the plant-specific serine/threonine protein phosphatase BSU1 and BSU1 inactivates BIN2 by dephosphorylation at
pTyr200. Unphosphorylated BES1 and BZR1 accumulate in the nucleus where they bind to the promoters of BRtarget genes, leading to diverse cellular en developmental responses.
14
General introduction: Brassinosteroid biosynthesis and signalling in plants
BR binding induces autophosphorylation of BRI1 in vivo and a number of the
phosphorylation sites have been identified and studied (Wang et al., 2005a).
Characterization of these sites revealed that phosphorylation of the kinase domain is very
important for kinase function and downstream BR signalling (Wang et al., 2005a).
Furthermore, phosphorylation of the carboxy-terminal domain plays a critical role in the
activation of events downstream of BRI1 (Wang et al., 2005b). Binding of BR also leads to
dimerization of BRI1 with the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1
(BAK1) that is subsequentely phosphorylated (Fig. 2). These phosporylation events lead to
the dissociation of the repressor BRI1 KINASE INHIBITOR 1 (BKI1) from BRI1 (Li et
al., 2002; Nam and Li, 2002; Wang and Chory, 2006). BRI1 and BAK1 are not only
localized to the plasma membrane but also in endosomes. Co-expression of BRI1 and
BAK1 in protoplasts of cowpea and Arabidopsis led to endocytosis of both receptors
(Russinova et al., 2004). In addition, Geldner et al. (2007) observed localization of BRI1GFP in the endosomes of root cells of Arabidopsis that was independent of BR. Plants
treated with Brefeldin A (BFA), which inhibits trafficking from the early to late endosomal
compartments and vacuoles, enhanced the accumulation of BRI1 in endosomal
compartments. Interestingly, similar to a BR treatment, BFA on its own was able to induce
strong dephosphorylation of BES1 and suppressed DWARF4 (DWF4) gene expression. This
suggests that BRI1 can signal from these endosomal compartments and is not just
transported. BRI1 in the endosome might exist in an activated form because the negative
regulator BKI1 is only present at the plasma membrane (Geldner et al., 2007). Recently,
Song et al. (2009) demonstrated that the MEMBRANE STEROID-BINDING PROTEIN 1
(MSBP1) interacts with the extracellular domain of BAK1 in a BL-independent manner.
Like BAK1, MSBP1 is localized in the plasma membrane and endosomal compartments.
MSBP1 accelerates BAK1 endocytosis and MSBP1 expression reduced the interaction
between BRI1 and BAK1 in vivo. This suggests that MSBP1 acts as a negative factor in the
BR signalling pathway.
Another class of BRI1 substrates that have been identified are members of the BRSIGNALING KINASES (BSKs) and represent the next step in the signalling cascade
towards the nucleus (Tang et al., 2008). Recently Kim et al. (2009) showed that
phosphorylation of BSK1 a member of the BSK family, facilitates interaction with and
presumably activation of the plant-specific serine/threonine protein phosphatase BRI1
SUPPRESSOR 1 (BSU1). BSU1 is regarded as a positive regulator of the BR signalling
pathway and encodes a serine-threonine protein phosphatase with a N-terminal Kelchrepeat domain and a C-terminal phosphatase domain (Mora-Garcia et al., 2004).
15
Chapter 1
Dephosphorylation of the GSK3/SHAGGY-like kinase BR-INSENSITIVE 2 (BIN2) by
BSU1 inactivates BIN2 that acts as a negative regulator of the BR signalling pathway.
BIN2’s role as negative regulator is performed through the phosphorylation of the closely
related transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAZOLE
RESISTANT 1 (BZR1, He et al., 2002; Yin et al., 2002). Peng et al. (2010) recently
showed that BIN2 interacts directly with BZR1 via a 12 amino acid BIN2-docking motif
(DM) adjacent to the C-terminus of BZR1. A treatment with the proteasome inhibitor
MG132 increased the accumulation of phosphorylated BZR1 protein and this suggests that
phosphorylation of BZR1 and BES1 by BIN2 promotes the degradation of BZR1 and BES1
(He et al., 2002). Peng et al. (2010) reinforced this finding by demonstrating that
Arabidopsis lines expressing BZR1-GFP without the BIN2-DM could not be
phosphorylated and led to accumulation of BZR1. Vert & Chory (2006) demonstrated that
phosphorylation inhibited the DNA-binding activity and transcription activation potential of
BES1. A study in Arabidopsis demonstrated that 14-3-3 proteins interact with
phosphorylated BZR1 (Gampala et al., 2007). Binding by 14-3-3 proteins was shown to
increase the cytoplasmic retention of phosphorylated BES1 and mutation of a BIN2
phosphorylation site in BZR1 was shown to abolish 14-3-3 binding, leading to increased
nuclear localization of the BZR1 protein. Similar results have been obtained with the rice
homolog OsBZR1 (Bai et al., 2007). Taken together this suggests that BIN2-mediated
phosphorylation inhibits BZR1 and BES1 by at least three ways: degradation by the
proteasome, loss of DNA binding activity and cytoplasmic retention. Until recently, it was
believed that BSU1 activated BES1 and BZR1 directly by dephosphorylation. However
Kim et al. (2009) demonstrated that BSU1 does not dephosphorylate the BES1 and BZR1
transcription factors. In contrary, it dephosphorylates BIN2 and as a result,
unphosphorylated BZR1/BES1 accumulate in the nucleus where they regulate the
expression of BR-responsive genes. Thus, the effect of BSU1 on the phosphorylation status
of BES1 and BZR1 is indirect and runs via inhibition of BIN2.
BES1 and BZR1 belong to a group of transcription factors which are only found in plants
(Li and Deng, 2005). BZR1 represses the transcription of several BR biosynthetic genes
such as CPD and DWF4 by directly binding to their promoters via a motif called the BR
response element (BRRE), thereby creating a feedback mechanism (He et al., 2005). BES1
has been found to interact with the basic helix-loop-helix transcription factor BES1Interacting Myc-like protein 1 (BIM1) to synergistically bind to E box sequences present in
many BR-induced promoters such as the SAUR-AC1 promoter (Yin et al., 2005). Another
target of BES1 is MYB30 (Li et al., 2008). Interestingly, MYB30 was shown to bind to a
conserved MYB-binding site in BR target gene promoters creating a new mechanism in
which BES1 and MYB30 cooperatively regulate BR gene expression during the early
16
General introduction: Brassinosteroid biosynthesis and signalling in plants
stages of plant development. Yu et al. (2008) identified two JmjN/C domain-containing
histone demethylases as putative BES1 partners, ELF6 (EARLY FLOWERING 6) and
REF6 (RELATIVE OF EARLY FLOWERING 6). It therefore appears that the BES1 and
BZR1 proteins act as hubs, interacting with proteins from diverse developmental pathways
to activate and/or repress various target genes. This central role of BES1 and BZR1 is also
reflected by the pleiotropic phenotype of BR mutants.
Several proteins of the BR signalling pathway such as the GSK3-like kinases and 14-3-3
proteins are also conserved in mammals. Striking parallels with WINGLESS (Wnt),
TRANSFORMING GROWTH FACTOR BETA (TGF beta), and RECEPTOR TYROSINE
KINASE (RTK) signalling in animals have been proposed (Haubrick and Assmann, 2006).
In similarity to the above described interactions between BIN2, BES1/BZR1 and 14-3-3
proteins in BR signaling, Wang et al. (2004) showed that phosphorylation of the HEATSHOCK TRANSCRIPTION FACTOR 1 (HSF1) by EXTRACELLULAR SIGNALREGULATED KINASE 1 (ERK1) and GSK3, and subsequent association of 14-3-3
proteins led to cytoplasmic sequestration of HSF1 and inhibition of HSF1 to bind DNA
under certain growth conditions. The BRI1 receptor is a serine/threonine kinase that lacks
tyrosine kinase activity. Most animal receptor-like kinases (RLK) have tyrosine kinase
activity, with the exception of the TRANSFORMING GROWTH FACTOR β (TGF-β)
receptors. Thus a potential split of RLKs is suggested prior to the evolutionary separation of
plants and animals (Bishop and Koncz, 2002). Ehsan et al. (2005) reported that Arabidopsis
TGF-β RECEPTOR INTERACTING PROTEIN (TRIP-1), a protein with high sequence
similarity to a mammalian substrate of the TGF-β RII receptor kinase, as another
phosphorylation target of BRI1.
Plant GSK3-like kinases
The initial goal of our project was to clarify the role of the GSK3-like kinase GSK1 from
Arabidopsis in tolerance against sodium stress. Therefore, we will take a closer look on
what is known about GSK3-like kinases, with emphasis on their roles in plants. The
Arabidopsis genome encodes ten GSK3-like kinases and together with the other plant
GSK3-like kinases they can be classified into four subgroups based on phylogenetic
analyses of the amino acid- and cDNA sequences (Dornelas et al., 1998; Dornelas et al.,
1999; Jonak and Hirt, 2002; Yoo et al., 2006). In plants, members of the GSK3-like kinase
family seem to have both similar and distinct functions. The protein sequences of
Arabidopsis GSK3-like kinases (ASKs) are highly conserved throughout the kinase
17
Chapter 1
domain, in contrast to the N- and C-terminal regions that are highly variable. Possibly this
variability in the terminal regions might lead to specific functions of ASKs.
For many plant GSK3-like kinases it is still elusive in what kind of pathway(s) they act
(Table 1). A role for the three group II members in BR signalling is well established
nowadays (Chapter 2; Vert and Chory, 2006; Koh et al., 2007; Kim et al., 2009; Yan et al.,
2009). However, even in triple knock-outs for group II GSKs a substantial amount of BES1
remains phosphorylated, suggesting that other ASKs play a role in BR signalling as well
(Vert and Chory, 2006; Yan et al., 2009). De Rybel et al. (2009) discovered a small
molecule, called bikinin, which inhibits the activity of all members of group I and II and the
group III member ASKθ. Inhibition of these seven GSKs activates BR responses, indicating
the involvement of additional Arabidopsis GSKs in BR signalling. Recently Rozhon et al.
(2010) provided stong evidence that ASKθ acts as a negative regulator in the BR signalling
pathway as well.
Interestingly, the group I members ASK11 and ASK12 also seem to be involved in floral
patterning at several developmental stages (Dornelas et al., 2000). Group II GSK3-like
kinases might also be involved in NaCl stress, although results of Piao et al. (1999; 2001)
conflicts with results obtained by others (Chapter 2; Koh et al., 2007; Zeng et al., 2009).
Piao et al. (2001) demonstrated that ectopic expression of Glycogen Synthase Kinase 1
(GSK1) in Arabidopsis leads to expression of multiple stress-induced genes and increased
salt tolerance . However Koh et al. (2007) showed that an insertion mutant of rice OsGSK1,
is more tolerant to salt stress and Zeng et al. (2009) showed that the semi-dominant mutant
bin2-1 is very sensitive to salt. In Medicago sativa WIG seems to be involved in wounding
and MSK4 in carbohydrate metabolism (Jonak et al., 2000; Kempa et al., 2007).
It can be concluded that the majority of GSK proteins in Arabidopsis are involved in BR
signalling. It is tempting to speculate that other roles inferred for GSKs in Arabidopsis and
other species are direct and indirect effects of changes in BR signalling as well.
Regulation of brassinosteroid levels
BRs can be classified into C27, C28 and C29 steroids depending on the length of the sidechain. Not only the steroidal skeleton, but also its side-chain is metabolized by
modifications such as hydroxylation, dehydrogenation and glycosylation (Bajguz, 2007).
Both BR metabolism and catabolism seem to play an important role in the control of
bioactive BR levels. For instance the cytochrome P450 monooxygenases CYP72B1
(BAS1) and CYP72C1 both catalyze the hydroxylation of the bioactive BRs brassinolide
and catasterone generating inactive products (Turk et al., 2003; Nakamura et al., 2005).
18
ASKκ/ASK41
ASKδ/ASK42
MSK4
ASKβ/ASK31
ASKθ/ASK32
BSKθ
NSK6
NSK59
NSK91
NSK111
PSK6
PSK7
WIG
IV
III
II
Arabidopsis thaliana
Arabidopsis thaliana
Brassica napus
Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum
Petunia hybrida
Petunia hybrida
Medicago sativa
Arabidopsis thaliana
Arabidopsis thaliana
Medicago sativa
AJ002280
Y07822
Y12674
Y08607
AJ002315
AJ224163
AJ002314
AJ224164
AJ224165
AJ295939
X79279
AC079732
AF432225
Y13437
DQ060684
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Oryza sativa
Oryza sativa
Petunia hybrida
Petunia hybrida
X94938
X94939
X99696
ASKζ/ASK23
BIN2/ASKη/ASK21
GSK1/ASKι/ASK22
GhBIN2-B
GhBIN2-C
GhBIN2-D
GhBIN2-E
OSKη
OSKGSK1
PSK8
PSK9
Dornelas et al., 1999; Vert & Chory 2006; Chapter 2
Dornelas et al., 1999; Li et al.
Dornelas et al., 1999; Vert & Chory 2006; Chapter 2
Sun et al., 2004
Sun et al., 2004
Sun et al., 2004
Sun et al., 2004
Dornelas et al., 1999
Koh et al., 2007
Chapter 4
Chapter 4
Tichtisnky et al., 1998; Charrier et al., 2002
Tichtisnky et al., 1998; De Rybel et al., 2009
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Tichtisnky et al., 1998
Jonak et al., 2000
Charrier et al., 2002
Charrier et al., 2002
Kempa et al., 2007
high expression in aerial organs, embryogenesis, expression tip cotyledon and weak in roots
weak expression in aerial organs, embryogenesis and roots
weak expression in aerial organs, tip cotyledon and roots
consistently expressed throughout the plant
highest expression in hypocotyls, leaf buds, immature bolls and ovules
consistently expressed throughout the plant
weak expression in aerial organs, tip cotyledon and roots
not determined
young shoots and roots, calli, developing seeds and highest in panicles
not determined
not determined
flower buds, pollen; upregulated in the dark
flower buds, pollen, roots
not determined
mainly in pollen and anther
not determined
not determined
not determined
stamen (highest), ovary, pollen
stamen (highest),leaves,sepals,petals,styles,ovary,stigma, pollen
roots,stems flowers, leaves (low) after wounding strongly upregulated
very low expression in roots, leaves,inflorescence stems,flowers
very low expression in roots, leaves,inflorescence stems,flowers; upregulated by NaCl or PEG
in roots: amyloplasts, plastids, in epidermis and cortex cells; plastid of leaf mesophyll cells
not determined
possibly in BR signalling
not determined
not determined
not determined
not determined
not determined
probably in development, reproduction
probably in development
wounding
probably in reproduction
probably in abiotic stress
NaCl stress, carbohydrate metabolism
Einzenberger et al., 1995
Decroocq-Ferrant et al., 1995
Pay et al., 1993
Pay et al., 1993
Pay et al., 1993
BR signalling
BR signalling, NaCl stress
BR signalling, NaCl stress
BR signalling
BR signalling
BR signalling
BR signalling
not determined
BR signalling, abiotic stress
probably in BR signalling
not determined
probably development
development
Nicotiana tabacum
Petunia hybrida
probably development
probably development
probably development
Medicago sativa
Medicago sativa
Medicago sativa
Dornelas et al., 1999; Dornelas et al., 2000; Kim et al., 2009
Dornelas et al., 1999; Dornelas et al., 2000; Kim et al., 2009
Charrier et al., 2002
BR signalling, flower patterning
BR signalling, flower patterning
BR signalling
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
X77763
X83619
X68410
X68409
Reference
Expressed
weak expression in aerial organs and hypocotyl, high floral meristem
weak expression in aerial organs, tip cotyledon and hypocotyl, high floral meristem
consititutively expressed in roots, leaves infloresescence stems, siliques, seedlings, flowers,
upregulated by NaCL
constituitve expression in leaves, petioles,roots, stems, nodes, flower buds, developing
flowers, (un)opened mature flowers,seed pods, various stage of somatic embryos
constituitve expression in roots, stems and nodes, flower buds, globular embryos
weak expression in roots and stems, moderately in nodes, high in early flower development
gradually decreased in later stages and absent after fertilization, moderate in globular
embryos and decreased in later stages
high expression corolla, ovary, during pollen development; low expression in leaves
stems,roots, ovules, embry sac and highest expression in leaves, flowers,
ovaries and flower buds
Function
Species
NTK-1
PSK4
I
X68411
MSK2
MSK3
MSK1
Accesion no.
X75432
X75431
AL163792
Group
ASKα/ASK11
ASKγ/ASK12
ASKε/ASK13
GSK3 like kinases
Table 1. Plant GSK3-like kinases
General introduction: Brassinosteroid biosynthesis and signalling in plants
19
Chapter 1
Nomura et al. (2001) suggested that C-6 oxidation as well as steroid side chain
hydroxylation at C-22 and C-23 are potential rate-limiting reactions in BR biosynthesis in
Arabidopsis, tomato and pea. Several experiments have shown that the BR signalling
pathway tightly controls the biosynthesis of BRs. In a bri1-5 mutant background, the BR
biosynthesis genes CPD, DET2 and DWF4 are upregulated, while CYP72B1 is
downregulated (Choe et al., 2001).
Severe bri1 alleles as well as the semi-dominant allele of BIN2 accumulate high levels of
catasterone and brassinolide (Noguchi et al., 1999; Choe et al., 2002). Wang et al. (2002)
demonstrated that a dominant mutation in BZR1 reduced the level of BRs and CPD. In
addition, He et al. (2005) demonstrated that BZR1 repressed the transcription of several BR
biosynthetic genes such as CPD and DWF4. This suggests that BZR1 acts as a repressor on
the BR synthesis pathway. Taken together these data indicate that BR levels are regulated
by a feedback mechanism mediated by the BR signalling pathway.
Transport of brassinosteroids
The distribution of BRs in plants is widespread; with higher levels found in young growing
tissue (such as shoot tip and young internodes) in comparison to older vegetative tissue.
The highest levels of bioactive BRs are found in pollen and immature seeds (Bajguz and
Tretyn, 2003; Symons and Reid, 2004).
Several studies have shown that a number of BR biosynthesis enzymes are membranebound and since steroid hormones are relatively hydrophobic, BR synthesis is thought to
take place on or near internal cell membranes such as the endoplasmatic reticulum (Klahre
et al., 1998; Shimada et al., 2001). Assuming that BRs are synthesized within the cell,
transport out of the cell is required if BRs are perceived at the exterior cell surface by the
BRI1 receptor. Yamamoto et al. (2001) observed that cultured Zinnia elegans callus tissue
secreted BRs in the medium, supporting the idea that BRs are transported out of the cell. At
the moment it is unclear how BR transport takes place, but perhaps through conjugation
with fatty acids or glucose or via proteinaceous transporters BRs are transported out of the
cell (reviewed by Symons et al., 2008). Since signalling can also occur via endosomal
BRI1, there seems no absolute requirement for BR transport (Geldner et al., 2007).
Although short-distance transport of BRs appears likely, is it questionable whether BRs are
transported over a longer distance. Numerous studies have tried to answer this question, but
the obtained results are often contrary. Application of radio-labelled BRs to the roots of
rice, wheat and cucumber plantlets showed that exogenous applied BRs were transported
into the shoot (Nishikawa et al., 1994). Furthermore, Arabidopsis BR biosynthesis mutants
were rescued when they were grown on media containing bioactive BRs (Choe et al.,
20
General introduction: Brassinosteroid biosynthesis and signalling in plants
1998). On the other hand exogenous BRs applied to leaves of pea, rice, cucumber and
wheat were relatively immobile (Yokota et al., 1992; Nishikawa et al., 1994). With respect
to the feeding experiments it is doubtful whether exogenous applied BRs do reflect
endogenous BRs in plants as grafting experiments in pea showed that BRs are not
transported from the root to shoot or vice versa. In addition, removing the apical bud did
not result in any significant changes in BR levels in either the stem or leaf tissues of pea
and removal of the three youngest expanded leafs did not substantially alter endogenous BR
levels in the stem or apical tissues (Symons and Reid, 2004). More evidence that plants do
not undergo long distance transport of BRs is derived from the unstable BR-deficient dx
mutant of tomato. Revertant sectors in dx did not restore the mutant phenotype. Again, the
immobility of BRs was also revealed by reciprocal grafting experiments using dx and wild
type plants (Bishop et al., 1996; Montoya et al., 2005). Interestingly, depletion of active
BRs through overexpression of the catabolic enzyme BAS1 in the epidermis of wild type
Arabidopsis reduced growth, suggesting that growth depends on local BR biosynthesis
(Savaldi-Goldstein et al., 2007). Considering all studies, it seems most likely that BRs are
produced at the location where they are needed and that the BR levels in plant tissues are
rather regulated through tissue-specific control of BR biosynthesis, catabolism and
inactivation than via long-distance travelling.
Physiological responses to brassinosteroids
BRs are involved in various growth- and developmental processes such as cell division and
elongation, vascular differentiation, abiotic- and biotic stresses, photomorphogenesis,
senescence and fertility (Clouse et al., 1996; Li et al., 1996; Szekeres et al., 1996; Bajguz
and Hayat, 2009).
The major cause of dwarfism is reduced cell size and several studies proved the
involvement of BRs in cell elongation (Kauschmann et al., 1996; Azpiroz et al., 1998). For
example, BR application promotes the elongation of stems, petioles and flower peduncles
in dicotyledons as well as coleoptiles and mesocotyls in monocots (reviewed by Clouse and
Sasse, 1998). Savaldi-Goldstein et al. (2007) showed that expression of the BRI1 receptor
or BR biosynthesis gene CPD in the epidermis of Arabidopsis BR mutants rescues their
dwarf phenotype, but not when expressed in underlying tissues. Their results suggest that
BR signalling in the epidermis is an important factor in leaf growth. Reinhardt et al. (2007)
showed that DWF4 expression in the leaf margin cells of a dwf4 mutant restored leaf shape,
but not leaf size. This insinuates that BRs play a role in leaf shape.
21
Chapter 1
In several plant species BRs were shown to upregulate the expression of XYLOGLUCAN
ENDOTRANSGLYCOLASES/HYDROLASES (XETs or XHTs), which are a group of
enzymes with cell-wall modifying activities (Zurek and Clouse, 1994; Xu et al., 1996;
Koka et al., 2000). As (BL-induced) cell expansion requires cell wall loosening it is
imaginable that these enzymes are induced. Microarray analysis in Arabidopsis showed that
also other genes involved in cell expansion and cell wall organization such as expansins,
extensins and pectin modifying enzymes are upregulated after BL treatment (Goda et al.,
2002). These data suggest that BRs play an important role in the regulation of cell wall
expansion, possibly through controlling the expression or the activity of wall modifying
factors. Also a role of BRs in microtubule organization has been implicated, as brassinolide
was able to restore the microtubule organisation and rescue cell elongation in the
Arabidopsis bul1 mutant (Catterou et al., 2001).
BRs also seem to play a role in cell division. Nakajima et al. (1996) showed that addition of
epi-brassinolide to Chinese cabbage mesophyll protoplasts increased the cell division rate.
This is further supported by Oh & Clouse (1998) who observed acceleration of the cell
division rate in leaf mesophyll protoplasts of Petunia hybrida under optimal auxin and
cytokinin conditions. They showed that a high concentration of BR rather had an inhibitory
effect on cell division and that the effect of BR is dependent on the auxin/cytokinin ratio.
However, microscopic examination of leaves of BR biosynthesis and BR signalling mutants
of Arabidopsis led to some suspicion as the dwarfish phenotype was due to a reduced cell
size and not a consequence of a decreased number of cells (Kauschmann et al., 1996).
Further studies need to clarify the role of BRs in cell division.
Several experiments suggest that BRs are involved in vascular differentiation. Cano
Delgado et al. (2004) identified three genes, designated BRI1-LIKE RECEPTOR KINASE 1
(BRL1), BRL2 and BRL3, which have a high sequence identity with BRI1. BRL1 and BRL3
were shown to complement a bri1 mutant when expressed under control of the BRI1
promoter and both can bind to brassinolide with high affinity. A brl1 mutant showed an
increase in phloem and a decrease in xylem differentiated cells. Knocking out of brl1 in a
weak bri1 mutant background enhanced the vascular defects. A bri1brl1brl3 mutant was
smaller than the strong bri1 allele (bri1-101) and the vasculature defects were enhanced.
Altogether this implies that BR signalling in provascular cells through BRL1, BRL3 and
BRI1 receptors contributes to the maintenance of proper xylem/phloem ratios. In addition,
Nagata et al. (2001) reported that brassinazole, a BR biosynthesis inhibitor, inhibited the
development of secondary xylem in cress.
Brassinolide was shown to accelerate senescence in leaves of mung bean seedlings and in
the detached cotyledons of cucumber seedlings (reviewed by Clouse and Sasse, 1998). BR
biosynthesis and signalling mutants have an expanded lifespan when compared to normal
22
General introduction: Brassinosteroid biosynthesis and signalling in plants
plants, supporting the idea that BRs accelerate senescence in plants. To clarify the role of
BRs in senescence it would be valuable to examine the effect of BR application on
senescence-associated mutants of Arabidopsis and to study the expression of senescenceassociated genes in BR mutants.
Another common characteristic of most BR biosynthesis and signalling mutants is reduced
fertility or male sterility. In Prunus avium brassinolide was shown to improve elongation of
pollen tubes (Hewitt et al., 1985). The cpd mutant was shown to be male sterile as its pollen
were unable to elongate during germination (Szekeres et al., 1996). The dwf4 mutant on the
other hand has viable pollen, but they cannot reach the stigma because the stamen filaments
failed to elongate and are shorter than the carpels which results in the deposition of pollen
on the ovary wall rather than onto the stigmatic surface (Choe et al., 1998). The only
mutant with stamens longer than the gynoecium with wild-type fertility is dwf5-1 (Choe et
al., 2000). All other identified dwf5 alleles and other BR biosynthesis mutants have stamens
which are shorter than the gynoecium. Although dwf5-1 plants had siliques with more seeds
than the other BR mutants, seeds did not develop normally and exogenous application of
BR was shown to improve germination.
Another prominent feature of several BR biosynthesis mutants is their de-etiolated
phenotype; dark grown seedlings having the appearance of light-grown seedlings with short
hypocotyls and open cotyledons. Arabidopsis BR biosynthesis mutants such as cpd and
det2 possess a de-etiolated phenotype when grown in the dark and they stimulate the
expression of light-regulated genes (Chory et al., 1991; Szekeres et al., 1996). BR
biosynthesis mutants of tomato and rice also develop a de-etiolated phenotype in the dark,
but this is not the case for BR biosynthesis mutants of pea (reviewed by Fujioka and
Yokota, 2003). This suggests that the role of BRs in photomorphogenesis might differ
among plant species. Bancos et al. (2006) demonstrated that the expression of the
Arabidopsis BR biosynthesis genes CPD and CYP85A2 are under diurnal regulation. The
expression profile of CPD is circadian regulated and dependent on light. In addition, the
BR-catabolyzing enzymes CYP72B1 and CYP72C1 are repressed by light (Turk et al.,
2003; Nakamura et al., 2005). As brassinolide was shown to accumulate in the light, it
might be that the level of active BRs is influenced by the diurnal expression of both ratelimiting biosynthetic enzymes and enzymes that inactivate BRs.
There are several reports published describing the relationship between BRs and abiotic
stresses such as salt, heavy metals, high and low temperature stress and drought (reviewed
by Bajguz and Hayat, 2009). The role of BRs in pathogenic defence has also been
investigated. For instance the addition of BR containing extract of Lychnis viscaria L. seeds
yielded an increased resistance of tobacco, cucumber and tomato to viral and fungal
23
Chapter 1
pathogens (Roth et al., 2000). Furthermore, Nakashita et al. (2003) demonstrated that
brassinolide independently of salicylic acid (SA) enhanced the resistance to the viral
pathogen tobacco mosaic virus (TMV), the fungal pathogen Odium sp. and the bacterial
pathogen Pseudomonas syringae pv. tabaci. In rice, brassinolide enhanced the resistance
against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas
oryzae pv. Oryzae. This suggests that brassinolide acts as inducer of a broad range of
disease resistances in rice and tobacco and possibly other plant species.
Cross talk with other plant hormones
Interactions of BRs with other plant hormones are well known. BRs were shown to induce
the expression of OPDA-REDUCTASE 1 (OPR3), an enzyme involved in jasmonate
biosynthesis and induction of OPR3 gene expression by membrane damage was impaired in
the BR- biosynthesis mutant cabbage 1 (cbb1, Schaller et al., 2000). This suggests
intertwinement of the jasmonate and BR biosynthesis pathway.
BRs and gibberellins (GA) were shown to antagonistically regulate the expression of the
GA responsive gene GASA1 and the GA-repressible gene GA5 (Bouquin et al., 2001).
Shimada et al. (2006) reported that mutation of the rice SPINDLY gene (OsSPY), a negative
regulator of the GA signalling pathway, reduced the expression of several BR biosynthesis
genes and OsBRI1. Wang et al. (2009) reported that GA-induced OsGSR1 was repressed
upon BR treatment. Transgenic plants in which OsGSR1 expression was down regulated by
RNA interference had phenotypes similar to BR mutants, had a reduced level of
endogenous BRs and were rescued upon a brassinolide treatment. Interestingly, OsGSR1
interacted with DIM, a BR biosynthetic enzyme, suggesting that GA directly activate BR
biosynthesis via OsGSR1. Further analysis should reveal the cross talk mechanisms
between BRs and GA.
Microarray analysis has shown that several BR responsive genes are also regulated by ABA
(Nemhauser et al., 2006). Germination of det2 and bri1-1 mutants is more strongly
inhibited by abscisic acid (ABA) than wild type seeds (Steber and McCourt, 2001). This
suggest that ABA and BRs work antagonistic in seed germination and that a BR signal is
needed to overcome the inhibition of germination by ABA. Subsequently, Zhang et al.
(2009) showed that in Arabidopsis seedlings ABA rapidly induced the expression of the
BR-suppressed genes CPD and DWF4 and also phosphorylation of BES1 was enhanced. In
the absence of BIN2, ABA failed to induce BES1 phosphorylation. Taken together, their
results suggest that the ABA signalling pathway cross talks with the BR signalling pathway
downstream of the BRI1 receptor but upstream of BES1.
24
General introduction: Brassinosteroid biosynthesis and signalling in plants
The synergistic action of BRs and auxin in cell growth and development is a well-known
phenomenon (reviewed by Mandava, 1988; Halliday, 2004; Hardtke et al., 2007). Recently
Ibañes et al. (2009) showed that auxin transport coupled to BR signalling is required to set
the number and arrangement of plant-shoot vasculature. Several studies showed that BRs
and auxins regulate a similar set of target genes such as genes belonging to the AUX/IAA,
SAUR and GH3 families (Nakamura et al., 2003; Goda et al., 2004; Nemhauser et al.,
2004). Most of the BR/auxin target genes are coordinately regulated, so if a gene is
repressed by auxin it is usually also repressed by BR. AUX/IAA genes were expressed at a
lower level in the BR biosynthesis mutant det2 and in the BR signalling mutant bri1 and
AUX/IAA expression could be restored after BL application in the det2 mutant. In addition
some auxin-responsive genes were up regulated in the BES1-D mutant (Yin et al., 2002).
Nemhauser et al. (2004) observed that the TGTCTC auxin-responsive element is enriched
in brassinolide-regulated gene promoter sequences. The core ARF-binding element TGTC
was enriched in promoters of brassinolide, auxin and dual regulated genes. DR5, a wellcharacterized synthetic element containing ARF binding sites, was not only induced by
auxin, but also by BRs and both hormones are required for full induction (Nemhauser et al.,
2004). Like auxin, BRs promote primary root growth at low concentrations (Mussig et al.,
2003). In the Arabidopsis mutant brevis radix (brx) impaired auxin-responsive gene
expression was observed which could be restored upon a brassinolide treatment. BRX
expression was strongly induced by auxin, but mildly repressed by brassinolide. This
suggests that BR levels are rate-limiting for auxin-induced transcriptional responses. BRX
was shown to regulate the expression of the BR biosynthesis gene CPD, suggesting a
feedback loop that involves the BRX transcriptional regulator. Chandler et al. (2009)
showed that BIM1, formerly known to interact with BES1, controls embryonic patterning
via its interaction with DORNROESCHEN (DRN) and DORNROESCHEN-LIKE
(DRNL). Given that DRN is also involved in auxin signalling it is conceivable that the
auxin and BR signalling pathways control embryonic patterning via DRN. Work by Vert et
al. (2008) showed that the negative regulator of the BR pathway, BIN2, binds and
phosphorylates the AUXIN RESPONSE FACTOR 2 (ARF2). They suggest that repressor
ARFs, such as ARF2 may compete with activator ARFs for binding auxin responsive
elements (AuxRes). Phosphorylation of repressor ARFs by BIN2 would inactivate them
leaving the AuxRes more accessible for activator ARFs. De Grauwe et al. (2005) proposed
that ethylene, auxin and BRs all play a role in the formation of the apical hook. Gendron et
al. (2008) discovered that a compound named brassinopride (BRP) inhibits BR action and
promotes ethylene action. Their results suggest that active downstream BR signalling is
required for ethylene-induced apical hook formation (Li et al., 2004). ARF2 protein levels
25
Chapter 1
were also decreased upon ethylene treatment but accumulate in the light and transcription
of HOOKLESS1 (HLS1), a N-actetyltransferase, was up regulated by ethylene but down
regulated by light. All together this insinuates that ARF2 and possibly other ARF repressors
are at the convergence of BRs, ethylene, light, and auxin signalling networks.
Aims and outline of this thesis
The goal of this PhD research project was to investigate the role of the GSK3-like kinase
GSK1 and other group II GSK3-like kinases (BIN2 and ASKζ) in Na+ stress. In Chapter 2
we analysed the effect of Na+ stress in several insertion mutants. In contrast to results of
others we could not detect a role in tolerance against Na+ stress for GSK1 or for the other
group II members. In a yeast two hybrid assay, GSK1 interacted with a member of the
BES1/BZR1 family. As BIN2 negatively regulates the BR signalling pathway by
phosphorylating the transcription factors BES1 and BZR1, it prompted us to investigate
whether the group II GSK3-like kinases act redundantly in the BR signalling pathway.
In order to learn more about the mode of actions of GSK1 and the other group II GSK3-like
kinases in BR signalling and possibly in other signal transduction pathways we tried to
isolate the protein complexes with help of various Tandem Affinity Purification (TAP)
methods described in Chapter 3.
Within our petunia collection we found several mutant families that resemble sterol/BR
biosynthesis and signalling mutants. In Chapter 4 we characterized and analysed petunia
sterol/BR biosynthesis mutants. For four families, the mutant phenotype was caused by the
insertion of transposable elements in the BR biosynthesis gene CPD. Furthermore we report
the identification of a bri1 mutant in petunia and isolated several homologs of key
regulators of the Arabidopsis BR signalling pathway from petunia.
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Chapter 2
Group II GSK3/SHAGGY-like kinases act redundantly
in the Arabidopsis brassinosteroid signalling pathway
Nathalie Verhoef, Annegret Roß, Ronald Koes and Erik Souer
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
Abstract
Brassinosteroids (BR) are steroid hormones that are essential for plant growth and
development. In Arabidopsis, BRs bind to the leucine-rich repeat (LRR) receptor
serine/threonine kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), which initiates a
phosphorylation cascade leading to a final BR response. The group II GSK3-like kinase
BR-INSENSITIVE 2 (BIN2) negatively regulates the BR signalling pathway by
phosphorylating the transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and
BRASSINAZOLE RESISTANT 1 (BZR1). In this study we characterized two other group
II GSK3-like kinases from Arabidopsis, named Glycogen Synthase Kinase 1 (GSK1) and
Arabidopsis GSK3-like kinase ζ (ASKζ). Mutant analysis and their interaction with
members of the BES1/BZR1 family allude to a redundant role of group II GSK3-like
kinases in the BR signalling pathway. In contrast to prior research we could not detect a
role for GSK1 in tolerance against Na+ stress, neither for the other group II members.
Introduction
GLYCOGEN SYNTHASE KINASE-3 (GSK3) is a highly conserved serine/threonine
protein kinase. It was originally identified as a key regulator in glycogen synthesis in
mammals (Embi et al., 1980), but subsequent work showed its involvement in an array of
processes such as cell proliferation, cell fate specification, differentiation and apoptosis
(Frame and Cohen, 2001). Purification and molecular cloning revealed two isomers,
GSK3α and GSK3β, that have 97% homology within their catalytic kinase domains. It was
generally assumed that GSK3α and GSK3β have the same biochemical and physiological
functions, but with the year’s closer examination invalidate this statement (Ruel et al.,
1993; Hoeflich et al., 2000).
Although the role of GSK3 in mammals and its homolog SHAGGY in Drosophila
melanogaster (Siegfried et al., 1992) has been extensively studied, little is known about the
specific functions of GSK3-like kinases in plants. In higher plant species such as
Arabidopsis thaliana (Dornelas et al., 1999; Li et al., 2001), Medicago sativa (Pay et al.,
1993), Oryza sativa (Koh et al., 2007), Nicotiana tabacum (Einzenberger et al., 1995) and
Petunia hybrida (Decroocq-Ferrant et al., 1995) several GSK3-like kinases have been
identified. In Arabidopsis ten GSK3-like kinases, also known as Arabidopsis GSK3-like
kinases (ASKs), have been identified. These ten ASK genes can be classified into four
subgroups based on phylogenetic analyses of amino acid- and cDNA sequences (Dornelas
et al., 1998; Dornelas et al., 1999; Jonak and Hirt, 2002). The protein sequences of ASKs
37
Chapter 2
are highly conserved throughout the kinase domain, in contrast to the N- and C- terminal
regions that are highly variable. This variability might point to specific functions for each
of the GSK3-like kinases in a variety of plant processes. For some of the GSK3-like kinases
specific roles were assigned. ASK11 and ASK12 have been reported to play a role in
flower development (Dornelas et al., 2000), while GSK1 seems to play a role in Na+
signalling (Piao et al., 1999; 2001). The best-studied GSK3-like kinase in plants is BRINSENSITIVE2 (BIN2 also known as ASKη/ UCU1/DWF12/AtSK2-1), a member of
subgroup II, which is involved in BR signalling in Arabidopsis (Li et al., 2001).
Brassinosteroids (BRs) are steroidal hormones that are important for growth and
development in plants. Deficiency in BR biosynthesis or signalling causes dramatic growth
defects including dwarfism, reduced apical dominance, male sterility, delayed flowering
and senescence, and photomorphogenesis in the dark (Li et al., 1996; Szekeres et al., 1996;
Azpiroz et al., 1998). In Arabidopsis, BRs are perceived at the plasma membrane by
BRASSINOSTEROID INSENSITIVE 1 (BRI1), which encodes a leucine-rich repeat
(LRR) receptor serine/threonine kinase. As a result, BRI1 can dimerize and
transphosphorylate the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)
and dissociate from BRI1 KINASE INHIBITOR 1 (BKI1), a repressor of BRI1 (Li et al.,
2002; Nam and Li, 2002; Russinova et al., 2004; Gampala et al., 2007; Wang et al., 2008a).
Recently, Kim et al. (2009) showed that BRI1 mediated phosphorylation of the BRSIGNALING KINASE 1 (BSK1) results in interaction and presumably activation of the
plant-specific serine/threonine protein phosphatase BRI1 SUPPRESSOR 1 (BSU1). BSU1
in turn inactivates BIN2 by dephosphorylation at pTyr200. BIN2 negatively regulates the
BR signalling pathway by phosphorylating closely related members of a plant-specific
transcription factor family BES1/BZR1. Phosphorylated BES1 and BZR1 is inactivated by
protein degradation, cytoplasmic retention and/or inhibition of their DNA-binding activities
(He et al., 2002; Yin et al., 2002; Vert and Chory, 2006; Gampala et al., 2007; Ryu et al.,
2007; Peng et al., 2010).
The last decade a number of papers was published on the mode of action of BIN2, but the
other ASKs fell into oblivion. In this study we have analysed the group II ASKs from
Arabidopsis. Mutant analysis and interaction with the BES1 and BZR1 transcription factors
point to a redundant role for these kinases in BR signalling, results that were confirmed by
other scientists as well (Vert and Chory, 2006; Kim et al., 2009; Yan et al., 2009).
Furthermore, in contrast to previous reports (Piao et al., 1999; 2001) we could not establish
a role for these kinases in tolerance against Na+ stress.
38
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
Results
Redundancy between GSK1, ASKζ and BIN2
Within this project we intended to study the role of the GSK3-like kinase GSK1 in salt
tolerance. Previous work by Piao et al. (1999; 2001) implied a role for GSK1 in salt
tolerance as over-expression led to an increased tolerance in Arabidopsis. Phylogenetic
analysis placed GSK1 in subgroup II together with ASKζ and BIN2 (Jonak and Hirt,
2002). To unravel the role(s) of GSK1 and its closest homolog ASKζ, we identified lossof-function alleles for both genes (Fig. 1a and b). Single loss-of-function mutants in GSK1
and ASKζ did not show any visible phenotypes neither did the double mutant. However, a
triple insertion mutant for all group II ASKs, GSK1, ASKζ and BIN2, showed aberrant
growth of the leaves and long bending petioles (Fig. 1c). Since a phenotype is only apparent
in a insertion mutant of all group II ASKs, we assume that BIN2, GSK1 and ASKζ act
redundantly.
BIN2 plays an important role in the brassinosteroid (BR) signalling pathway and thus
GSK1 and ASKζ might function in this pathway too. Asami et al. (2000) reported the
finding of a specific BR biosynthetic inhibitor brassinazole (brz) and we would expect that
single or double group II ask loss-of-function mutants would be sensitive to brz whereas the
triple mutant should show resistance to brz. Indeed, Arabidopsis wild type, single and
double mutant seedlings treated with brz showed a strong reduction in hypocotyl length
(Fig. 1d) and the thickness of the cell wall was strongly increased (Fig. 1e). Inhibition of
BR biosynthesis by brz in the triple mutant background did hardly affect hypocotyl
elongation. However, brz still had some reducing effect on the longitudinal growth of the
triple insertion mutant, suggesting that other GSK3s still partly inhibited the BR signalling
pathway. Temporarily switching on genes with help of the estrogen receptor-based
inducible XVE system was shown to be an effective molecular tool (Zuo et al., 2000; Papdi
et al., 2008; Pre et al., 2008). As the triple insertion mutant displayed strong resistance
against brz (Fig. 1d), we tested whether induction of either one of the transgenes in a triple
mutant background would re-establish sensitivity to brz. Group II ASK transcripts were
clearly detected after induction by estradiol, although gene expression varied between
independent lines (Fig. 1f). In the non-induced transgenic lines no transcript was observed,
indicating that the XVE system is reliable to switch on ASKs. In contrast to our
expectations, not all transgenic lines of pER8:BIN2dom, pER8:BIN2 and pER8:GSK1 were
sensitive to brz after induction (Fig. 1g). In fact none of the transgenic pER8:ASKζ lines
analyzed in figure 1f were sensitive to brz and for pER8:BIN2 only one line was found (Fig.
1g).
39
Chapter 2
A
D
BIN2
Wt Col
gsk1
askζ
gsk1/askζ
bin2/gsk1/askζ
200 bp
ATG
STOP
DMSO
ASKζ
ATG
STOP
GSK1
1 μM brz
STOP
W
t
gs Col
as k1
k
gs ζ
k
bi 1/a
n2 sk
W /gs ζ
t k
bi Ws 1/a
n2
sk
ζ
ATG
B
E
+
-
1 μM brz
GSK1
Wt Col
ASKζ
BIN2
bin2/gsk1/askζ
ACTIN
C
F
pER8:BIN2L8137 pER8:BIN2domL8138
Wt Col
gsk1
askζ
gsk1/askζ
14
17
18
9
19
14
21
19
- +- +- +- +
- +- +- +- +
pER8:ASKζL8140
pER8:GSK1L8139
24
20
34
37 39
- + - +- +- + - +
5
6
9
11 12
- + - + - + - + - + estradiol
ASK
ACTIN
Wt Ws
bin2/gsk1/askζ bin2
bin2/gsk1/askζ background
G
Wt Col
+
-
+
+
bin2/gsk1/askζ
pER8:BIN2doma
+
-
+
-
+
+
+
+
pER8:BIN2b
+
-
+
+
pER8:GSK1c
+
-
+
+
estradiol
1 µM brz
Figure 1. Analysis of insertion mutants in group II ASKs A) Schematic representation showing the T-DNA
integration site (triangle) in BIN2 (At4G18710), GSK1 (At1G06390) and ASKζ (At2G30980). Solid bars indicate
exons, thin lines introns. B) RT-PCR analysis on total RNA isolated from leaves showing expression of BIN2,
GSK1 and ASKζ in various mutant backgrounds. ACTIN was used as control. C) Phenotype of bin2, gsk1 and askζ
single loss-of-function mutants, the gsk1/askζ double mutant and the group II ask triple insertion mutant grown
under short-day conditions. D) The effect of brassinazole (brz) on cell elongation in hypocotyls of ask mutant and
wild type seedlings. E) Light microscopy of stems of brassinazole treated and non-treated wild type and triple ask
mutant seedlings. Bar = 100 µM. F) Independent transgenic lines were screened for their ability to express group
II ASK transgenes in the presence or absence of the inducer estradiol. RT-PCR analysis on total RNA isolated
from seedlings was performed to determine ASK expression (Upper row of panels). ACTIN was used as control
(Lower row of panels). G) Restored brassinazole sensitivity in a subset of lines after induced expression of various
group II ASK genes in mutant seedlings. a, L8138-9; b, L8137-14; c, L8139-13.
40
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
A gain of function mutation in GSK1 and ASKζ results into a dwarfish phenotype
In a screen to search for BR insensitive (semi)dwarf mutants, several semi-dominant
mutants in BIN2 were identified (Li et al., 2001; Choe et al., 2002; Li and Nam, 2002;
Perez-Perez et al., 2002). bin2-1 carries a missense mutation in the part encoding the kinase
domain that results in the conversion of a glutamic acid (E) at amino acid 263 into a lysin
(K), leading to a more active BIN2 kinase (Li and Nam, 2002). In order to test if this amino
acid change also increases the activity of GSK1 or ASKζ, a similar mutation was
introduced in GSK1 and ASKζ (Fig. 2a). These dominant constructs were expressed under
control of a CaMV35S promoter in Arabidopsis.
Indeed, some transformants expressing GSK1dom or ASKζdom have a dwarfish phenotype
that is morphologically comparable to the previously characterized bin2-1 mutant (Fig. 2b).
Furthermore, the level of transgene expression correlates with the severity of the phenotype
(Fig. 2c). These results suggest that all three group II kinases can negatively regulate the
BR signalling pathway.
A
B
KpnI
L G T P T R E E
cttggtactccaactcgtgaagaa
cttggtaccccaactcgcaaagaa
L G T P T R K E
a
b
c
d
e
f
Wt Col
35S GSK1dom
35S GSK1dom
KpnI
L G T P T R E E
cttggtactccaactcgcgaagaa
cttggtaccccaactcgcaaagaa
L G T P T R K E
C
a
b
c
g
d
e
f
Wt Col
35S ASKζdom
35S ASKζdom
g
g
GSK1/ASKζ
Wt Col
ACTIN
Figure 2. Gain-of-function mutants of GSK1 and ASKζ have a dwarfish phenotype. A) Sequence comparison
of wild type and dominant constructs of GSK1 and ASKζ. The introduced KpnI site used for cloning (see Material
and Methods) as well as the nucleotide(s) resulting in the desired amino acid change, glutamic acid (E) into lysin
(K), are indicated in green. B) Over-expression of GSK1dom and ASKζdom under control of a 35S promoter in
Arabidopsis leads to dwarfism. C) RT-PCR analysis on total RNA isolated from leaves of the plants shown in B
(a-g). ACTIN was used as control.
41
Chapter 2
GSK1 and ASKζ bind to members of the BZR1/BES1 family
Our data suggest that GSK1 and ASKζ function redundantly with BIN2 as negative
regulators of the BR signalling pathway. To further explore the function(s) of GSK1, a
yeast two-hybrid approach was used to find interacting partners. A full-length GSK1
protein was used as bait to screen a two-hybrid cDNA library made from mature
Arabidopsis leaves and roots. Only one interacting clone was found and interestingly this
clone contained the C-terminal part of the BEH2 protein (Fig. 3a). BEH2 is a homolog of
BES1 and BZR1, two plant specific transcription factors that have been shown to interact
with BIN2 (Zhao et al., 2002). As the BES1/BZR1 family only consists of six genes, twohybrid constructs of the complete family were generated and screened for interaction with
BIN2, GSK1 and ASKζ. Like BIN2, also GSK1 and ASKζ strongly interact with BZR1
and BES1 (Fig. 3b). Noteworthy, in contrast to GSK1 and BIN2, ASKζ seem to (weakly)
B
A
MAAGGGGGGG
RRRRAITAKI
VLKALCLEAG
ISGTPTNFST
HGSPVSSSFP
IASSIPANLP
GSKRKLTSEQ
SSPTRRAGHQ
RWINFQSTAP
DWGMSGMNGR
EVGVEDLELT
GSSSGRTPTW
YSGLRAQGNY
WIVEDDGTTY
NSSIQPSPQS
SPSRYDGNPS
PLRISNSAPV
LPNGGSLHVL
TPPTIPECDE
TSPTFNLVQQ
GAEFEFENGT
LGGTKARCX
KERENNKKRE
KLPKHCDNNE
RKGFKPPASD
SAFPSPAPSY
SYLLLPFLHN
TPPLSSPTSR
RHPLFAISAP
SEEDSIEDSG
TSMAIDMKRS
VKPWEGEMIH
G
a
G l4B
al D
G 4BD
al G 4BD AS
al - K
4B G ζ
S
G D-B K1
al
G 4B IN2
al D
G 4BD
al G 4BD AS
al - K
4B G ζ
D SK
-B 1
G
IN
al
2
G 4B
al D
4
G BD
al G 4BD AS
al - K
4B G ζ
D SK
-B 1
IN
G
al
2
G 4B
al D
4
G BD
al G 4BD AS
al - K
4B G ζ
D SK
-B 1
IN
2
interact with all members of the BZR1/BES1 family, but most strongly to BEH2, BES1 and
BZR1.
Gal4 AD-BZR1
Gal4 AD-BES1
Gal4 AD-BEH2
Gal4 AD-BEH1
Gal4 AD-BEH3
Gal4 AD-BEH4
Gal4 AD-δBEH2
Gal4 AD
-LT
-LTH + 3AT
-LTHA
-LT X-gal
Figure 3. GSK1 and ASKζ bind to members of the BES1/BZR1 transcription factor family. A) The complete
amino acid sequence of BEH2 is shown. The underlined part of the sequence indicates the part of BEH2 fished
from the yeast two-hybrid library using full-length GSK1 as bait. B) Yeast two-hybrid interaction of members of
the BES1/BZR1 family with GSK1, ASKζ and BIN2. For BEH2, two clones were used, the full-length sequence
(BEH2) and the part of the sequence that was picked up from the initial library screening with GSK1 (δBEH2, see
panel A). Interactions were measured by growth on medium lacking histidine with addition of 3-amino-1,2,4triazole (-LTH + 3-AT), lacking histidine and adenine (-LTHA) and by blue coloring using 5-bromo-4-chloro-3indolyl-β-D-galactoside (-LT X-gal).
The group II ASKs seem not involved in NaCl stress signalling
Previously it has been suggested that GSK1 plays an important role in NaCl signalling
(Piao et al., 1999; 2001). Over-expression of GSK1 enhanced the resistance to NaCl stress
and induced the expression of salt responsive genes like RD29 and AtCP1. We therefore
42
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
expected that the gsk1 mutant would be more sensitive to salt stress and considered this
might be the same for mutants in the other group II members, ASKζ and BIN2.
In order to check whether group II ASKs play a role in NaCl signalling, three-week-old
wild type and bin2/gsk1/askζ plants were exposed to a high NaCl concentration (Fig. 4a).
Whereas the leaves of untreated wild type and bin2/gsk1/askζ plants remained green, NaCl
stress caused yellowing of the rosette leaves and growth was retarded. However no clear
difference in response to NaCl between wild type and the triple bin2/gsk1/askζ plants was
observed. Since this experiment was performed on soil, the actual exposure to NaCl is
uncertain. A more accurate experiment to check the influence of NaCl was performed by
growing wild type, gsk1 and bin2/gsk1/askζ plants on hydroponics for three weeks,
followed by exposure to various NaCl concentrations (Fig. 4b). As a control we used a
mutant in the HKT1 gene (Rus et al., 2001). HKT1 encodes a Na+ transporter, which is
involved in the recirculation of Na+ from shoots to roots (Berthomieu et al., 2003). As
predicted, hkt1 is hypersensitive to NaCl treatment. In contrast, wild type Arabidopsis, gsk1
and bin2/gsk1/askζ plants did not show any morphological change upon a mild NaCl
treatment.
A
0 mM
C
200 mM
4,0
Wt Col
gsk1 mutant
bin2/gsk1/askζ
3,5
bin2/gsk1/askζ
B
Wt Col
gsk1
bin2/gsk1/askζ
rootgrowth in cm
Wt Col
3,0
2,5
2,0
1,5
1,0
hkt1
0,5
0 mM
0,0
0mM
25mM
NaCL
25 mM
50 mM
Figure 4. GSK1 and the other two members of the group II ASK family seem not involved in NaCl stress
signalling. A) Phenotypic comparison of three-week-old soil grown wild type and bin2/gsk1/askζ plants treated
with 0 or 200 mM NaCl for a period of 10 days. B) NaCl treatment of wild type, gsk1, bin2/gsk1/askζ and hkt1
plantlets grown on hydroponics under short-day conditions. C) Root growth measurements of NaCl treated wild
type, gsk1 and bin2/gsk1/askζ plantlets on hydroponics under short-day conditions. Error-bars indicate the
standard error.
43
Chapter 2
Raising the NaCl concentrations affected plant growth, but no difference in phenotype was
encountered between the tested mutants and wild type (results not shown). As these
experiments are not very accurate, depending on visual inspection only, minor differences
in sensitivity to NaCl would have been missed. Therefore, classical root bending assays
(Wu et al., 1996) were performed to assess the NaCl tolerance of wild type and bin2, gsk1,
askζ, gsk1/askζ, bin2/gsk1/askζ mutant seedlings, but no obvious difference in root length
inhibition was observed (results not shown). Next a sensitive quantitative root assay
experiment was set up in which three week old wild type, gsk1 and bin2/gsk1/askζ plants
were grown on hydroponics, treated with 25 mM NaCl for 7 days, which incompletely
inhibited root growth, and root lengths were measured (Fig. 4c). The root growth of wild
type, gsk1 and bin2/gsk1/askζ was greatly reduced upon salt treatment, but no significant
difference in response to NaCl was encountered between wild type and the tested mutants.
Neither the single and double mutant seedlings of bin2, askζ and gsk1/askζ responded
differently when compared to wild type (data not shown). The fact that we consistently
found no difference in response to NaCl between wild type and the ask mutants questioned
the involvement of GSK1 and other Group II ASKs in salt stress signalling.
Discussion
The Arabidopsis genome contains ten GSK3-like kinases. In phylogenetic trees GSK1
clusters together with ASKζ and BIN2 to form the group II GSK3-like kinases (Jonak and
Hirt, 2002). BIN2 is well known as a negative regulator of BR signalling (Li et al., 2001).
We analysed loss-of-function alleles of all three genes, but in single mutants no visible
phenotype was detected. However, an insertion mutation of all group II ASKs displayed a
peculiar phenotype with abnormal stretched bending leaves and long petioles. Insensitivity
of the triple mutant to the BR biosynthesis inhibitor brz indicates that this phenotype is due
to constitutive BR sensing.
We also transformed estradiol inducible versions (Zuo et al., 2000) of group II GSKs into
the mutant background. Although most transformants nicely respond to the estradiol
treatment with expression of the transgene, only a subset of the transformants gained
sensitivity to brz treatment. It is unclear at the moment where this variability comes from.
Our initial goal was to use these transformants to study downstream effects of specific
group II GSK3-like kinase genes. A comparison by micro-array between GSK1, ASKζ and
BIN2 induced in the triple mutant background might reveal gene-specific down-stream
events.
44
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
Gain of function mutations in GSK1 and ASKζ resulted in a dwarf phenotype similar to the
previously described semi-dominant bin2-1 mutant (Li et al., 2001). Simultaneously,
similar results were obtained by Vert and Chory (2006) and Yan et al. (2009). In addition,
like ΒΙΝ2, GSK1 and ASKζ interacted with members of the BES1/BZR1 transcription
factor family. These data suggest that group II ASKs act redundantly as negative regulators
of BR signalling.
The triple insertion mutant turned out to be not completely insensitive to a treatment with
brz (Fig. 1d). This suggests that other GSK3s may be involved in the BR signalling
pathway. This idea is further supported by the observation that BES1 proteins are still
phosphorylated in a triple insertion mutant for all group II GSK3-like kinases (Vert and
Chory, 2006). Just recently Kim and co-workers (2009) demonstrated that not only group II
ASKs bind and phosphorylate BZR1, but also members of group I (ASK11-13).
Furthermore transgenic plants over-expressing ASK12 as well as a semi-dominant mutant
displayed similar dwarf phenotypes as seen before with BIN2. Altogether this suggests that
not only group II ASKs are involved in BR signalling, but at least also members of group I.
This is further supported by De Rybel et al. (2009) who identified a small molecule called
bikinin, that was shown to inhibit the activity of all group I and II ASK members and ASKθ
from group III. Inhibition of all seven ASKs activated BR responses and furthermore a
large overlap of downstream transcriptional regulation was observed between a bikinin and
BL treatment. Recently Rozhon et al. (2010) showed that bikinin could rescue the growth
phenotype of Arabidopsis seedlings with an enhanced ASKθ activity and inhibited ASKθ
mediated phosphorylation of BEH2. A negative role for ASKθ in the BR signalling
pathway was further confirmed by several phenotypic and molecular analyses that showed
that ASKθ is negatively regulated by BRI1 and that it phosphorylates several members of
the BES1/BZR1 family.
The initial aim of this project was to study the role of the GSK3-like kinase GSK1 in
tolerance towards salt. In contrast to previous research by Piao et al. (1999; 2001) we could
not establish a role for GSK1 in tolerance against Na+ stress. Neither was this the case for
the other group II GSK3-like kinases BIN2 and ASKζ. Piao et al. demonstrated that ectopic
expression of GSK1 in Arabidopsis leads to expression of multiple stress-induced genes
and salt tolerance was increased. Diametrically opposed, Koh et al. (2007) showed that a
insert mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, is more tolerant to salt
stress. Furthermore, NaCl significantly inhibited germination of BR deficient (det2-1) and
signalling mutants (bri1-301) in Arabidopsis (Zhang et al., 2009). This was also observed
when the repressor of the BRI1 receptor BKI1 was over-expressed. Plants over-expressing
BRI1 on the other hand were more resistant to NaCl. Moreover, Zeng et al. (2009) showed
45
Chapter 2
that the semi-dominant mutant bin2-1 was very sensitive to salt. Therefore, all data point to
a positive role for BRs in the tolerance to salt stress. It therefore is puzzling why Piao et al.
observed increased Na+ tolerance upon inhibition of BR signalling. One reason could be
that over-expression of GSK1 might have caused unforeseen sight effects.
Given the general picture that increased BR signalling improved Na+ tolerance we expected
that knocking out ASKs would result in a more salt resistant plant. However, even a triple
insertion mutation of the group II GSK3-like kinases did not result in more salt resistant
plants. It might be that also in salt stress signalling, additional redundancy among GSK3like kinases exists. In this respect it is noteworthy that in Medicago sativa a group IV
GSK3-like kinase was identified that is involved in stress tolerance (Kempa et al., 2007).
Analysis of higher order mutant combinations might shed light on the function of all
GSK3-like kinases in Arabidopsis.
Experimental procedures
Plant material and growth
Unless stated otherwise, Arabidopsis Columbia was used as wild type control for phenotype
comparisons in RNA analyses, brassinazole experiments and in NaCl stress assays. Seeds
of Arabidopsis Colombia, mutant and transgenic lines were sterilized in 10% bleach,
washed three times with sterile water, dissolved in 0.1% agarose and sewed on 0.8% (w/v)
agar plates containing 0.5X Murashige and Skoog (MS) salts, 1.5% sucrose at pH 5.7-5.9.
After stratification for 2 days at 4 °C, seeds were germinated in a climate chamber at 22 °C
under a 10-hr-light/14-hr-dark photoperiod. Later, seedlings were transferred to soil or
grown in hydroculture in a half strength Hoagland solution (3 mM KNO3, 2 mM Ca(NO3)2
x 4 H2O, 1 mM NH4H2PO4, 0.5 mM MgSO4 x 7 H2O, 1 µM KCl, 25 µM H3BO3, 2 µM
MnSO4 x 4 H2O, 2 µM ZnSO4 x 7 H2O, 0.1 µM CuSO4 x 5 H2O, 0.1 µM (NH4)6Mo7O24 x
24 H2O, 20 µM Fe(Na)EDTA, 2 µM MnSO4 x H2O and 0.39 g/l MES adjusted to pH 5.5
with KOH). Plants were kept in either a climate chamber at 22 °C under a 10-hr-light/14hr-dark photoperiod or grown under standard greenhouse conditions as indicated.
Mutant isolation
The knock-out mutant for BIN2 (At4G18710, FLAG_593C09, Wassilawskija background)
was isolated from the FLAGdb Collection at INRA Versailles (http://urgv.
evry.inra.fr/projects/FLAGdb++/HTML/index.shtml). The knock-out mutants for GSK1
(At1G06390, SALK_062147, Colombia background) and ASKζ (At2G30980, SALK_
46
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
060220, Colombia ecotype) were obtained from the SALK Institute Collection (http://
signal.salk.edu/cgi-bin/tdnaexpress). The double gsk1/askζ and triple bin2/gsk1/askζ
mutants were obtained via cross-pollination and confirmed by PCR. In case of the triple
insertion mutant, offspring of several triple mutants that originated from independent
crossings were analyzed to circumvent any unwanted side effects of potential modifiers
originating from mixing different ecotypes.
Generation of gain-of-function mutants
The full length coding sequences of GSK1 (101-FW and 102-RV) and ASKζ (121-FW and
122-RV) were amplified via RT-PCR on Arabidopsis Colombia leaf mRNA. Amplification
products were cloned into the pMON999 (PET24; GSK1 and PET23; ASKζ, BgIII/Xba1)
and completely sequenced. The generation of the dominant constructs of GSK1 and ASKζ
into the Gateway pENTR4 vector (Invitrogen) was performed into two steps. First, the
5´part of GSK1 and ASKζ was amplified by PCR (M544-FW and M549-RV on PET24;
121-FW and M549-RV on PET23). These fragments were cloned as Nco1/Kpn1 fragments
into pENTR4 (PET69; 5´ GSK1dom and PET70; 5´ASKζdom). Then the 3’ part, containing
the glutamic acid (E) into lysin (K) substitution was generated (M550-FW and M551-RV
on PET24; M550-FW and M546-RV on PET23). These PCR fragments were cloned into
PET69 and PET70 to create PET72; GSK1dom and PET73; ASKζdom. Next, a Gateway LR
recombination reaction transferred the dominant cDNA constructs into destination vector
pK2GW7, which contains the 35S promoter and terminator (Karimi et al., 2002) and the
inserts were fully sequenced (PET75; 35SCaMV:GSK1dom and PET76;
35SCaMV:ASKζdom). Nucleotide sequences of primers are listed in table 1. The constructs
were introduced in Arabidopsis using Agrobacterium tumefaciens strain C58C1 (pMP90)
and the floral dip method (Clough and Bent, 1998).
Generation of CaMV35S constructs
The full length coding sequences of GSK1 (101-FW and 102-RV) and ASKζ (121-FW and
122-RV) were amplified via RT-PCR on Arabidopsis Colombia leaf mRNA. BIN2 was
amplified (144-FW and 145-RV) from a clone that was kindly gifted by Dr. Jianming Li.
Amplification products were cloned into the Gateway pENTR4 vector (Nco1/BamH1,
PET43; GSK1, PET44; ASKζ and PET78; BIN2) and fully sequenced. Next, a Gateway LR
reaction was performed to transfer the constructs into the destination vector pH2WG7
(Karimi et al., 2002), containing the 35S promoter and terminator (PET142;
47
Chapter 2
35SCaMV:GSK1, PET143; 35SCaMV:ASKζ and PET145; 35SCaMV:BIN2). The inserts
were sequenced and transformed via electroporation into the Escherichia coli strain XL1blue (Stragene) and afterwards in the Agrobacterium tumefaciens strain C58C1 (pMP90).
The plasmids were introduced in the Arabidopsis bin2/gsk1/askζ mutant using the floral
dip method (Clough and Bent, 1998). Nucleotide sequences of primers are listed in table 1.
Generation of the transient-expressing constructs
The full length coding sequences of GSK1 (101-FW and 102-RV) and ASKζ (121-FW and
122-RV) were amplified via RT-PCR on Arabidopsis leaf mRNA. Amplification products
were cloned into the pMON999 (PET24; GSK1 and PET23; ASKζ, BgIII/Xba1). BIN2 was
amplified from a clone (144-FW and 145-RV, PET42; BIN2) that was kindly gifted by Dr.
Jianming Li. Generation of BIN2dom into the Gateway pENTR4 vector (Invitrogen) was
performed into two steps. First, the 5´part of BIN2 was amplified by PCR using primers
144-FW and M549-RV on PET42 . This fragment was cloned as a Nco1/Kpn1 fragment
into pENTR4 (PET71; 5´BIN2dom). The 3’ part, containing the glutamic acid (E) into lysin
(K) substitution, was amplified with M550-FW and 145-RV. The PCR fragment was
cloned into PET71 to create PET74. The full length coding sequences of GSK1, ASKζ,
BIN2 and BIN2dom were amplified from PET24 (GSK1; M623-FW and 80-RV, cut with
XhoI/XbaI), PET23 (ASKζ; M624-FW and 85-RV, cut with XhoI/XbaI), PET42 (BIN2;
M621-FW and M622-RV, cut with SalI/SpeI) and PET74 (BIN2dom; M621-FW and M622RV, cutted with SalI/SpeI) and cloned in pER8 (XhoI/Spe1). The inserts were sequenced
and transformed via electroporation into the Escherichia coli strain XL1-blue (Stragene)
and afterwards in the Agrobacterium tumefaciens strain C58C1 (pMP90). The plasmids
were introduced in the Arabidopsis bin2/gsk1/askζ mutant using the floral dip method
(Clough and Bent, 1998). Nucleotide sequences of primers are listed in table 1.
Estradiol treatments with pER8 transgenic lines
T2 generation transgenic plants carrying the XVE cassette were selected on 0.8% (w/v)
agar plates containing 0.5X Murashige and Skoog (MS) salts, 1.5% sucrose and 30 mg/l
hygromycin for selection. After stratification for 2 days at 4 °C, seeds were germinated in a
climate chamber at 22 °C under a 16-hr-light/8-hr-dark photoperiod for 10 days, after
which 15-20 seedlings were transferred to 50 ml polypropylene tubes (Sarstedt) containing
10 ml 0.5X MS medium with 1.5% sucrose. The tubes were incubated on a shaker at 120
48
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
rpm for 4 additional days before treatment. Plants were either treated with 4 µM estradiol
(Sigma, dissolved in DMSO) or DMSO for 24 hours unless stated otherwise.
Brassinazole experiment
Brassinazole (Tokyo Chemical Industry) was dissolved in DMSO (1 mM) and stored at -20
°C. Seeds of Arabidopsis Colombia, mutant and transgenic lines were sterilized in 10%
bleach, washed three times with sterile water, dissolved in 0.1% agarose and sown in a
straight line on 0.8% (w/v) agar plates containing 0.5X Murashige and Skoog (MS) salts,
1.5% sucrose and 1 µM brassinazole or DMSO as control. The plates were wrapped in
aluminium foil and incubated for 48 hrs at 4 °C to stimulate equal germination. Plates were
placed in a vertical orientation for 7 days at 22 °C. The seedlings that were used for
microscopy were made transparent in 100% EtOH. A similar experiment was performed
with the pER8 transgenic lines, only seeds were sown on various plates with or without 4
µM estradiol, 1 µM brassinazole or DMSO as a control.
RT PCR analysis on ASK insertion mutants
Plant tissue was homogenized in liquid nitrogen and 1 ml Trizol (38% phenol, 0.8 M
guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M NaAc (pH5) and 5% glycerol)
was added after which the samples were extracted with 0.3 ml chloroform. The nucleic
acids were precipitated with an equal volume 2-propanol and washed with 75% EtOH. The
pellet was dissolved in 500 µl RNase free H2O. A DNase treatment was performed by
adding 50 µl DNase buffer (0.5M Tris/HCl pH 7.6; 0.1 M MgCl2) and 3 µl RNase free
DNase (Roche), followed by an incubation step for 30 min at 37°C. Next, a phenolchloroform extraction was performed and the RNA was precipitated again using 2propanol. The pellet was washed with 75% EtOH and dissolved in RNase free H2O. Total
RNA was quantified using the Nanodrop ND1000. cDNA was synthesized from total RNA
(2 µg, boiled for 1 min) using 100 Units M-MLV Reverse Transcriptase (Invitrogen), 2 mM
dNTPs, 100 mM DTT, 10X RT buffer and 10 µM oligo-dT primer (P0007) at 42°C for 1
hr. cDNA products were amplified using primers specific for GSK1 (M558–FW and M561RV) , ASKζ (M559-FW and M562-RV), BIN2 (M560-FW and M563-RV) and ACTIN
(M554-FW and M555-RV). The nucleotide sequences of the primers used in figure 1b are
shown in table 1. It was ensured that the amplification rate was in a linear range by using a
reduced number of amplification cycles (27 cycles for GSKs, 21 cycles for ACTIN). All
PCR products were separated by electrophoresis in a 1% (w/v) agarose.
49
Chapter 2
Table 1. Description of primers used for PCR
Primer
Gene
Sequence 5´-3´
M554-FW
M555-RV
85-FW
121-FW
122-RV
M546-RV
M559-FW
M562-RV
M624-FW
M726-FW
M727-RV
140-FW
141-RV
M730-FW
M731-RV
144-FW
145-RV
M560-FW
M563-RV
M621-FW
M622-RV
M728-FW
M729-RV
M732-FW
M733-RV
M549-RV
M550-FW
80-FW
101-FW
102-RV
M544-FW
M551-RV
M558–FW
M561-RV
M623–FW
M724-FW
M725-RV
P0007-RV
ACTIN
ACTIN
ASKζ
ASKζ
ASKζ
ASKζ
ASKζ
ASKζ
ASKζ
ASKζ
ASKζ
BEH3
BEH3
BES1
BES1
BIN2
BIN2
BIN2
BIN2
BIN2
BIN2
BIN2
BIN2
BZR1
BZR1
GSK
GSK
GSK1
GSK1
GSK1
GSK1
GSK1
GSK1
GSK1
GSK1
GSK1
GSK1
Oligo-dT
GGCCAACAGAGAGAAGATGACTC
CGAAAGCAGATTCGGGAAATGAAC
GCTCTAGACTAGGGTCCAGCTTGAAATG
GCCCATGGCTTCGATACCATG
GGGATCCCTAGGG TCCAGCTTGAAATG
GGGGTCGACCTAGGGTCCAGCTTGAAATGG
CGTCGTCCCGATATAGACAAC
CTAGGGTCCAGCTTGAAATGGA
GGCTCGAGATGACTTCGATACCATTGGGG
GTAATAGAGAGAGAACAAAATAGTCT
GTTGTCTATATCGGGACGACGTTTC
GGAATTCATGACGTCGGGGACTAGAACG
CCTCGAGTCATCTGGTTCTTGAGTTTCC
AAACGTACTTCTTCTTCAAAACGCG
GACGTCATCTTCTTCGAGGAATACG
GCCCATGGCTGATGATAAGGAGATGCCTG
GGGATCCTTAAGTTCCAGATTGATTCAAGAAG
CTGTGTCTCTATCGCCATGGCTG
GATTGATTCAAGAAGCTTAGAC
GGGTCGACATGGCTGATGATAAGGAGATG
GGACTAGTTTAAGTTCCAGATTGATTCAAG
AGAGAAGAGCTGGATTCACATGGTCT
TCAGCCATGGCGATAGAGACACAG
GTTGGAGAAGAAAGAGAGATTCTTC
TCATCGGGAAAACCAACAACAAACC
TTCGCGAGTTGGGGTACCAAGAAC
GTTCTTGGTACCCCAACTCGCAAAGAAAT
GCTCTAGATTAACTGTTTTGTAATCCTGTG
CCCCATGGCCTCATTACCATTG
GGGATCCTTAACTGTTTTGTAATCCTGTG
GGCCATGGCTCATTACCATTGGGG
GGCTCGAGTTAACTGTTTTGTAATCCTG
CGCCGTCCTGAATTGGATTCT
TTAACTGTTTTGTAATCCTGTG
GGCTCGAGATGGCCTCATTACCATTGGGG
GCAAAGAGAGAGGAAAAATTAGACT
GAATCCAATTCAGGACGGCGTTTC
GACTCGAGTCGACATCGATTTTTTTTTTTTTTT
Yeast two-hybrid
The full length coding sequences of GSK1 (101-FW and 102-RV, listed in table 1) and
ASKζ (121-FW and 122-RV, listed in table 1) were amplified via RT-PCR on Arabidopsis
leaf mRNA and cloned in pAS1 (Nco1/BamH1, Clontech). Dr. Jianming Li kindly gifted
BIN2 in pAS1 and BZR1 and BES1 in pACT. BEH1 (ABRC clone U17510), BEH2 (ABRC
50
Group II GSK3/SHAGGY-like kinases act redundantly in the brassinosteroid signalling pathway
clone U13164) and BEH4 (ABRC clone U13344) were cloned EcoRI/SalI in pAD-GAL4
(Stratagene), cut with EcoRI and Klenow was used to get it in frame. The full length coding
sequence of BEH3 (140-FW and 141-RV, listed in table 1) were amplified via RT-PCR on
Arabidopsis leaf mRNA and cloned in pAD-GAL4 (EcoRI/XhoI). The clones were
sequenced to ensure that no mutations were introduced after PCR amplification. The
plasmids were introduced into yeast strain PJ69 (James et al., 1996), which carries HIS3,
ADE2 and LACZ reporter genes driven by distinct GAL4-responsive promoters. For the
yeast two-hybrid screen, GSK1 was used as a bait against a cDNA library from the ABRC
stock center (CD4-10) that was prepared from mature Arabidopsis leaves and roots. Library
screen was performed as described previously (Quatrocchio et al., 2006) yielding
approximately 1 x 105 transformants. The two-hybrid screen was optimized by adding 3amino-triazole, an enzymatic inhibitor of His3. Plasmid DNA was isolated from positive
(HIS, ADE) colonies, transformed in Escherichia coli, sequenced and subsequently
reintroduced in PJ69, either with the empty pBD-GAL4 vector or with the bait plasmid
expressing GSK1. Only plasmids that specified a HIS, ADE phenotype when cotransformed with the bait vector, but not when co-transformed with the empty pBD-GAL4,
were considered as positives and analyzed further. The yeast two hybrid assay between the
BES1/BZR1 family and members of the GSK3/SHAGGY like family was performed on the
same manner.
NaCl stress assays
In order to analyse the effect of NaCl stress on Arabidopsis Colombia and
bin2/gsk1/askζ plants grown on soil, three-week-old plantlets were soaked every three days
in 0 or 200 mM NaCl for a period of 10 days under greenhouse conditions.
To test the effect of a NaCl treatment on Arabidopsis Colombia, gsk1, bin2/gsk1/askζ and
hkt1 in hydroculture, seeds were sown directly in agarplugs (Araponics) in a hydroponic
growing system (Araponics). After a cold treatment of 48 hrs at 4°C to synchronise
germination, seeds were germinated in a climate chamber at 22 °C under a 10-hr-light/14hr-dark photoperiod in half strength Hoagland solution. After 3 weeks, plants of
approximate equal size were transferred to one litre pots and subjected to half strength
Hoagland solution supplemented with 0, 25 or 50 mM NaCl. Hoagland solutions were
refreshed every week. Pictures were taken after an additional growth of 4 weeks.
For the root measurement experiment, Arabidopsis Colombia, gsk1, bin2/gsk1/askζ plants
of approximate equal size were transferred to one litre pots after 3 weeks. After a total of 30
days, plants were subjected to half strength Hoagland solution supplemented with 25 mM
51
Chapter 2
NaCl or further cultivated on standard Hoagland solution. After 3 days of treatment, root
tips were dipped in active coal powder. After another 3 days, root growth was measured as
the length of the unstained segment. Per treatment 12 plants were analysed, which were
divided over a total of 4 pots.
Acknowledgements
We thank Maartje Kuijpers for plant care. BIN2, BES1 and BZR1 clones were a kind gift
of Jianming Li. This work was supported by a grant of the Netherlands Organisation for
Scientific Research (NWO).
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55
Chapter 3
Towards identification of group II ASKs
containing complexes in Arabidopsis
Nathalie Verhoef and Erik Souer
Towards identification of group II ASKs containing complexes in Arabidopsis
Abstract
The two mammalian glycogen synthase kinase 3 (GSK3) genes play a role in diverse
processes like metabolism, development and disease. In Arabidopsis there are ten
GSK3/SHAGGY-like kinases (ASKs) that have been implicated in stress and
brassinosteroid (BR) signalling. The group II ASKs, including its most famous member
BIN2, seem to be involved in the repression of BR signalling (Chapter 2). At the start of
this project it was unknown by which mechanism group II ASKs are regulated in the BR
signalling pathway. Furthermore group II ASKs might have additional roles in other
signalling pathways. The setting in which group II ASK proteins reside within the cell
might give answers to these questions. Therefore, we sought to reveal group II ASK
complexes within the plant cell. For this purpose we fused the group II ASK members to
various Tandem Affinity Purification (TAP) tags that were available. Although in most
cases we found fusion protein expression, the final isolation of the protein complex of plant
extracts appeared problematic.
Introduction
Several methods have been developed to discover interactions between a known protein
and unknown partner proteins. Commonly used methodology to identify protein-protein
interactions are complex immuno-precipitation (Co-IP) and yeast two-hybrid analysis.
Yeast two-hybrid interaction requires protein processing and binding in a setting that is
incomparable with the natural habitat where the studied protein normally resides in. A
disadvantage of Co-IP is that for every protein a new antibody needs to be generated.
Specificity and capacity of these antibodies to detect low levels of protein is sometimes
insufficient. The development of several synthetic affinity tags for which high quality
antibodies are available circumvents these disadvantages. Purification of protein complexes
using antibodies against affinity tags can be performed directly from plant extracts.
Furthermore, indirect interactions can be revealed, whereas yeast two-hybrid only identifies
direct interactions between bait and prey protein. However, fusion of an affinity tag to a
protein yields an artificial molecule, for which biological activity has to be proven. Full
complementation of the corresponding mutant by the fusion protein is the preferred method
to do so.
The ideal affinity tag is small, allows rapid purification to maintain protein (complex)
integrity and conserve post-translational modifications. Examples of epitope tags that are
used nowadays are Green Fluorescent Protein (GFP), Glutathione-S transferase (GST),
59
Chapter 3
FLAG, myc and HIS (Reviewed by Terpe, 2003). Although many of these tags are very
well suited to recognise the studied protein in extracts or even in living cells, purification of
a complex where the protein of interest belongs to, is often hampered by low yield and copurification of proteins that are not genuine interactors.
A
D
contaminant
TAPi
Target protein
Protein A
partners
contaminant
TAPa
TEV cleavage site
Target protein
Protein A
partners
6x HIS
9x myc
CBP domain
B
TAPi purification
C
First affinity step
Reversed TAPi purification
First affinity step
IgG beads
E
TAPa purification
First affinity step
IgG beads
Cal beads
TEV cleavage step
Native elution (EGTA)
Second affinity step
Second affinity step
3C cleavage site
3C cleavage step
Second affinity step
Cal beads
α-Myc
Ni beads
Native elution (EGTA)
Elution via low pH
Figure 1. Schematic representation of TAPi and TAPa purification. A) The TAPi tag consists of two IgGbinding units of protein A from Staphylococcus aureus (ProtA) and the Calmodulin Binding Peptide (CBP) that
are fused in tandem and separated by a TEV protease cleavage site. B) TAPi tagged proteins can be recovered by
affinity selection on an IgG-matrix. After washing, TEV protease is added to release the bound material. The
eluate is then incubated with calmodulin-coated beads in the presence of calcium. After washing, the bounded
material is released with EGTA. C) An alternative Reversed TAPi method has been used by us (see Results).
Here, protein extracts are first incubated with calmodulin-coated beads in the presence of calcium. After washing,
the bounded material is released with EGTA. Next the eluate is incubated with IgG beads, washed and elution is
performed with a low pH buffer. D) The TAPa tag consists of two IgG-binding domains of protein A, a 3C
cleavage site (3C), nine myc epitopes and six histidine repeats. E) Protein extracts are first incubated with IgG
beads. After washing, TAPa-tagged proteins are cleaved by the low-temperature active rhinovirus 3C protease
(3C). The eluate is either incubated with α-myc beads or in our case with nickel coated beads. In the latter case the
complex can be eluted with a imidazole containing buffer.
60
Towards identification of group II ASKs containing complexes in Arabidopsis
The generation of Tandem Affinity Purification (TAP) tags containing two or more
epitopes separated by a cleavable protease site was believed to improve complex
purification significantly. The first TAP purification was demonstrated in yeast and later
this approach was used to purify protein complexes from many other organisms including
plants (Puig et al., 2001; Rohila et al., 2004; Witte et al., 2004; Rubio et al., 2005; Rohila
et al., 2006). The original TAP tag consists of the IgG binding domain of protein A from
Staphylococcus aureus and the Calmodulin-Binding Peptide (CBP) which are fused in
tandem and separated by a tobacco etch virus (TEV) cleavage site (Puig et al., 2001). Later
on this tag (TAPi) was renovated to improve protein purification from plants (Rohila et al.,
2004). This tag still contained the same affinity tags and cleavage site, but the duplicated
regions of the tandem protein A domains were made non-identical and a nuclear
localization signal (NLS) was removed from the CBP domain. In addition, catalase intron 1
from castor bean was introduced for higher gene expression in plants. As the TEV cleavage
should be performed at a rather undesirable high temperature (16 ˚C), Rubio et al. (2005)
developed an alternative TAP tag (TAPa) in which the TEV cleavage site was replaced by
the human rhinovirus 3C protease cleavage site which is optimally cleaved at 4°C, allowing
purification under conditions that usually stabilize protein complexes (Fig. 1d,e). The 3C
cleavage site is also more specific as it consists of an eight amino acid sequence instead of
the six residues present in the TEV cleavage site. Furthermore they replaced the CBP
domain by nine myc epitopes and six histidine repeats, enabling protein purification via two
different strategies.
Protein-protein interaction studies proved to be useful in the study of several signalling
pathways in plants (Xinga and Chen, 2006; Batelli et al., 2007; Van Leene et al., 2008).
The signalling cascade downstream of the plant hormone brassinosteroid (BR) now
constitutes a pathway that runs from the plasma membrane to activation of target promoters
in the nucleus (Kim et al., 2009). BRs are perceived at the plasma membrane by the
leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1, Li
and Chory, 1997). Upon BR activation, BRI1 dimerize and transphosphorylate with the coreceptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and dissociate from the
repressor BRI1 KINASE INHIBITOR 1 (BKI1), leading to BRI1 activation and
phosphorylation of BR SIGNALING KINASES (BSKs; Li et al., 2002; Nam and Li, 2002;
Wang and Chory, 2006; Tang et al., 2008). Downstream of the BSKs the GSK3/SHAGGYlike kinase BR-INSENSITIVE 2 (BIN2) acts a negative regulator by phosphorylating two
closely related transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and
BRASSINAZOLE RESISTANT 1 (BZR1) (He et al., 2002; Yin et al., 2002). Kim et al.
(2009) showed that phosphorylation of BSK1 a member of the (BSKs), interacts with and
61
Chapter 3
presumably activates the plant-specific serine/threonine protein phosphatase BRI1
SUPPRESSOR 1 (BSU1), which in turn inactivates BIN2 by dephosphorylation at
pTyr200. As a result unphosphorylated BZR1/BES1 accumulate in the nucleus where they
regulate the expression of BR-responsive genes. Next to BIN2, 5 other Arabidopsis
SHAGGY-like protein kinases (ASKs) act redundantly with BIN2 in the Arabidopsis BR
signalling pathway (Chapter 2; Vert and Chory, 2006; Kim et al., 2009; Yan et al., 2009;
Rozhon et al., 2010).
In order to learn more about the mode of action of GSK3/SHAGGY-like kinases, we
transformed Arabidopsis with different TAP tagged versions of group II members for
subsequent analysis of interacting proteins in plants. Here, we present an analysis of the
efficiency of complex purification of various TAP fusions.
Results
Molecular and phenotypical characterization of TAPi-GSK1/ASKζ transgenic lines
In order to isolate proteins interacting with ASKζ and GSK1 we used the improved
Tandem Affinity Purification tag (TAPi tag) made by Rohila et al. (2004) as this tag had
already successfully been used for the isolation of protein complexes from plants.
Transgenic Arabidopsis lines were generated containing a N-terminal fusion of the TAPi
tag to both ASKζ and GSK1 (N-TAPiASKζ and N-TAPiGSK1, Fig. 2a). GSK1 was also
fused with the C-TAPi tag at its carboxyl end (C-TAPiGSK1, Fig. 2a). As a negative
control to use during complex isolation along with the TAPi tagged fusions, the C-TAPi tag
alone was transformed to Arabidopsis. All TAPi derived constructs were over-expressed in
Arabidopsis Colombia under control of the strong constitutive CaMV35S promoter.
Independent lines of the T2 generation were screened for their ability to express the fusion
protein and for several transgenic lines containing N-TAPiGSK1, N-TAPiASKζ and CTAPi constructs high levels of fusion-protein were detected (Fig. 2b-d). The PAP antibody
cross-reacted with proteins of the expected size; the TAPi tag alone had a size of ~20 kDa
and N-TAPiGSK1 and N-TAPiASKζ had a size of ~78 kDa. In the C-TAPiGSK1
transgenic lines no accumulation of fusion-protein could be detected (data not shown).
Since we could detect the fusion mRNA in the C-TAPiGSK1 transformed T2 lines, it seems
that the tag is somehow detrimental for synthesis or stability of the C-terminal tagged
fusion protein (data not shown). Visually, all characterized transgenic lines were
indistinguishable from untransformed Arabidopsis Colombia (Fig. 2e).
62
T35S
TEV
CBP domain
attR1::Cmr::ccdB::attR2
08
2x IgG BD
35S:C-TAPi
35S
T35S
attR1::Cmr::ccdB::attR2
CBP domain
TEV
C
N
-T
AP
iA
N
SK
-T
ζ J8
AP
10
iA
0N
SK
3
-T
ζ J8
AP
10
iA
0N
S
6
-T
Kζ
J
AP
81
00
W iAS
-7
tC
Kζ
J
81
ol
0
35S
D
C
-T
A
C Pi J
-T
J
A 81
C Pi J 01-T
81 1
0
A
C Pi J 1-4
-T
81
0
A
C Pi J 1-5
-T
8
AP 101
C
-T i J81 -6
0
A
W Pi J 1-8
t C 81
ol 01-9
N
-T
AP
B
35S:N-TAPi
N
A
iG
SK
-T
1 J8
AP
09
iG
7N
6
SK
-T
1 J8
AP
09
iG
7W
10
SK
tC
1 J8
ol
09
7
-1
3
Towards identification of group II ASKs containing complexes in Arabidopsis
2x IgG BD
Figure 2. Molecular and phenotypic analysis of TAPi-GSK1/ASKζ transgenic lines. A) Schematic drawing of
the N-TAPi and C-TAPi expression cassettes under control of the CaMV35S promoter. ASKζ and GSK1 will
replace the region between the two attR recombination sites after Gateway recombination. B-D) From several
independent N-TAPiGSK1 (B), N-TAPiASKζ (C) and C-TAPi (D) lines protein was extracted from leaves and
loaded on a SDS-PAGE gel followed by Western blotting. The Western blot was immuno-probed with the
peroxidase anti peroxidase antibody (PAP) that recognizes the protein A part of the TAPi tag.
TAPi purification with N-TAPiGSK1
Initially, a small-scale TAP tag purification (~3 gram leaf tissue) was performed with plants
expressing N-TAPiGSK1 or C-TAPi. Protein extracts were incubated with IgG beads and
after washing incubated with TEV protease. In the second purification step, the TEV eluate
was incubated with calmodulin-coated beads and subsequently eluted by using an EGTAcontaining buffer (Fig. 1b). In order to follow the purification process, samples were taken
after each step and subjected to Western blot analysis using the peroxidase anti peroxidase
antibody (PAP) or the anti calmodulin binding protein antibody (CBP) to follow the
presence or absence of the epitopes. N-TAPiGSK1 as well as C-TAPi proteins bound quite
efficient to the IgG beads, but after the TEV protease cleavage both proteins could not be
detected (Fig. 3a,b). In the case of C-TAPi, cleavage did work as we could detect the
protein A part that remained attached to IgG beads after cleaving and, after concentration of
the final C-TAPi eluate, the CBP part that was clipped of the original fusion protein (Fig.
3a).
After N-TAPiGSK1 purification no N-TAPiGSK1 protein was observed, neither could we
detect the cleaved protein A part bound to the IgG beads (Fig. 3b). Although we succeeded
to perform the TAPi purification with C-TAPi, analysis of flow through fractions and
eluates revealed that a lot of the fusion protein was lost during the purification, especially
during the TEV protease cleavage step. During purification of N-TAPiGSK1 all fusion
protein seems to be lost after the TEV protease cleavage and second purification step.
63
Chapter 3
B
First affinity step
Ex
tra
c
Bo t (1
un )
d
Fi ed
to
na
Ig
le
G
lu
at
(2
e
)
(5
)
Ex
tra
c
Bo t (1
un )
de
d
El
to
ua
Ig
te
G
af
(2
te
Ig
)
r
G
be TEV
ad
cl
s
ea
(4
va
)
g
e
(3
)
e
Ex
tra
c
Bo t (1
un )
d
Fi ed
to
na
Ig
le
G
lu
at
(2
e
)
(5
)
tra
c
Bo t (1
un )
de
d
El
to
ua
Ig
te
G
af
(2
te
Ig
)
r
G
be TEV
ad
cl
s
ea
(4
va
)
g
Ex
Input 1
C
(3
)
A
IgG beads
2
4
TEV cleavage step
TAPiGSK1
TAPiGSK1
3
Second affinity step
TAPi
Protein A
part
Cal beads
TAPi
*
~
35S:TAPiJ8321-5
CBP part
*
~
Native elution (EGTA)
35S:N-TAPiGSK1J8300-4
5
Figure 3. Inmuno-blot of different fractions obtained during TAPi purification of N-TAPiGSK1 and CTAPi. A-B) A TAPi purification was performed using protein extracts from C-TAPi (A) and N-TAPiGSK1 (B)
transformants. Several fractions, indicated above each lane, were separated on a 12,5% SDS-PAGE gel. The
Western blot was probed with either CBP (*) or PAP (~) antibodies. The numbering corresponds to fractions that
were collected during purification as depicted in C (C) TAPi purification scheme. The numbering refers to
samples on the immuno-blots shown in A and B : 1. Input protein extract (40 µg); 2. IgG bound fraction prior to
TEV cleavage (10x); 3. Eluate after protease TEV cleavage (20x); 4. IgG beads after the TEV protease cleavage
step (20x); 5. Eluate from calmodulin beads using EGTA containing buffer followed by a concentration step
(250x). x indicate the amount of protein loaded relative to input sample.
In order to find out why a lot of fusion protein was lost during the TEV protease step,
several experiments were performed. Rohila et al. (2004) reported that binding of a certain
protease to the IgG beads might have a negative effect on the purification of certain TAP
tagged proteins. Addition of the protease inhibitor E64 increased their protein recovery, but
in our case the amount of fusion protein was not visibly changed with or without the
addition of E64 (data not shown) at the stage when the C-TAPi or N-TAPiGSK1 was
bound to the IgG beads.
Next, we determined how efficiently TEV protease cleaved C-TAPi and N-TAPiGSK1.
Protein extracts were incubated with IgG coated beads and after washing TEV protease was
added. At several time points a sample was taken and immuno-probed with the PAP or
CBP antibody (Fig. 4). Looking at the C-TAPi cleavage, most of the fusion proteins were
cleaved within 1 hour. Cleavage of N-TAPiGSK1 on the other hand was less efficient.
After 1 hour of treatment still a lot of fusion protein was not cleaved. Due to the high
background of the immuno-blot incubated with CBP antibody we failed to detect the
cleaved N-TAPiGSK1 proteins. So, although a large fraction of the N-TAPiGSK1 fusion
protein is cleaved it could be that the released CBP-GSK1 fusion is degraded by a protease
in the extract.
64
Towards identification of group II ASKs containing complexes in Arabidopsis
N-TAPiGSK1J8293-6
60
30
45
15
pu
0
In
60
30
45
15
0
In
pu
t
t
C-TAPiJ8321-5
min
TAPGSK1
CBP
TAP
Protein A part
Protein A part
0
CBP part
TAPGSK1
PAP
TAP
Protein A part
Protein A part
Figure 4. TEV does not cleave N-TAPiGSK1 efficiently. Protein extracts from transgenic plants containing NTAPiGSK1 and C-TAPi (negative control) were incubated with IgG coated beads. During the TEV cleavage
several samples were taken and separated on a 12,5% SDS-PAGE gel. The Western blot was immuno-probed with
either the CBP or PAP antibody. The input extract contained 40 µg protein. In the TEV with IgG beads fractions,
50x the amount of protein was loaded relative to the input.
Reverse TAPi purification with N-TAPiGSK1
As optimilization of the TAPi purification was unsuccesfull we reasoned it might be
possible to perform TAPi purification in reverse ommiting the TEV cleavage step (Fig. 5).
The protein extract was first incubated with the calmodulin coated beads and then the
second affinity purification step was performed with IgG beads. To release the proteins
from the beads without using TEV cleavage, other ways of eluting were tested such as
boiling and by using the gentle Ag/Ab buffer (Pierce). The best results were obtained with a
low pH buffer (Fig. 5). The reverse TAPi purification was more efficient in comparison to
the TAPi purification. C-TAPi binding and elution from the calmodulin beads went very
well, but in the case of N-TAPiGSK1 not all fusion protein did bind to beads or was
released from the beads. Elution from the IgG beads went successful, but the amount of
fusion protein at the end was too low to detect it on SDS PAGE after Coomassie staining.
65
Chapter 3
(6
)
B
In
pu
t(
C 1)
al
be
Fi ad
na s e
le
lu
at
lu
Fi
na ate e (
le
1 2)
Pr lua (3)
ot
te
A
2
(4
C
)
al
be
ad
C
al
s
be (5)
Ig ad
s
G
be flow
ad
th
r
s
(7 oug
)
h
In
pu
t(
C 1)
al
be
Fi ads
na
l e elua
lu
te
Fi
(2
na ate
)
le
1
Pr lua (3)
te
ot
2
A
(4
C
)
al
be
ad
C
s
al
(
5)
be
Ig ads
G
be flow
ad
th
r
s
(7 oug
)
h
(6
)
A
Input 1
C
First affinity step
Cal beads
6
5
Native elution (EGTA)
TAPiGSK1
Cal beads
2
second affinity step
IgG beads
TAP
Elution via low pH
7
C:TAPiJ8321-5
N-TAPiGSK1J8293-6
IgG beads
3,4
Figure 5. Inmuno-blot of different fractions obtained during the reverse TAPi purification of N-TAPiGSK1
and C-TAPi. A-B) A reverse TAPi purification was performed with protein extracts from N-TAPiGSK1 (A) and
C-TAPi (B) transformants. Several fractions, indicated above each lane, were separated on a 12,5% SDS-PAGE
gel. The Western blot was probed with the CBP antibody. The numbering corresponds to fractions that were
collected during purification as depicted in C (C) Reverse TAPi purification scheme. The numbering refers to
samples on the immuno-blots shown in A and B : 1. Input protein extract (40 µg); 2. Eluate after from calmodulin
beads using EGTA containing buffer (6,25x); 3,4. 2x Eluate from IgG beads using a low pH buffer followed by a
concentration step (250x); 5. Calmodulin beads after elution (6,25x); 6. Unbound protein after calmodulin bead
incubation (18,75x); 7. IgG beads after elution (100x). x indicate the amount of protein loaded relative to extract
sample. Prot A (1 ng) was loaded to quantify the amount of detected protein.
Molecular and phenotypic characterization of TAPa-GSK1/ASKζ transgenic lines
Rubio et al. (2005) constructed another version of the TAP tag, the TAPa tag. They
improved the tag by substituting the TEV cleavage site by the human rhinovirus 3C
protease cleavage site and replacing the CBP domain by nine myc epitopes and six histidine
repeats, enabling protein purification via two different purification strategies (Fig. 6a). We
generated transgenic plants expressing N-TAPaGSK1, C-TAPaGSK1 and C-TAPaASKζ.
As a negative control we kindly received N-TAPaGFP seeds from Alois Schweighofer
(Max-Planck-Institute of Molecular Plant Physiology). All TAPa derived constructs were
expressed in Arabidopsis Colombia under control of the CaMV35S promoter.
Transformants were selected, self-pollinated and all transgenic lines had a similar
phenotype as wild type Colombia (Fig. 6b). Independent lines of the T2 generation were
screened for their ability to express the fusion protein. Several transgenic lines containing
N-TAPaGSK1, C-TAPaGSK1 or C-TAPaASKζ constructs produced fusion-proteins,
however protein levels were lower compared to the amounts produced in TAPi expressing
lines (Fig. 6c). The ASK fusions had a size of ~100 kDa while N-TAPaGFP had a size of
~75 kDa (Fig. 6c,d) which corresponds well to the expected size.
66
Towards identification of group II ASKs containing complexes in Arabidopsis
A
B
N-TAPa
2x 35S TMVU1Ω
2x IgG BD
6x HIS
3C
N-TAPaGSK1L8079
Nos ter
9x myc
attR1::Cmr::ccdB::attR2
C-TAPa
2x 35S TMVU1Ω
3C
C-TAPaASKζL8082
Nos ter
2x IgG BD
Wt Col
0
D
C
C
N
-T
AP
aG
SK
-T
AP
1 L8
07
N aAS
9
-T
AP Kζ M
80
aG
34
W
t C FP
M
ol
80
7
08
3
9x myc
W
tC
o
N l
-T
A
C PiG
-T
A SK
N Pi L8 1 L80
-T
77
07
A
8
N PaG
-T
AP SK
a 1L
C
-T GS 807
9
A
K
C PaA 1 L80
-T
80
AP SK
C a ζ L8
08
-T AS
1
AP K
aG ζ L8
SK 082
1 L8
attR1::Cmr::ccdB::attR2
6x HIS
Figure 6. Molecular and phenotypic analysis of TAPa-GSK1/ASKζ transgenic lines. A) Schematic drawing of
the N-TAPa and C-TAPa expression cassettes under control of the CaMV35S promoter. ASKζ and GSK1 will
replace the region between the two AttR recombination sites after Gateway recombination. B) Phenotype of
transgenic lines expressing N-TAPaGSK1, C-TAPaASKζ and wild type Colombia grown under short-day
conditions. C-D) Immunoblots containing leaf protein extracts from several selected lines containing NTAPaGSK1, C-TAPaASKζ or N-TAPaGFP constructs compared to transformants expressing TAPi fusions. The
immunoblot was probed with the PAP antibody.
Are TAP tagged versions of ASKs still functional?
In order to check whether ASK fusions were still functional, we expressed N-TAPaGSK1,
C-TAPaGSK1 or C-TAPaASKζ in triple bin2/gsk1/askζ mutant background (Chapter 2) and
checked whether they could rescue the mutant phenotype. Several independent lines of the
T2 generation containing N-TAPaGSK1, C-TAPaGSK1 or C-TAPaASKζ constructs
produced fusion-proteins (Fig. 7a). Unfortunately, all transgenic lines still appeared to have
a similar phenotype as the triple insertion mutant (Fig. 7b). As the bin2/gsk1/askζ mutant
phenotype is not always easy to score visually, we used sensitivity towards the BR inhibitor
brassinazole (brz) as an assay for complementation of the mutant phenotype. Again, in none
of the transgenic plants sensitivity to brz was restored (Fig. 7c). This suggests that either the
TAP tag disturbs the function of the ASKs or the used CaMV35S promoter did not express
the transgene at the proper time and/or place. The latter could be ruled out as expression of
the ASKζ and BIN2 cDNAs under control of the CaMV35S promoter in the triple mutant
background restored their sensitivity towards brz (Fig. 7d). Unfortunately, due to poor plant
transformation we only possessed one transformant that expressed GSK1.
67
Chapter 3
A
N-TAPaGSK1M8111
3
5
6a
7b
B
Wt
a
b
e
f
d
c
C-TAPaASKζΜ8112
2
4
10c
13d
Wt
bin2/gsk1/askζ
Wild type
C-TAPaGSK1M8113
2
D
3
7e
10f
Wild typeL8211-2
-
+
Wt
35S:ASKζL8251-8a
-
Wild typeL8211-2
-
1 µM brz
1 µM brz
-
+
-
35S:BIN2L8253-2a
+
Wild typeL8211-2
+
-
+
35S:GSK1L8250-1a
-
+
35S:ASKζL8251-12a
+
bin2/gsk1/askζL8215-2
-
35S:BIN2L8253-3a
-
+
+
bin2/gsk1/askζL8215-2
-
+
bin2/gsk1/askζL8215-2
-
+
Figure 7. The TAPa tag disturbs the function of ASKs. A) Immunoblots containing leaf protein extracts from
several independent T2 generation lines expressing N-TAPaGSK1M8111, C-TAPaASKζ M8112 or C-TAPaGSK1M8113 in
triple mutant background. Wt Col was used as control. The immunoblot was probed with the myc antibody. B)
Phenotype of transgenic lines expressing N-TAPaGSK1M8111, C-TAPaASKζ M8112 or C-TAPaGSK1M8113 in triple
mutant background and wild type Colombia grown under short-day conditions. C) No restored brassinazole
sensitivity after expression of N-TAPaGSK1M8111, C-TAPaASKζM8112 or C-TAPaGSK1M8113 genes in triple mutant
seedlings. The characters (a-f) depicted in B and C refers to samples on the immuno-blots shown in A. D)
Restored brassinazole sensitivity after expression of individual group II ASK genes in triple mutant seedlings.
Constructs were over-expressed in triple mutant background.
68
a
Towards identification of group II ASKs containing complexes in Arabidopsis
C
Wild type
bin2/gsk1/askζ
a
b
Wild type
bin2/gsk1/askζ
c
d
Wild type
bin2/gsk1/askζ
e
f
DMSO
1 µM brz
DMSO
1 µM brz
DMSO
1 µM brz
69
Chapter 3
Although GSK1 expression was high (data not shown), the T2 generation seedlings were
less sensitive to a brz treatment than wild type seedlings and thus CaMV35S GSK1 did not
fully complemented the mutant phenotype. (Fig. 7d). Taken together, at least in the case of
ASKζ and BIN2 it seems that the TAP tag itself has a detrimental effect on the function of
tra
ct
(1
)
be
El ads
ua
te bef
or
a
Fi
na fter e e
le
3C l u t
io
lu
cl
n
at
e
(2
e
)
(4 ava
)
ge
(3
)
IgG beads
First affinity step
2
G
TAPaASKζ
TAPaGSK1
Input 1
D
Ig
C
Ex
B
Ex
tra
c
Ig t (1
G
)
be
El ads
ua
te bef
or
a
Fi
na fter e e
le
3C l u t
io
lu
cl
n
at
e
(2
e
)
(4 ava
)
ge
(3
)
A
Ex
tra
c
Ig t (1
G
)
be
El ads
ua
te bef
or
a
Fi
na fter e e
le
3C lut
io
lu
cl
n
at
e
(2
e
)
(4 ava
)
ge
(3
)
these ASKs.
3C cleavage step
TAPaGFP
IgG beads
3
N-TAPaGSK1L8079-1
C-TAPaASKζL8082-5
N-TAPaGFP M8071
Second affinity step
Ni beads
Ni beads
4
Figure 8. Inmuno-blot of different fractions obtained during the TAPa purification of N-TAPaGSK1, CTAPaASKζ and N-TAPaGFP. A-C) A TAPa purification was performed with protein extracts from NTAPaGSK1 (A) and C-TAPaASKζ (Β) and N-TAPaGFP (C) transformants. Several fractions, indicated above
each lane, were separated on a 12,5% SDS-PAGE gel. The Western blot was probed with α-myc. The numbering
corresponds to fractions that were collected during purification as depicted in D (D) TAPa purification scheme.
The numbering refers to samples on the immuno-blots shown in A , B and C: 1. Input protein extract (40 µg); 2.
IgG bound fraction prior to 3C protease cleavage (2,5x); 3. Eluate after 3C protease cleavage (6,25x); 4. Eluate
from Ni beads using imidazole containing buffer followed by a concentration step (100x). x indicate the amount of
protein loaded relative to extract sample.
TAPa purification with N-TAPaGSK1 and C-TAPaASKζ
Although the TAPa tagged proteins do not seem to be functional we still pursued to test if
we could purify the fusion protein and anything that might be associated with it. A largescale TAPa tag purification (~15 gram leaf tissue) was performed with plants expressing NTAPaGSK1, C-TAPaASKζ and N-TAPaGFP. Total protein was isolated and incubated
with IgG beads. After washing the beads were incubated with 3C protease and the collected
eluate was incubated with nickel-coated resin. Final elution was performed with an
imidazole-containing buffer and the eluate was concentrated. After each step samples were
taken to evaluate the efficiency of the purification (Fig. 8). Fusion protein was lost in the
IgG binding step and during the 3C cleavage step, but most protein seem to be lost during
the second affinity purification step (Fig. 8). Although we succeeded to purify both ASKs
70
Towards identification of group II ASKs containing complexes in Arabidopsis
and silver staining revealed bands that most probably correspond to both ASKs, the protein
level was still too low to detect it by mass spectrometry.
Discussion
Almost a decade ago Puig et al. (2001) developed the Tandem Affinity Purification (TAP)
method to rapidly purify protein complexes under native conditions from yeast. Although
this tag has been proven to be useful, only a limited number of articles have been published
about TAP purifications in plants suggesting that this method is not totally trouble-free. In
order to optimize TAP purification from plants, scientists developed over the years several
plant-adapted versions of the TAP tag, such as TAPi, TAPa and GS (Rohila et al., 2004;
Rubio et al., 2005; Van Leene et al., 2008).
In chapter 2 we showed that group II GSK3-like kinases are involved in BR signalling and
discussed about a possible function of GSK3-like kinases in salt stress signalling. In order
to learn more about the functions of group II GSK3-like kinases we wanted to perform TAP
purifications with the GSK3-like kinases GSK1 and ASKζ. In transgenic plants harbouring
the C-TAPiGSK1 construct we could not detect any fusion-protein. Probably the TAPi tag
at the C-terminus of GSK1 disturbs the synthesis or stability of the fusion-protein
emphasising the necessity for making both N and C-terminal fusions when using epitope
tags.
In chapter 2 we observed that gain of function mutants of GSK1 and ASKζ develop a
dwarfish phenotype. All transgenic plants carrying TAP tagged versions of GSK1 and ASKζ
turned out to have a normal phenotype. Li et al. (2002) observed for a fraction of
transformants a dwarfish phenotype when they over-expressed BIN2. In our hands,
expression of BIN2 under control of the CaMV35S promoter only sporadically leads to
dwarfism, depending on high transcript levels. Taken this into account it could be that the
level of expression in the transgenic lines was too low to generate a dwarfish phenotype or
that we should screen more transformants. Nevertheless, the lack of phenotypic effects in
lines over-expressing group II GSK3-like kinases seems not to be a good indication for
functionality of the protein.
TAPa tagged GSK1 or ASKζ had a normal phenotype too. Here we checked the
functionality of the tagged ASKs by expressing all three constructs in the triple
bin2/gsk1/askζ mutant background (described in chapter 2). None of the transgenic lines
could rescue the mutant phenotype and insensitivity towards the BR inhibitor brz was
comparable to the triple mutant control. The CaMV35S promoter worked properly as sole
expression of ASKζ and BIN2 under control of the CaMV35S promoter in the triple mutant
71
Chapter 3
background resulted in sensitivity towards a brz treatment. It thus seems that the TAPa tag
has a detrimental effect on the function and/or folding of the ASKs and might disrupt
certain ASK-protein interactions. We did not test fusions with TAPi for activity because
similar antibiotic resistance markers in the T-DNA tagged mutants and the TAPi vectors
made generation of these transformants difficult. At least our results show that testing
complementation of the corresponding mutant by the fusion proteins is a required
experiment.
Although there are doubts about the activity of the fusion-proteins it is not to be said that
these proteins do not end up in their natural complexes or at least contact a subset of their
natural partners. Also, as the fusion protein itself is translated well as indicated by Western
blots they can serve at least as pilots for testing the feasibility of different TAP tag
purifications. In a pilot TAPi purification with N-TAPiGSK1 and C-TAPi as a control we
succeeded to purify the CBP part of the TAPi tag. The N-TAPiGSK1 purification however
failed; we could not detect any purified CBP-GSK1 in the final eluate nor in the eluate after
the TEV cleavage. Somehow the fusion-protein completely disappeared during the TEV
cleavage, as we could not detect any fusion-protein in the flow through after binding to the
IgG beads, neither attached to IgG beads after the TEV protease cleavage. This experiment
was repeated several times with the same result. Specificity of the TEV protease has been
questioned as some proteins might contain an endogenous TEV protease cleavage site or a
degenerate site that also can be recognized by the TEV protease. In the peptide sequence of
GSK1 we did not spot a TEV protease cleavage site, so it seems not likely that GSK1 is
cleaved. Rohila et al. (2004) encountered that some TAP purifications were less successful
due to binding of a certain protease to the IgG beads. To overcome this problem they added
the protease inhibitor E64 during the TEV cleavage step. The addition of E64 in our TAPi
purifications did not increase protein recovery. As we could detect cleavage of CBP-GSK1
from the IgG coated beads, it is most likely that GSK1 is somehow degraded after it is
cleaved. Several attempts to optimize the TAPi purification did not help and our last shot to
use the TAPi tag was to try to reverse the purification steps thereby omitting cleavage with
TEV protease. Although we were able to purify our protein of interest, the amount was too
low to detect it on a SDS PAGE gel stained with Coomassie blue. In addition, silver
staining revealed a high background of contaminating proteins.
As the normal and reverse TAPi purifications did not look promising, we switched to the
TAPa system developed by Rubio (2005). In this tag the rather undesirable TEV cleavage
site is replaced by the human rhinovirus 3C protease cleavage site which is efficiently
cleaved at 4 °C. Furthermore, the cleavage site is more specific as it consists of an eight
amino acid sequence instead of six, thereby lowering the chance that your protein of
interest and its bindings partners are cleaved. In addition, they replaced the CBP domain by
72
Towards identification of group II ASKs containing complexes in Arabidopsis
nine myc epitopes and six histidine repeats, allowing one to chose between metal affinity
chromatography or myc epitope immunoprecipitation. The tag-protein level was
considerably lower in plants expressing N-TAPaGSK1, C-TAPaGSK1 and C-TAPaASKζ,
compared to those containing the TAPi fusions. Although we succeeded to purify our
ASKs, the purification was not optimal as a lot of our fusion protein was lost during the
purification steps. Silver staining of the final TAP eluates revealed bands that corresponded
to the size of the ASKs. However mass spectrometry used to identify the ASKs and
possible interactors failed because the protein levels were too low.
Unfortunately there was no time to further optimize the TAPa purification. Although the
TAPa tag seems to have a detrimental effect on the ASKs it still may be partially functional
and thus might be used for identification of binding partners. In general, there are several
ideas that might help to successfully purify TAP tagged complexes in the future. First,
purification of binding partners might work better when performed in a mutant background
as there no competition with the endogenous protein. Second, the amount of protein extract
should be increased and the best way to do this is to perform multiple TAPa purifications in
parallel rather then to perform one large purification. Third, although it is preferable to
perform the TAPa purification from whole plantlets it might be easier to perform a
purification from cell suspensions. Cell suspensions are easy to handle, grow fast and one
has an unlimited supply of cells that grow equally. However a disadvantage could be that
your protein of interest is involved in certain biological processes that do not take place in
cell suspensions. In that case certain bindings partners will not be revealed. Fourth, it might
help to perform protein cross-linking to stabilize the protein in its complex before the start
of the purification (Rohila et al., 2004). This is especially worthwhile when the interaction
is weak or transient.
Although there are still several possibilities that might help to improve the TAPa
purification it is questionable whether the TAP purification technique is the best way to
reveal protein complexes. The fact that in these ten years only a few articles are published
about TAP purifications, mainly by the scientist that developed the tags themselves, it is
questionable whether this technology is a foregone conclusion. At least the TAP tag seems
not superior to other, single tag methods like GFP, MYC or FLAG that have been used
successfully by a large number of researchers nowadays.
73
Chapter 3
Experimental procedures
Plant material and growth
Seeds of Arabidopsis Colombia and transgenic lines were sterilized in 10% bleach, washed
three times with sterile water, dissolved in 0.1% agarose and sewed on 0.8% (w/v) agar
plates containing 0.5X Murashige and Skoog (MS) salts, 1.5% sucrose at pH 5.7-5.9. The
seeds were germinated in the climate chamber at 22 °C under a 10-hr-light/14-hr-dark
photoperiod. Later, seedlings were transferred to soil and growth was continued in either a
climate chamber at 22 °C under a 10-hr-light/14-hr-dark photoperiod or under standard
greenhouse conditions.
Brassinazole experiment
Brassinazole (Tokyo Chemical Industry) was dissolved in DMSO (1 mM) and stored at -20
°C. Seeds of Arabidopsis Colombia, mutant and transgenic lines were sterilized in 10%
bleach, washed three times with sterile water, dissolved in 0.1% agarose and sown in a
straight line on 0.8% (w/v) agar plates containing 0.5X Murashige and Skoog (MS) salts,
1.5% sucrose and 1 µM brassinazole or DMSO as control. The plates were wrapped in
aluminium foil and incubated for 48 hrs at 4 °C to stimulate equal germination. Plates were
placed in a vertical orientation for 7 days at 22 °C.
Generation of TAPi/TAPa fusion constructs
To obtain N-tagged GSK1/ASKζ fusion proteins, full length coding sequences of GSK1 (NTAPi, 101-FW and 102-RV; N-TAPa M544-FW and M551-RV) and ASKζ (121-FW and
122-RV) were amplified by RT-PCR on Arabidopsis leaf total RNA. In order to generate
the C-tagged GSK1/ASKζ fusions, the coding sequences of GSK1 (101-FW and 123-RV)
and ASKζ (121-FW and M547-RV) were amplified without STOP codon. The
amplification products were cloned into the Gateway pENTR4 vector (Nco1/BamH1;
PET43 GSK1+
ASKζ
−STOP
, PET44 ASKζ +
STOP
, PET63 GSK1
+ STOP
STOP
, PET46 GSK1-
STOP
and Nco1/Xho; PET65
). Next, a Gateway LR reaction was performed to transfer
PET43 (PET50) and PET44 (PET51) in the pN-TAPi destination vector and PET46
(PET49) in the pC-TAPi destination vector. The pN-TAPi and pC-TAPi constructs were
kindly provided by Dr. Michael Fromm (University of Nebraska, Lincoln, USA). The pNTAPa and pC-TAPa plasmids were obtained from the Arabidopsis Biological Resource
74
Towards identification of group II ASKs containing complexes in Arabidopsis
Center (ABRC). PET63 (NcoI/BamHI, PET66) was cloned in pN-TAPa and PET46
(NcoI/XhoI, PET68) and PET65 (NcoI/XhoI, PET67) in pC-TAPa. The complete inserts of
all clones and the fusion boundaries with the epitopes in the final clones were sequenced.
Clones were transformed via electroporation into the Escherichia coli strain XL1-blue
(Stragene) and afterwards in the Agrobacterium tumefaciens strain C58C1 (pMP90). The
plasmids were introduced in the Arabidopsis Colombia using the floral dip method (Clough
and Bent, 1998). The nucleotide sequences of the primers used in this experiment are listed
in table 1. As a control for the TAPa system, we kindly received N-TAPaGFP seeds from
Alois Schweighofer (Max-Planck-Institute of Molecular Plant Physiology).
Fast plant protein extraction method
The fast plant protein extraction method was used to screen individual transformants.
Tissue was ground in liquid nitrogen and transferred into a frozen 1.5 ml eppendorf tube to
a volume of ~ 50 μl. To the tissue, 100 μl of 1 x SDS sample buffer (0.0625 M Tris-HCl,
2% SDS, 10% glycerol, 0.15 M β-mercapto-ethanol, 0.025% bromophenol blue, pH
adjusted to 6.8 with HCl) was added. After vigorous mixing, the extract was boiled for 10
min. After a short centrifugation, 10 μl of extract was loaded on a 12.5 % (w/v) SDS PAGE
gel. Protein extracts were stored at -20 ºC.
Western analysis, immuno-blotting and detection
Proteins were separated by SDS-PAGE and subsequently transferred to Immobilon PVDF
membrane (Millipore) using the BIO-RAD semi-dry blotting system. In case of using the
peroxidase anti peroxidase antibody (PAP, Sigma) and α-myc antibodies, blots were
blocked first for 1 hour in 5% milk powder (Marvel) in 1x TBST (20 mM Tris-HCl at pH
7.6, 140 mM NaCl and 0.5 ml Tween20). Blots incubated with the anti calmodulin binding
protein antibody (CBP, Upstate) were blocked first o/n. After blocking, blots were briefly
washed in 1x TBST and incubated with the PAP antibody (1/7500, 5% milk) or the a-myc
antibody (1/2000, 5% milk in 1x TBST) overnight at 4°C or with the CBP antibody (1/5000
in 5% milk) for 1 hr at room temperature. Blots immunoprobed with CBP were incubated
with the secondary anti-rabbit IgG-peroxidise antibody (1/5000, Promega) and those
immunoprobed with α-myc with the secondary anti-mouse IgG- peroxidase antibody
(1/7500, Promega) or anti-mouse IgG-AP (1/7500, Promega) for 1 hour in 1x TBST and 5
% milk. After washing, proteins were visualized according the manufacturers protocol by
either using the Amersham ECL kit or the BCIP/NBT color development substrate
(Promega).
75
Chapter 3
TAPi purification method
The TAPi purification was based on the protocol described by Rohila et al. (2004). Leaf
tissue (15 gram, fresh weight) was ground in liquid nitrogen and thawed in ~25 ml TAPi
buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM EDTA, 20 mM NaF, 10 mM βmercaptoethanol, 0.1% IGEPAL and 1x complete protease inhibitor cocktail EDTA free
from Roche), filtered through miracloth (Calbiochem), and centrifuged for 10 min at 4 °C.
The supernatant was incubated with 500 µl washed IgG beads (IgG Sepharose 6 Fast Flow
beads, GE Healthcare) for 2 hrs at 4 °C with gentle rotation. The beads were loaded onto a
disposable column (Altech Extract-Clean Reservoirs) and washed three times with 10 ml
TAPi buffer lacking protease inhibitors and once with 10 ml of cleavage buffer (TAPi
buffer with 1 mM DTT). Next a cleavage step was performed for 2 hrs at 16 °C with gentle
rotation, in which the IgG beads were incubated with 0.5 ml cleavage buffer and 100 U
AcTEV protease (Intvitrogen) and 1 µM E-64 protease inhibitor (Calbiochem). The
supernatant was recovered by centrifugation and mixed with 100 µl washed calmodulin
beads (Stratagene), 3 volumes of calmodulin binding buffer (10 mM Tris HCl pH 8.0, 150
mM NaCl, 1 mM MgAc, 1 mM imidazole, 2 mM CaCl2, 10 mM β-mercaptoethanol, 0.1%
IGEPAL) and 2 mM CaCl2. The beads were washed three times with calmodulin binding
buffer for 5 min and eluted two times with 0.5 ml calmodulin elution buffer (10 mM Tris
HCl pH 8.0, 150 mM NaCl, 1 mM MgAc, 1 mM imidazole, 10 mM β-mercaptoethanol,
0.1% IGEPAL and 10 mM EGTA). The eluates were concentrated to 50 µl with help of an
Amicon centrifugal filter unit (Millipore) separated on a 12.5% SDS-PAGE gel, blotted and
immunoprobed with PAP or CBP antibody.
To test cleavage of the fusion proteins (Fig. 4), total protein was isolated from 7.5 gram leaf
tissue. Supernatant was collected filtered through miracloth (Calbiochem), and centrifuged
for 10 min at 4 °C. The extract was incubated with 150 µl washed IgG beads for 2 hrs at 4
°C with gentle rotation. The beads were washed and 0.5 ml TEV buffer was added. Prior to
the addition of 100 U AcTEV protease and 1 µM E-64 a sample was taken (0 min) The
cleavage step was performed at 16 °C with gentle rotation and every 15 minutes a sample
was taken. The samples were loaded on a 12.5% SDS-PAGE gel, blotted and incubated
with PAP or CBP antibodies.
76
Towards identification of group II ASKs containing complexes in Arabidopsis
Reverse TAPi purification method
Leaf tissue (15g, fresh weight) was grind in liquid nitrogen and thawed in ~25 ml
calmodulin binding buffer (10 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM MgAc, 1 mM
imidazole, 2 mM CaCl2, 10 mM β-mercaptoethanol, 0.1% IGEPAL and 1x complete
protease inhibitor cocktail EDTA free from Roche), filtered through miracloth
(Calbiochem), and centrifuged for 10 min at 4 °C. The supernatant was incubated with 500
µl washed calmodulin beads (Stratagene) for 2 hrs at 4 °C with gentle rotation. The
supernatant including the beads were loaded onto a disposable column (Altech ExtractClean Reservoirs) and washed five times with 10 ml calmodulin binding buffer. Next, the
beads were incubated for 5 min with 2 ml calmodulin elution buffer (10 mM Tris HCl pH
8.0, 150 mM NaCl, 1 mM MgAc, 1 mM imidazole, 10 mM β-mercaptoethanol, 0.1%
IGEPAL and 10 mM EGTA). The eluate was collected and the elution step was repeated.
To the eluate (4 ml) 20 mM NaF, 2 mM EDTA and 1x complete protease inhibitor cocktail
(EDTA free) was added. After addition of 400 µl washed IgG beads (IgG Sepharose 6 Fast
Flow beads, GE Healthcare) the mixture was rotated for 2 hrs at 4 °C. The beads were
loaded onto a column (Altech Extract-Clean Reservoirs) and washed three times with 10 ml
TAPi buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM EDTA, 20 mM NaF, 10 mM
β-mercaptoethanol, 0.1% IGEPAL and 1x complete protease inhibitor cocktail EDTA free).
The beads were incubated for a few minutes with 2 ml low pH elution buffer (0.1M
glycine-HCl pH 3.0) and this step was repeated. The eluate was concentrated to 50 µl on an
Amicon centrifugal filter unit (Millipore), separated on a 12.5% SDS-PAGE gel, blotted
and immuno-detected using an PAP or CBP antibody.
TAPa purification method
The alternative TAP was based on the protocol described by Rubio et al (2005). Leaf tissue
(15 gram fresh weight) was ground in liquid nitrogen and thawed in ~25 ml TAPa buffer
(50 mM Tris HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% IGEPAL and 1x complete
protease inhibitor cocktail EDTA free from Roche), filtered through miracloth
(Calbiochem), and centrifuged for 10 min at 4 °C. The extracts were incubated with 500 µl
washed IgG beads (IgG Sepharose 6 Fast Flow beads, GE Healthcare) for 2 hrs at 4 °C with
gentle rotation. After centrifugation at 300 rpm for 2 min at 4 °C, the IgG beads were
recovered and washed three times with 10 ml TAPa buffer without protease inhibitors and
once with 10 ml of cleavage buffer (TAPa buffer with 1 mM DTT). Next, a cleavage step
was performed for 2 hrs at 4 °C with gentle rotation, in which the IgG beads were incubated
77
Chapter 3
with 5 ml cleavage buffer and 25 µl (50 U) 3C protease (PreScission Protease, GE
Healthcare). The supernatant was recovered by centrifugation and mixed with 1 ml washed
Ni-Nt agarose beads (Qiagen). The beads were washed three times with 10 ml TAPa buffer
without protease inhibitors and the eluate was recovered by adding 5 ml of imidazole
containing buffer (TAPa buffer with 0.05 M imidazole), concentrated to 50 µl by using an
Amicon centrifugal filter unit (Millipore), separated on a 12.5% SDS-PAGE gel and
immuno-detected using a PAP or α-myc antibody.
Table 1. Description of primers used for PCR
Primer
Gene
Sequence 5´-3´
121-FW
122-RV
M547-RV
101-FW
102-RV
123-RV
M544-FW
M551-RV
ASKζ
ASKζ
ASKζ
GSK1
GSK1
GSK1
GSK1
GSK1
GCCCATGGCTTCGATACCATG
GGGATCCCTAGGG TCCAGCTTGAAATG
GGGGTCGACGGTCCAGCTTGAAATGGAAAG
CCCCATGGCCTCATTACCATTG
GGGATCCTTAACTGTTTTGTAATCCTGTG
GGGATCCAGGGTCCAGCTTGAAATGG
GGCCATGGCTCATTACCATTGGGG
GGCTCGAGTTAACTGTTTTGTAATCCTG
Acknowledgements
We thank Maartje Kuijpers for plant care. This work was supported by a grant of the
Netherlands Organisation for Scientific Research (NWO).
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79
Chapter 4
Brassinosteroid biosynthesis and signalling in petunia
Nathalie Verhoef, Michiel Vandenbussche, Gert-Jan de Boer,
Tom Gerats, Ronald Koes and Erik Souer
Brassinosteroid biosynthesis and signalling in petunia
Abstract
Brassinosteroids (BRs) are steroidal plant hormones that play an important role in the
growth and development of plants. The biosynthesis of sterols and BRs and the signalling
cascade they induce in plants has been elucidated largely through metabolic studies and the
analysis of mutants in Arabidopsis and rice. Only fragmentary details about BR signalling
in other plant species are known. Here we used a forward genetics strategy in petunia, by
which we identified 19 petunia families with phenotypic alterations typical for BR
deficiency mutants. In five families we disclosed the tagged genes as the petunia BR
biosynthesis gene CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARF (CPD) and
the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor. In addition, we cloned several
homologs of key regulators of the BR signalling pathway from petunia based on homology
with their Arabidopsis counterparts, including the BRI1 receptor, a member of the BRI1EMS-SUPPRESSOR 1 (BES1) / BRASSINOZOLE RESISTANT 1 (BZR1) transcription
factor family (PhBEH2) and two GSK3-like kinases (PSK8 and PSK9). PhBEH2 was
shown to interact with PSK8 and 14-3-3 proteins in yeast revealing similar interactions as
during BR signalling in Arabidopsis. Interestingly, in yeast PhBEH2 also interacted with
proteins implicated in other signalling pathways. This suggests that PhBEH2 might
function as an important hub in the cross talk between diverse signalling pathways.
Introduction
The importance of steroidal hormones in mammalian development is already known for
almost eight decades. The idea that plant steroids have hormonal functions arose when
scientists reported the isolation of brassinolide, a steroid with strong plant growthpromoting activity, from bee-collected pollen of Brassica napus L (Grove et al., 1979). The
identification of several brassinosteroid (BR) biosynthesis- and signalling mutants helped to
some extent to unravel the BR biosynthesis and signalling pathway and nowadays BRs are
accepted as the sixth class of plant phytohormones.
BRs are widely distributed among the plant kingdom and so far they have been found in
gymnosperms, monocots, dicots, a pteridophyte, a bryophyte and a chlorophyte (Bajguz
and Tretyn, 2003). BRs are involved in various growth- and developmental processes such
as cell division and elongation, vascular differentiation, abiotic- and biotic stresses,
photomorphogenesis, germination, senescence and fertility (Clouse et al., 1996; Li et al.,
1996; Szekeres et al., 1996; Bajguz and Hayat, 2009).
83
Chapter 4
Epoxidase
SMT1
Lupeol synthase
Squalene-2,3epooxide
Squalene
Mevalonate
24-Methylenecycloartenol
Cycloartenol
CYP51G1
CYP710A
Stigmasterol
DIM
Sitosterol
DWF5
DWF7
5-Dehydroavenasterol
Isofucosterol
Cycloeucalenol
δ 7-Avenasterol
Cyclopropyl isomerase
Obutusifoliol
24-Ethylidenelophenol
membranes
CYP51G1
δ8,14-Sterol
SMT2
FK
24-Methylenecholestrol
DWF5
5-Dehydroepisterol
DWF7
24-Methylene
lophenol
Episterol
HYD1
4α-Methylfecosterol
DIM
Campesterol
Campest-4-en3β-one
DWF4
22α-Hydroxy-5−
campesterol
Campest-4en-3-one
DWF4
SAX1
DET2 5α-Campestan-
6-Oxocampestenol
DWF4
DWF4
DET2 22α-Hydroxy-5α22α-Hydroxycampest-4-en-3-one
campestan-3-one
DWF4
6-Deoxocathasterone
CYP90
C1/D1
CYP90C1/D1
6α-Hydroxy
campestenol
Campestenol
3-one
Cathasterone
CYP90C1/D1
6-Deoxoteasterone
CYP90C1/D1
CYP90C1/D1
CYP85A1/A2
Teasterone
3-Epi-6-deoxocathasterone
22,23-Dihydroxy
campesterol
22,23-Hydroxycampest-4-en-3-one
3-Dehydro-6-deoxoteasterone
CYP85A1/A2
3-Dehydroteasterone
CYP85A2
7-Oxateasterone
CYP90C1/D1
6-Deoxotyphasterol
6-Deoxycatasterone
CYP85A1/A2
6α-Hydroxycatasterone
CYP85A1/A2
Typhasterol
CYP85A2
7-Oxatyphasterol
Catasterone
CYP85A2
Brassinolide
Figure 1. The proposed sterol/brassinosteroid biosynthesis pathway in Arabidopsis thaliana. The metabolic
products are shown in boxes and the enzymes involved are depicted in red. Mevalonate is converted via multiple
steps into squalene (black dashed arrow). The light green arrows represent the early C-22 oxidation pathway. The
late C-6 oxidation pathway is visualized by red arrows and the early C-6 oxidation pathway by dark green arrows.
The sterol biosynthesis pathway can be divided into three domains (Fig. 1). In the first part,
the mevalonate derivative squalene is converted via multiple steps into 24-methylene
lophenol. Subsequently, the pathway diverges into two downstream branches, one branch
produces cellular membrane components, while in the second branch brassinosteroids are
synthesized from the precursor campesterol (Lindsey et al., 2003). Detailed studies on the
BR biosynthesis route in Catharanthus roseus and Arabidopsis thaliana led to the
identification of two parallel branched routes, better known as the early and late C-6
oxidation pathways. Recent studies demonstrated that these two pathways are linked to
each other, implying that they are not totally autonomous. Although the early and late C-6
oxidation pathways are found throughout the plant kingdom, it seems that for species such
as tomato and tobacco the late C-6 oxidation pathway is more prevalent (Nomura et al.,
2001). Upstream of the C-6 oxidation pathways Fujioka et al. (2002) revealed an early C-
84
Brassinosteroid biosynthesis and signalling in petunia
22 oxidation pathway. It thus seems that, due to the existence and intertwinement of several
biosynthesis pathways, plants are able to synthesize BRs in several ways.
A number of sterol and BR biosynthesis mutants have been described, predominantly in
Arabidopsis, and many of the genes have been cloned and studied (Choe et al., 1998;
Klahre et al., 1998; Mathur et al., 1998; Choe et al., 1999a; Choe et al., 2001; Clouse,
2002b). BR biosynthesis mutants and some sterol mutants such as dwarf7 (dwf7), diminuto
(dim) and dwarf5 (dwf5) can be rescued by exogenous application of BR. However,
mutants that are affected in genes functioning in early steps of the sterol biosynthesis
pathway such as FACKEL (FK), STEROL METHYL TRANFERASES 1 & 2 (SMT1, SMT2)
and HYDRA1 (HYD1) are embryo lethal and can not be rescued by application of
exogenous BR. Presumably, the absence of these sterols interferes with membrane
integrity.
BR signalling has largely been elaborated by studies in Arabidopsis. Yet, it is still unclear
to what extent these findings can be extrapolated to other plant species. The currently
known BR signalling pathway in Arabidopsis runs from membrane to nucleus (Kim et al.,
2009). Despite the identification of these key components, it seems unlikely that all
components of the Arabidopsis BR signalling pathway are identified. Moreover, how the
well-known crosstalk between BR signalling and other hormone signalling pathways is
achieved is not completely clear.
In Arabidopsis, BRs are perceived by the plasma membrane located leucine-rich repeat
receptor-like kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) that dimerizes with
the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) thereby inducing
transphosphorylation. BRI1 then dissociates from its repressor BRI1 KINASE INHIBITOR
1 (BKI1, Li and Chory, 1997; Li et al., 2002; Nam and Li, 2002; Russinova et al., 2004;
Wang et al., 2008a). Subsequently, BRI1 phosphorylates BR SIGNALING KINASE 1
(BSK1), which in turn phosporylates and activates the plant-specific serine/threonine
protein phosphatase BRI1 SUPPRESSOR1 (BSU1, Tang et al., 2008). BSU1 inactivates a
negative regulator of BR signalling, the Arabidopsis GSK3/SHAGGY-like kinase
BRASINOSTEROID INSENSITIVE 2 (BIN2) by dephosphorylation at pTyr200 (Kim et
al., 2009). BIN2 performs its negative effect on BR signalling by phosphorylating two
closely related plant-specific transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and
BRASSINOZOLE RESISTANT 1 (BZR1) resulting in inhibition of their activity through
proteasome mediated degradation, cytoplasmic retention by 14-3-3 proteins and inhibition
of their DNA-binding and transcriptional activities (Li et al., 2001; He et al., 2002; Yin et
al., 2002; Vert and Chory, 2006; Gampala et al., 2007; Ryu et al., 2007; Peng et al., 2010).
Interestingly, other Arabidopsis GSK3-like protein kinases (ASKs) play a redundant role
with BIN2 in the BR signalling pathway (Chapter 2; Vert and Chory, 2006; Kim et al.,
85
Chapter 4
2009; Yan et al., 2009; Rozhon et al., 2010). In other plant species such as Brassica napus,
Medicago sativa, Nicotiana tabacum, Oryza sativa, Gossypium hirsutum and Petunia
hybrida several GSK3-like kinases have been found. In Oryza sativa and Gossypium
hirsutum group II GSK3-like kinases, to which BIN2 belongs, have been described
although their role in the BR signalling pathway still need to be demonstrated (Sun et al.,
2004; Sun and Allen, 2005; Koh et al., 2007).
To reveal to which extent the biosynthesis of steroids and BRs and the signalling cascades
downstream in plants are conserved we browsed our petunia collection for mutants that
exhibit a typical BR deficiency phenotype. Here, we describe the identification and partial
characterisation of a number of these petunia mutants. To reveal if our current knowledge
on BR biosynthesis and signalling can be extrapolated to petunia we initiated an analysis of
BR signalling in petunia. We isolated several petunia homologs of key components of the
BR signalling pathway in Arabidopsis and determined if a similar network of interaction
between these factors can occur. In addition, we identified several components of other
hormone pathways that interacted with the petunia homolog of a member of the
BES1/BZR1 family of transcription factors. These interactions reveal new mechanisms, not
recognised from Arabidopsis research that suggest extensive cross talk between BR
signalling and other hormone signalling pathways.
Results
Identification of petunia sterol/BR biosynthesis and signalling mutants
In order to study BR biosynthesis and signalling in petunia we visually screened
populations of the line W138. This petunia line contains more than 200 copies of the active
transposable element dTph1, leading to a high incidence of mutations (Gerats et al., 1990;
van Houwelingen et al., 1998). We identified four mutants (Table 1) that phenocopy
sterol/BR biosynthesis and signalling mutants from other species. Apart from these four, we
received 15 mutant families with similar phenotypes that were identified in a running large
scale transposon tagging experiment using petunia W138 at Enza Zaden (Enkhuizen)
(Table 1). Due to their appearance with reduced stature and dark round shiny leaves we
named these mutants compact disc (cd).
In order to identify whether these mutants are BR biosynthesis or signalling mutants,
individual mutant plants were sprayed 2-3 times a week with 1 µM 24-epibrassinolide or
with a control solution under standard greenhouse conditions. Out of 19 mutant families, 18
clearly responded to spraying with 24-epibrassinolide by elongation of internodes, petioles
and leaves (Fig. 2). Complementation of the mutant phenotype in these plants indicates that
86
Brassinosteroid biosynthesis and signalling in petunia
these are sterol/BR biosynthesis mutants. Only one mutant family, cd10P2020, did not
respond to exogenous application of 24-epibrassinolide and thus likely harbours a dTph1
insertion in a BR signalling gene.
Table 1 Overview of cd alleles and results of complementation experiment by brassinolide treatment
Locus
Allele
Origin
Complemented by BRa
cd1
cd1-W503
VU
yes
cd1-K2078
Nijmegen
yes
cd2-W2254
VU
yes
cd2-P2013
ENZA
yes
cd2-P2015
ENZA
yes
cd2-P2018
ENZA
yes
cd3-D2213
VU
yes
cd3-P2014
ENZA
yes
cd9-P2016
ENZA
yes
cd9-P2019
ENZA
yes
cd9-P2021
ENZA
yes
cd9-P2022
ENZA
yes
cd9-P2023
ENZA
yes
cd9-P2024
ENZA
yes
cd9-P2025
ENZA
yes
cd9-P2026
ENZA
yes
cd9-P2027
ENZA
yes
cd9-P2028
ENZA
yes
cd9-P2016
ENZA
yes
cd10-P2020
ENZA
no
cd2b
cd3
cd9c
cd10
a
b
Complementation was tested by spraying with 1 µM 24-epibrassinolide.
Due to reduced fertility, cd2 allelism was confirmed molecularly as independent insertions in PhCPD (see text).
c
cd9P2016, cd9P2019, cd9P2022, cd9P2024, cd9P2025, cd9P2026, cd9P2027 and cd9P2028 are derived from a common ancestor
and thus might represent identical alleles.
Cross-pollination between the mutant families revealed that the 19 mutants could be
assigned to five different complementation groups (see Table 1). Ten out of 16 families
received from Enza Zaden harbour a dTph1 insertion in the same BR biosynthesis gene,
CD9. As some of these families are derived from a common ancestor (see Table 1) it is
likely that they represent only three different alleles.
As the mutated gene in cd2W2254 is known (see below), we were able to detect independent
transposon insertions in the same gene in cd2P2013, cd2P2015 and cd2P2018 and consider these
allelic. As can be derived from Table 1, the total number of sterol/BR biosynthesis genes hit
is four, while the remaining mutant cd10 represents a signalling mutant. All mutants have a
short robust stature with thick, small, round and dark-green leaves. The severity of the
dwarfish phenotype slightly differed among the complementation groups. The cd10 mutants
remained extremely small and did not flower after staying for months in the greenhouse.
87
Chapter 4
cd2 mutants were intermediate in size and occasionally flowered. The growth of the cd3
and cd9 mutants was clearly slower compared to wild type, but they ultimately flowered
and fertility appeared to be normal. In comparison to cd3 and cd9, cd1 mutants had a more
severe phenotype and were less fertile.
-
+
-
P2013
+
P2023
-
+
Brassinolide
P2020
Figure 2. Effect of 24-epibrassinolide on BR biosynthesis/signalling mutants. Plants were sprayed with 1 µM
24-epibrassinolide (+) or with a control solvent (-). Two BR biosynthesis mutants cd2
shown. The signalling mutant cd10
P2020
P2013
and cd9
P2023
are
did not respond to a brassinolide treatment.
Isolation of BR biosynthesis genes
We presumed that at least the majority of our cd mutants are insertions in genes known
from research on BR mutants in other species. Therefore, a candidate gene approach might
lead to identification of the mutated genes. As our knowledge about the molecular players
involved in BR biosynthesis in petunia is blank we started out to clone gene fragments of
known BR biosynthesis genes. Using a combination of database mining and primer design
based on sequences of Solanaceae species (eg. tomato, tobacco, potato etc. Supplemental
data, Table 1) we were able to clone gene and/or cDNA fragments of petunia homologs of
the Arabidopsis BR biosynthesis genes CPD, DWF5, CYP85A, DIM, DWF7, SMT2,
DETIOLATED2 (DET2), DWARF4 (DWF4) and CYP90C1 (ROT3) and a number of
signalling genes (see below).
Four BR biosynthesis mutant families are cpd mutants
Most mutant alleles in W138 are due to insertions of dTph1 transposons. We used the
obtained sequence data of petunia BR biosynthesis genes to determine in each cd mutant if
88
Brassinosteroid biosynthesis and signalling in petunia
13
20
2 P 15
2
0
W
ild 18
-ty
pe
PhCPD
2P
D
P2013
cd
2P
cd
-4
*
94
94
21
21
2M
2M
cd
cd
cd
20
C
A
-4
~
any of these known genes is disrupted by a dTph1 insertion. A PCR was performed on
DNA isolated from these mutants using gene-specific and/or transposon specific primers
When DNA isolated from cd2W2254 (Fig. 3a) and three mutants from Enza Zaden (cd2P2013,
P2015, P2018
) was used as a template, these PCRs identified dTph1 insertions at different sites
in the petunia homolog of the BR biosynthesis gene CPD (Fig. 3c,d,e). The dTph1
transposon insertion in allele cd2W2254 was found in the first exon, about 80 nucleotides
after the ATG. The other three mutants (cd2P2013, P2015, P2018) carry a dTph1 insertion at
different sites in the second exon (Fig. 3d). Plants in progeny of cd2W2254 occasionally gave
rise to revertant shoots (Fig. 3b). PCR and sequence analysis on genomic DNA extracted
from a revertant shoot (cd2M2194-4) revealed excision of dTph1 from CPD, leaving a 6 bp
footprint (Fig. 3c,d,e). Furthermore, a stable cpd mutant (cd2M2194-1) was identified in which
excision of dTph1 created an 8bp footprint (Fig. 3d,e).
dTph1 insertion
W2254
P2015
E
P2018
200 bp
cd2W2254
Wild-type
cd2K2056
cd2M2194-1
cd2M2194-4~
TCTCCTCCTCCG
TGCA
TCTCCTCCTCCG------dTph1-------CTCCTCCGTGCA
TCTCCTCCTCCC
GTCCTCCGTGCA
TCTCCTCCTAG
TCCTCCGTGCA
+ 8bp
+ 6bp
cd2P2013
B
revertant
shoot
Wild-type
cd2P2013
CCATGCGAGTGG
ACTG
CCATGCGAGTGG------dTph1-------GCGAGTGGACTG
cd2P2015
Wild-type
cd2P2015
ATATGTGCAGAT
AACG
ATATGTGCAGAT------dTph1-------GTGCAGATAACG
cd2P2018
dwarf part
Wild-type
cd2P2018
CCTACCGCAAGG
CCAT
CCTACCGCAAGG------dTph1-------CCGCAAGGCCAT
Figure 3. Four BR biosynthesis mutant families are defective in the CPD gene. A) Mutant phenotype of a
W2254
W2254
M2194-4
plant B) A cd2
plant that develops a revertant shoot (cd2
) C) PCR with CPD specific primers
cd2
on genomic DNA extracted from plants carrying various cpd alleles. D) Schematic drawing of CPD and mutant
alleles. Boxes represent exons and the thin lines represent introns. The triangles indicate the dTph1 transposon
insertions in the indicated alleles. E) Sequence analysis of the dTph1 insertions in various cpd alleles. The red
sequence indicates the target site duplication (TSD). * indicates dwarf part and ~ the revertant shoot.
For the other three loci cd1, cd3 and cd9 we were unable to detect transposon insertions in
the putative petunia homologs of DWARF5, CYP85, DET3, DIM, DWARF7, SMT2, DET2,
DWARF4 or ROT3. It must be said that we currently lack the complete sequences of these
89
Chapter 4
genes. As we miss 3’ sequences (DIM), 5’ sequences (DWARF5, CYP85, DWARF7, SMT2)
or both (DET3, DET2, DWARF4, ROT3) it cannot be excluded that there are dTph1
insertions present in these parts of the gene.
Identification of mutant alleles of BR biosynthesis genes by reverse genetics
To identify other mutants in petunia homologs of BR biosynthesis genes from Arabidopsis
we screened the 3D indexed petunia insertion database as described by Vandenbussche et
al. (2008). A mutant plant was identified that harbours a dTph1 insertion in the first exon of
a gene homologous to the Arabidopsis sterol biosynthesis gene DIM. Remarkably, in
progeny, plants homozygous for the mutant dimL2006 allele could not be identified. All seeds
seemed to germinate when sown on agar plates and no aberrant plantlets could be identified
in progeny. Furthermore, reciprocal crosses revealed that the allele is passed on via the
mother and father. At the moment it is unclear whether DIM interferes with seed
development or that the DIM gene is linked to a lethal allele of another gene. Another
possible explanation could be that DIM is recently duplicated in petunia and thus our PCR
primers amplify two genes.
Apart from this we also identified insertions in genes homologous to the Arabidopsis BR
biosynthesis genes DET2 and CYP85A1 (BR6OX1). The insertion in phcyp85A1L2228 is
located in intron 2. As no phenotypic alterations are seen in plants homozygous for the
insertion allele, we presume that the small dTph1 element is spliced from the pre-mRNA
along with intron 2. The insertion in the PhDET2 gene (phdet2L2224) is located in exon 1.
However, also in this case plants homozygous for the insertion allele do not show any
morphological aberrations, suggesting that DET2 is redundant in petunia.
Isolation of a BRI1 homolog from petunia
In Arabidopsis, BRs are perceived by the leucine-rich repeat receptor-like kinase BRI1 (Li
and Chory, 1997). BRI1 orthologs have been identified in species such as tomato (CU-3),
pea (LKA), rice (OsBRI1), barley (HvBRI1) and cotton (GhBRI1) (Yamamuro et al., 2000;
Montoya et al., 2002; Chono et al., 2003; Nomura et al., 2003; Sun et al., 2004). From the
Petunia hybrida EST collection we retrieved a sequence that showed high similarity to the
3´ end of BRI1 (GenBank accession number CV294320). The full-length mRNA sequence
of W138 PhBRI1 was obtained via RACE-PCR and somatic insertion-mediated-PCR
(SOTI-PCR, see Materials and Methods). Comparison of the derived amino acid sequence
of PhBRI1 with AtBRI1, OsBRI1, LeBRI1 and NbBRI1 revealed 63,3%, 53,4%, 84,1%
and 91,3% identity, respectively (Fig. 4a). Similar to the other identified BRIs, PhBRI1 has
90
Brassinosteroid biosynthesis and signalling in petunia
an Leucine-Rich Repeat (LRR) domain, a putative leucine-zipper motif, a transmembrane
domain to anchor the protein in the plasma membrane, and a cytoplasmic kinase domain
(Fig. 4b and Supplementary data Fig. 1). The BRI1 extracellular domain of petunia contains
25 tandem amino-terminal LRRs, which is interrupted by a 68 amino acid non-repetitive
island between the 21st and 22nd LRR.
Identification of a petunia mutant defected in BRI1
As described above, only one of the identified mutant families (cd10 P2020) did not respond
to exogenous application of 24-epibrassinolide and thus most likely harboured a dTph1
insertion in a BR signalling gene (Fig. 2a). One of the prime candidates for being hit in
cd10P2020 is the homolog of the Arabidopsis BR receptor, BRI1. Indeed, PCR analysis on
genomic DNA with numerous PhBRI1 specific primers resulted in a larger PCR fragment
in cd10 plants in comparison to wild type when using primers that amplified the 5’ part of
the gene (Fig. 4c). Sequence analysis of the 5’ part of PhBRI1 in cd10 revealed that the
dTph1 transposable element was inserted in the 3rd leucine-rich repeat sequence motif (Fig.
4b, c).
A
B
PhBRI1
A tC LV 1
Putative Leucine zipper motif 68 amino acid (aa) Transmembrane domain
non-repetitive island
kinase domain
Leucine-rich repeats
(LRR) 1-21
LRR 22-25
H vB R I1
1000
O sB R I1
A tB R I1
100 aa
968
Cd10
P hB R I1
1000
N bB R I1
C
W
1000
13
cd 8
1
cd 0 P2
10 020
P2 02 4
05
P sB R I1
dTph1 insertion
906
N tB R I1
D
Wild-type
cd10P2020-4
TAATGATGTTTC
CAGC
TAATGATGTTTC--dTph1--GATGTTTCCAGC
BRI1
1000
LeB R I1
0,1
Figure 4. Characterization of the BRI1 receptor from petunia. A) Phylogenetic tree constructed using derived
amino acid sequences of the BRI1 homolog isolated from petunia and various other species (At: Arabiopsis
thaliana; Hv: Hordeum vulgare; Os: Oryzia sativa; Le: Lycoperscicon esculentum; Nb: Nicotiana benthamiana;
Nt: Nicotiana tabacum; Ps: Pisum sativa and Ph: Petunia hybrida) The derived amino acid sequence of the
leucine-rich repeat receptor-like kinase CLAVATA1 from Arabidopsis was used as an outgroup. Genbank
accession numbers are recited in Supplemental Table 2. B) Schematic drawing of PhBRI1 with its predicted
functional domains. The triangle indicates the dTph1 transposon insertion in cd10. C) PCR with BRI1 specific
primers on genomic DNA extracted from cd10P2020and wild type plants. D) Sequence analysis of the dTph1
insertion in cd10. The red sequence indicates the target site duplication (TSD).
91
Chapter 4
Identification of a BEH2 homolog in petunia
To further explore the BR signalling cascade in petunia, we were aiming to identify
signalling components downstream of PhBRI1. In Arabidopsis, the BES1/BZR1
transcription factor family plays an important role in the regulation of BR-responsive genes
(Zhao et al., 2002). A PCR-based approach was performed to identify potential petunia
homologs. Primers were developed based on the sequences of all six BES1/BZR1 family
members of Arabidopsis and a nested RACE-PCR on petunia leaf cDNA yielded a PCR
fragment of the desired size. Sequence analysis revealed high sequence identity with
members of the BES1/BZR1 family. The 5´ end was isolated by SOTI-PCR (see material
and methods, data not shown). The derived amino acid sequence has highest sequence
identity to the BES1/BZR1 family member BES1 HOMOLOG 2 (AtBEH2) (64,2%) and
therefore we named the clone PhBEH2 (Fig. 5a,b). Like the previously characterized
BES1/BZR1 family members from Arabidopsis, PhBEH2 has a putative bipartite nuclear
localization signal (NLS) at its N-terminus (Fig. 5a). The central part of PhBEH2 is rich in
serine/threonine residues that match with the phosphorylation sites for GSK3 kinases
(Wang et al., 2002). At the C-terminus of PhBEH2 a WEGE core sequence was found
which is similar to the BIN2-docking motif (DM) of BZR1 (Peng et al., 2010). Furthermore
a PEST motif was identified that seems to play an important role in protein degradation as
BES1 and BZR1 proteins were stabilized in bes1-D and bzr1-D mutants carrying a
mutation in this motif (Wang et al., 2002; Yin et al., 2002).
PhBEH2 bind to a GSK3/SHAGGY like kinase and 14-3-3 proteins
To identify proteins that interact with PhBEH2, a yeast two-hybrid screen was performed.
As many transcription factors carry activation domains, auto-activation of the full-length
cDNA of PhBEH2 on yeast reporters was tested first. Indeed, introduction of the PhBES1GAL4 binding domain fusion resulted in some activation of the histidine reporter, but
selection on histidine and adenine resulted in no growth (Supplemental data Fig. 3)
Subsequently, the full-length cDNA of PhBEH2 was used as bait to screen a two-hybrid
cDNA library made from young petunia inflorescences and twelve interacting proteins were
found (Fig. 5c,d). Sequence analysis showed that five of these proteins were 14-3-3
proteins. Another interesting interacting partner had high similarity to the GSK3-like kinase
BIN2 from Arabidopsis (Fig. 5c,d). Other interaction partners include proteins homologous
to SECRET AGENT (SEC) that encodes an O-linked N-acetylglucosamine transferase
(OGT), ARIA (ARMADILLO Repeat protein Interacting with ABF2), TPL (TOPLESS)
92
Brassinosteroid biosynthesis and signalling in petunia
and two RRM (RNA Recognition Motif) proteins of which one is a homolog of FPA
(Flowering Protein A, Fig. 5c,d).
A
B
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
1
1
1
1
1
1
1
MKRFFYNSSEEERKKKAYSSKKMTSDGATSTS-AAAAAAAMATRRKPSWRERENNRRRER
----------------------MTSDGATSTSAAAAAAAAAAARRKPSWRERENNRRRER
----------------------MT---------AAGGGGGSATGRLPTWKERENNKRRER
----------------------MAAG-------GGGGGGGSSSGRTPTWKERENNKKRER
----------------------MT---------ASGGGSTAATGRMPTWKERENNKKRER
---------------------------------------MTSGTRTPTWKERENNKRRER
---------------------------------------MTSGTRMPTWRERENNKRRER
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
60
39
30
32
30
22
22
RRRAVAAKIYTGLRAQGNYNLPKHCDNNEVLKALCSEAGWVVEEDGTTYRKGHKPLPGDM
RRRAVAAKIYTGLRAQGDYNLPKHCDNNEVLKALCVEAGWVVEEDGTTYRKGCKPLPGEI
RRRAIAAKIFTGLRAQGNFKLPKHCDNNEVLKALCIEAGWIVEDDGTTYRKGHRRPPIEY
RRRAITAKIYSGLRAQGNYKLPKHCDNNEVLKALCLEAGWIVEDDGTTYRKGFKPPASDI
RRRAIAAKIFTGLRSQGNYKLPKHCDNNEVLKALCLEAGWIVHEDGTTYRKGSRP----RRRAIAAKIFAGLRIHGNFKLPKHCDNNEVLKALCNEAGWTVEDDGTTYRKGC-KPMDRRRRAIAAKIFTGLRMYGNYELPKHCDNNEVLKALCNEAGWIVEPDGTTYRKGCSRPVER-
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
120
99
90
92
85
80
81
AGSSSRATPYSSHNQSPLSSTFDSPILSYQVSPSSSSFPSPSRV--GDPHN--ISTIFPF
AGTSSRVTPYSSQNQSPLSSAFQSPIPSYQVSPSSSSFPSPSR---GEPNNNMSSTFFPF
AVSSMDISACSSIQPSPMSSSFPSPVPSYHASPTSSSFPSPSR---CDGNP--SSYILPF
SGTPTNFSTNSSIQPSPQSSAFPSPAPSYHGSPVSSSFPSPSR---YDGNPS-SYLLLPF
---TETTVPCSSIQLSPQSSAFQSPIPSYQASPSSSSYPSPTR---FDPNQS-STYLIPY
-----MDLMNGSTSASPCSSYQHSPRASYNPSPSSSSFPSPTN------PFGDANSLIPW
-----MEIGGGSATASPCSSYQPSPCASYNPSPGSSNFMSPASSSFANLTSGDGQSLIPW
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
176
156
145
148
138
129
136
LRN--GGIPS------SLPP----LRISNSAPVTPPVSSPTSRNPKP------------LRN--GGIPS------SLPS----LRISNSCPVTPPVSSPTSKNPKP------------LHNL-ASIPF------SLPP----LRISNSAPVTPPPSSPT-RGSQP------------LHNIASSIPA------NLPP----LRISNSAPVTPPLSSPTSRGSKR------------LQN--LASSG------NLAP----LRISNSAPVTPPISSPRRSNPR-------------LKNLSSNSPS------KLP---FFHGNSISAPVTPPLAR--------------------LKHLSTTSSSSASSSSRLPNYLYIPGGSISAPVTPPLSSPTARTPRMNTDWQQLNNSFFV
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
211
191
180
185
172
159
196
---LPTWESFTKQSMSMAAKQSMTSLNYPFYAVSAPASPTHHRQFHAPATIPECDESDSS
---LPNWESIAKQSMAIA-KQSMASFNYPFYAVSAPASPTHRHQFHTPATIPECDESDSS
---KPVWESFSRG--------PLHSFHHPIFAASAPSSPT-RRQYSKPATIPECDESDAA
---KLTSEQLPNGG-------SLHVLRHPLFAISAPSSPTRRAGHQTPPTIPECDESEED
---LPRWQS-------------------SNFPVSAPSSPTRRLHHYT--SIPECDESDVS
---SPTRDQVTIPD--------SGWLSGMQTPQSGPSSPTFSLVSRNPF-FDKEAFKMGSSTPPSPTRQIIPD--------SEWFSGIQLAQSVPASPTFSLVSQNPFGFKEEAASAAG
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
268
247
228
235
208
206
248
A tB E H 1
A tB E S 1
1000
A tB Z R 1
AtBES1
AtBZR1
PhBEH2
AtBEH2
AtBEH1
AtBEH3
AtBEH4
316
295
281
277
244
248
292
* * * * *
*
686
A tB E H 3
1000
A tB E H 4
* * * *
P hB EH2
840
0,1
A tB E H 2
** * *
* *
C
* *
1
8
16
17
1
8
16
17
1
8
16
17
25
36
41
60
25
36
41
60
25
36
41
60
67
73
78
81
67
73
78
81
67
73
78
81
AD
AD
-LT X-gal
AD
-LTH
-LTHA
D
*TVD-SGHWISFQKFAQQQPFSASMVPTSPTFNLVKPAPQQLSPNTAAIQ----------*
* *
Clone
Type
Arabidopsis homolog
TVD-SGHWISFQKFAQQQPFSASMVPTSPTFNLVKPAPQQMSPNTAAFQ----------SVE-SARWVSFQTLAP------SAAPTSPTFNLVKPVIPQQNILLDALSGRGAFGWGEAA
SIEDSGRWINFQ----------STAPTSPTFNLVQQTSMAIDMKRSDWGMSG-------TVD-SCRWGNFQSVNVS-----QTCPPSPTFNLVGKSVSSVG------------------DCNSPMWTPGQSGNCS-----PAIPAGVDQNSDVPMADGMT-AEFAFG----------GGGGSRMWTPGQSGTCS-----PAIPPGADQTADVPMSEAVAPPEFAFG-----------
1
14-3-3
GRF1/GF14χ (AT4G09000)
8
14-3-3
16
RNA binding
GRF1/GF14κ (AT5G65430)
FPA (AT2G43410)
17
OGT
SEC (AT3g04240)
25
ARMADILLO
ARIA (AT5G16050)
36
WD40
TOPLESS/related1 (AT1G80490)
41
14-3-3
GRF5/GF14ε (AT5G16050)
60
unknown
67
14-3-3
GRF2/GF14ω (AT1G78300)
73
14-3-3
78
GSK3/SHAGGY
GRF5/GF14ε (AT5G16050)
BIN2 (AT1G06390)
81
RNA binding
AT4G00830
EIGQSSEFKFENSQVKPWEGERIH-DVAMEDLELTLGN-GKAHS
EIGQSSEFKFENSQVKPWEGERIH-DVGMEDLELTLGN-GKARG
QRGHASEFDLESCKVKAWEGERIH-EVAVDDLELTLGA-GKAHA
MNGRGAEFEFENGTVKPWEGEMIH-EVGVEDLELTLGG-TKARC
----------VDVSVKPWEGEKIH-DVGIDDLELTLGHNTKGRG
-----CNAMAANGMVKPWEGERIHGECVSDDLELTLGN-SRTR-----SN---TNGLVKAWEGERIHEESGSDDLELTLGN-SSTR-
Figure 5. Characterization of PhBEH2 and the identification of binding partners by a yeast two-hybrid
screen. A) Deduced amino acid alignment of PhBEH2 and the six members of the BES1/BZR1 family from
Arabidopsis. Identical residues in four out of seven sequences are indicated in black, similar amino acids in gray.
Dashed lines represent gaps to facilitate alignment. A putative nuclear localization signal (straight black line),
putative phosphorylation sites by GSK-3 kinases (*), 14-3-3 binding site (red dashed line), PEST motif (black
dashed line) and BIN2-DM (straight red line) are shown. Ph, petunia; At, Arabidopsis. B) Phylogenetic tree
constructed using derived amino acid sequences of PhBEH2 and the BES1/BZR1 family from Arabidopsis.
Genbank accession numbers are recited in Supplemental Table 2. C) The full-length sequence of PhBEH2 was
fused to the yeast GAL4-binding domain and screened against a cDNA library made from young petunia
inflorescences. Interactions were measured by growth on medium lacking histidine (-LTH), histidine and adenine
(-LTHA) and blue colouring using 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (-LT X-gal). D) Classification of
the positive clones from (C). As indicated, for some proteins a homolog with known function is found in
Arabidopsis.
93
Chapter 4
Isolation and characterization of two group II members of the GSK3/SHAGGY-like family
from petunia
One of the interacting partners of PhBEH2 was a protein that has strong homology to the
GSK3-like kinase BIN2 from Arabidopsis and we named it PSK8 (Fig. 6a and Supplementary data Fig. 2). From a previous yeast two-hybrid screen performed in our laboratory,
using one of the transcription factors involved in flower pigmentation as bait, another
GSK3-like kinase was identified as an artefact (W. Verweij and F. Quatrocchio, personal
communication). As this clone missed the 5’ half of the cDNA, we performed SOTI-PCRs
(see materials and methods) to isolate the remaining part. We renamed this clone PSK9.
Both PSK8 and PSK9 are related to BIN2 as they group together with the other group II
ASKs (Fig. 6a). Kim et al. (2009) showed that the phosphatase BSU1 inactivates BIN2 by
dephosphorylation at pTyr 200. This tyrosine residue is also conserved in PSK8 and PSK9
(Supplemental Fig. 2).
To get more insight in the function of PSK8, a yeast two-hybrid screen was performed,
where full-length PSK8 was used as bait to screen a cDNA library made from young
petunia inflorescences. We identified eight different cDNAs that interacted with PSK8. The
interacting partners include two different LRR-extensins, REMORIN and several unknown
proteins. (Fig. 4b,c).
III
A
B
A S K 32
1
3
4
1
3
4
1
3
4
1
3
4
6
16
21
6
16
21
6
16
21
6
16
21
28
93
AD
28
93
AD
28
93
AD
28
93
AD
A S K 31
PSK6
MPK1
414
1000
PSK7
487
A S K 41
PSK4
I
1000
A S K 13
A S K 12
1000
A S K 42
A S K 11
A tA S K dzeta
0.1
1000
891
861
1000
910
PSK9
734
1000
-LTH
-LT
298
A tB IN 2
A tG S K 1
PSK8
C
IV
-LTHA
-LT X-gal
Clone
type
Arabidopsis homolog
1
unknown
At3g11590
3
GCN5-related N-acetyl transferase
At2g39020
4
REMORIN
AtREM6.3 (At1g53860)
6
LRR-extensin
AtLRX3 (At4g13340)
16
LRR-extensin
At1g26250
21
Dynein light chain
weak homology, no clear Arabidopsis homolog
28
unknown
no clear plant homolog
93
unknown
At3g63095
II
Figure 6. Phylogenetic analysis of PSK8/9 and the identification of PSK8 binding partners by a yeast twohybrid screen. A) Phylogenetic tree constructed of derived amino acid sequences of PSK8/9 and GSK3-like
kinases from Arabidopsis and petunia. MPK1 from Arabidopsis was used as an outgroup to construct the tree.
Genbank accession numbers are shown in Supplemental data Table 2. B) Interaction of PSK8 with eight different
clones in a yeast two-hybrid assay. Interactions were measured by growth on medium lacking histidine (-LTH),
histidine and adenine (-LTHA) and blue colouring using 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (-LT Xgal). C) Classification of the positive clones from (B). When a clear homolog in Arabidopsis is found their AGI
identifier is listed.
94
Brassinosteroid biosynthesis and signalling in petunia
Key regulators of the petunia/Arabidopsis BR pathway are functionally homologous
H
BZ 2
At R1
BE
AD S1
BE
At
Ph
H
BZ 2
At R1
BE
AD S1
BE
At
Ph
At
Ph
BE
H
BZ 2
At R1
BE
AD S1
PSK8 and PSK9 are highly homologous to the group II GSK3/SHAGGY-like kinases from
Arabidopsis and this prompted us to examine whether they also have functional homology.
In order to investigate this, a yeast two-hybrid between members of the BES1/BZR1 family
and the group II GSK3/SHAGGY like kinases from Arabidopsis and petunia was
performed. AtBIN2 interacted with AtBES1, AtBZR1 and also with BEH2 from petunia
(Fig. 7). A similar behaviour was found for the petunia BIN2 homolog PSK8. However,
PSK9 did not bind to PhBEH2 nor to the Arabidopsis members. As PhBEH2 is able bind to
PSK8, it seems that PSK9 does not interact with the BES1/BZR1 family and thus may not
be involved in BR signalling. However it seems more likely that the fusion between PSK9
and the GAL4 binding domain is somehow not functional. Our yeast-two hybrid results
suggest that group II GSK3/SHAGGY like kinases and members of the BES/BZR1 family
from Arabidopsis and petunia are structurally highly homologous.
PSK9
PSK8
AtBIN2
BD
-LT
-LTH
-LTHA
Figure 7. The BES1/BZR1 family and group II GSK3/SHAGGY like kinases from petunia and Arabidopsis
are functionally homologous. Full length sequences of AtBIN2, PSK8 and PSK9 were fused to the GAL4binding domain and AtBZR1, AtBES1 and PhBEH2 to the GAL4-activation domain and tested for interaction.
Interactions were measured by growth on medium lacking histidine (-LTH), histidine and adenine (-LTHA).
Discussion
The synthesis of BRs and the signalling cascade downstream the bioactive compounds has
been largely elucidated over the past ten years (Clouse, 2002a; Fujioka and Yokota, 2003;
Kim et al., 2009). However, as in many plant research areas these findings basically
originate from Arabidopsis. Partial conformation of evolutionary conservation as well as
additions came from several reports on BR biosynthesis and signalling in other species (eg.
Nomura et al., 2001; Montoya et al., 2002; Bai et al., 2007; Koh et al., 2007). We have
95
Chapter 4
used the resources that are available in petunia to study BR biosynthesis and signalling. The
initial results point to conserved genes and interactions, but also revealed potential new
relations between hormone pathways.
From our mutant collection we identified five different loci of which four are BR
biosynthesis genes. One locus, CD2, appeared to correspond to the Arabidopsis homolog of
CPD (Szekeres et al., 1996). At the moment it is unclear what the nature is of the remaining
three loci. As transposon insertions in the petunia homologs of DIM and DET2 did not
cause the typical BR deficiency phenotype, it seems unlikely that cd1, cd3 or cd9 mutants
carry insertions in these genes. By PCR analysis on DNA isolated from the three mutants
we were unable to detect dTph1 insertions in DWARF5, CYP85, DET3, DIM, DWARF7,
SMT2, DET2, DWARF4 or ROT3. However, due to the lack of complete gene sequences we
do not dare to claim that there are no insertions in those genes. If true however, the chance
that we have hit an unknown enzyme in BR biosynthesis seems high. Cloning of these
genes can then be accomplished using AFLP based transposon display (Van den Broeck et
al., 1998). In addition, we (in collaboration with Dr. Takao Yokota, Teikyo University,
Japan) are in the process of identifying the BR biosynthesis reaction intermediates that
accumulate in these mutants. These data might also give us clues about the nature of the
affected genes.
The single signalling locus that we identified, CD10, was shown to harbour an insertion in
the petunia homolog of the BR receptor, BRI1 (Clouse et al., 1996). The cd10 mutant
exhibits an extremely dwarfed stature and did not flower at all. BRI1 orthologs have been
identified in a number of species such as tomato, pea, rice, cotton and barley (Yamamuro et
al., 2000; Montoya et al., 2002; Chono et al., 2003; Nomura et al., 2003; Sun et al., 2004).
All bri1 mutants do flower except the osbri1 mutant from rice (Nakamura et al., 2006). The
strong mutants from rice and petunia might reflect complete loss of BRI1 function whereas
in other species either only partial loss-of-function alleles were identified and/or partial
redundancy in BRI1 function might exist.
PhBRI1 is most homologous to BRI1 from Lycoperscicon esculentum, Nicotiana
benthamiana and Nicotiana tabacum. In an amino acid alignment of BRI1 from petunia,
tomato, Arabidopsis and Nicotiana benthamiana the highest similarity was observed in the
crucial kinase domain (Supplementary data Fig. 1; Tang et al., 2008; Wang et al., 2008a).
The extracellular domain of PhBRI1 contains 25-tandem amino-terminal LRRs and a 68
amino acid non-repetitive island between the 21st and 22nd LRR. This island together with
LRR22 has proved to be important for BR binding by BRI1 in Arabidopsis (Kinoshita et
al., 2005). Thus it seems that the perception of the BR hormone is evolutionary well
conserved.
96
Brassinosteroid biosynthesis and signalling in petunia
Apart from BRI1, other key regulators of the Arabidopsis BR signalling pathway have been
identified from a number of species. In rice as well as in cotton gain-/loss-of-function of
BIN2 homologs suggest that they are involved in BR signalling (Sun et al., 2004; Sun and
Allen, 2005; Koh et al., 2007). In addition four genes (OsBZR1-4) were isolated from rice
that are homologous to the Arabidopsis BES1 and BZR1 transcription factors (Bai et al.,
2007). In order to reveal conservation in the BR signalling pathway among plant species,
we cloned several petunia homologs of key components of the BR signalling pathway in
Arabidopsis including the BRI1 receptor, a homolog of the BES1/BZR1 family and GSK3like kinases.
The BES1/BZR1 family homolog that we isolated from petunia showed highest sequence
identity to the BES1/BZR1 family member BEH2 (64,2%) and therefore we named this
clone PhBEH2. In Arabidopsis a BR treatment increased the accumulation of
dephosphorylated BZR1, BES1 and also BEH2 (Yin et al., 2005). Furthermore, the
bzr1/bes1 double mutant only exhibits a semi-dwarf phenotype. Therefore, it seems likely
that BEH2 and maybe other members of the BES1/BZR1 family are redundant to BES1 and
BZR1. In chapter 2 we showed that group II GSK3-like kinases interact with several
members of the BES1/BZR1 family, including AtBEH2, again insinuating that AtBEH2
also plays a role in the Arabidopsis BR signalling pathway.
The interaction spectrum of PhBEH2 in yeast also implies a role in BR signalling. First,
PhBEH2 is capable of interacting with Arabidopsis BIN2, a proven negative regulator of
BR signalling (Li et al., 2001). Second, the PhBEH2 protein interacted with several 14-3-3
proteins. Previous research in Arabidopsis showed that 14-3-3 proteins increase the
cytoplasmic retention of phosphorylated BES1. Mutation of a BIN2 phosphorylation site in
BZR1 was shown to abolish 14-3-3 binding, leading to increased nuclear localization of the
BZR1 protein (Gampala et al., 2007). Similar results have been obtained with the rice
homolog OsBZR1 (Bai et al., 2007). Like AtBZR1 and AtBES1, PhBEH2 also possesses
the conserved putative 14-3-3 binding site, suggesting that the regulation of the
BZR1/BES1 family by 14-3-3 proteins might be conserved among plants. Third, PhBEH2
interacted with a GSK3-like kinase (PSK8) that clustered together with the group II GSK3like kinases. Arabidopsis group II GSK3-like kinases have been shown to negatively
regulate BR signalling by phosphorylating BES1 and BZR1 (Chapter 2; Vert and Chory,
2006; Yan et al., 2009). Although PSK8 interacted with a member of the BES1/BZR1
family, more proof is needed to conclude whether this kinase plays a role in BR signalling.
For instance, phosphorylation of PhBEH2 by PSK8/9 should be demonstrated and mutants
might ultimately reveal its function. Next to PSK8 we also identified another group II
member (PSK9) in petunia. Solely based on homology it seems likely that PSK9 is
involved in BR signalling as well. The lack of interaction with PhBEH2 and with
97
Chapter 4
Arabidopsis BES1 and BZR1 is likely due to aberrant processing of the PSK9-GAL4 fusion
RNA and/or protein in yeast.
Our yeast two-hybrid screen with PhBEH2 revealed several interacting proteins that
function in diverse signal transduction pathways. This suggests that PhBEH2 could be an
important hub between BR and other signalling pathways. For instance, PhBEH2 interacted
with a protein that has high sequence homology to ARIA (ARMADILLO REPEAT
PROTEIN INTERACTING WITH ABF2), a positive regulator of the ABA signalling
cascade (Kim et al., 2004). ARIA modulates the transcriptional activity of ABF2, which
interacts with ABA-responsive elements in target promoters. ARIA seems only involved in
a subset of ABA-dependent processes, namely germination and growth of young seedlings,
but not for example in stomatal closure. Zhang et al. (2009) demonstrated that ABA rapidly
induced the expression of the BR-suppressed genes CPD and DWF4 and also enhanced the
phosphorylation of BES1. However in the absence of BIN2, ABA failed to induce BES1
phosphorylation. The interaction between PhBEH2 and PhARIA could be the link between
the ABA and BR signalling pathways.
Numerous reports point to a synergistic interaction between auxin and BR signalling.
Specificity of the identified interaction of PhBEH2 with a TOPLESS-related protein, a
transcriptional co-repressor of AUXIN RESPONSE FACTORS (ARFs) (Szemenyei et al.,
2008) seems doubtful, as the same protein was already identified as a possible partner in
other unrelated yeast 2-hybrid screens using various baits. However, recent findings point
to a more general role for TOPLESS(-related) proteins as transcriptional co-repressors in
diverse unrelated processes (Pauwels et al., 2010) and thus the discovered interactions
might be meaningful.
PhBEH2 interacts with two proteins containing RNA Recognition Motifs (RRM). One of
the RRM proteins is a homolog of FPA, a gene that regulates flowering time in Arabidopsis
via a pathway that is independent of day length. FPA negatively regulates the expression of
the floral repressor FLOWERING LOCUS C (Schomburg et al., 2001). Interestingly,
Domagalska et al. (2007) demonstrated that the BR signalling pathway plays an assisting
role in the repression of FLC and promotes flowering. This is consistent with the finding
that BR biosynthesis mutants as well as signalling mutants are late-flowering or, like our
cd10 (petunia bri1) mutant, non-flowering. In Arabidopsis two JmjN/C domain-containing
histone demethylases, ELF6 (early flowering 6) and REF6 (relative of early flowering 6),
were identified as BES1 partners (Yu et al., 2008). ELF6 was found to be a repressor in the
photoperiodic flowering pathway and REF6 negatively regulates the expression of FLC. In
addition many induced BR-induced genes such as cell wall-modifying enzymes involved in
cell elongation (like xyloglucan endotransglucosylases/hydrolases (XETs/XTHs),
expansins, and pectate lyases,) were down-regulated in ref6 and elf6 insertion mutants,
98
Brassinosteroid biosynthesis and signalling in petunia
suggesting that the interaction of BES1 with REF6/ELF6 positively regulates BR target
gene expression and promotes cell elongation. It could be that BRs control developmental
processes such as flowering and cell elongation via the interaction between BES1,
REF6/ELF6 and FPA. Additionally it has been shown that rice SVP-group MADS-box
proteins that repress floral meristem identity, work as negative regulators in BR responses,
however their roles are diversified and they function at different developmental stages (Lee
et al., 2008a). Further research by Lee et al. (2008b) suggest that SVP-group MADS-box
proteins repress BR responses by binding to the promoters of BR target genes.
Maybe one of the most interesting partners of PhBEH2 is a protein homologous to the
Arabidopsis gene SECRET AGENT (SEC) encoding an O-linked N-acetylglucosamine
transferase (OGT) (Hartweck et al., 2002; Hartweck et al., 2006). SEC catalyses the
transfer of a single O-linked-N-acetylglucosamine to specific serine/threonine residues of
proteins. SEC is partly redundant with another OGT in Arabidopsis called SPINDLY
(SPY). In Arabidopsis SPY negatively regulates the gibberellin (GA) pathway, but also
promotes cytokinin responses and interacts with the circadian clock protein GIGANTEA in
yeast (Tseng et al., 2004; Maymon et al., 2009). It seems that SEC is not involved in GA
signalling or only has a limited role, (Hartweck et al., 2006). Shimada et al. (2006) reported
that mutation of the rice SPINDLY gene (OsSPY) reduced the expression of several BR
biosynthesis genes and OsBRI1. As expected, the levels of sterol and BR compounds were
slightly up regulated in the OsSPY knock-out mutant. O-GlcNAcylation of target proteins
might affect their activity, localization, stability, and interactions with other proteins. The
fact that OGTs add acetylglucosamine to serine/threonine residues of proteins envisages a
situation where OGTs and GSKs compete for modification of these residues in the
BES/BZR family of proteins. Such a scenario, where an OGT competes with a kinase is a
widely recognised mechanism in animal research (Kamemura et al., 2002; Wang et al.,
2008b; Butkinaree et al., 2009). Most striking, there are specific examples where GSK3
phosphorylation and O-GlcNAcylation occurs on the same site of proteins (Wang et al.,
2007). The addition of acetylglucosamine by OGTs to BES1/BZR1 family members might
have role in localization, stability or activity of these proteins.
Although we identified some interesting links between BR signalling and other pathways
via PhBEH2 (and possibly via the other members of the BES1/BZR1 family) further
research should confirm these findings. For example, it would be interesting to see whether
PhSEC adds acetylglucosamine to PhBEH2 and if so what the consequences are for the
function of PhBEH2. Furthermore, it would be interesting to reveal whether ABA is
capable of inducing phosphorylation of BES1 in an aria mutant background and if the
expression of BR-suppressed genes is induced.
99
Chapter 4
Remarkably, we did not find any known homologs of the BR signalling pathway in the
yeast two-hybrid screen performed with PSK8. Using PhBEH2 as bait we isolated PSK8,
but using the same cDNA library the reverse interaction was not identified. It might be that
none of the BES/BZR1 family members present in the library is in frame with the GAL4
binding domain.
Nonetheless, our yeast two-hybrid screen with PSK8 might have revealed other roles of
group II GSK kinases that have not been recognised in other model species yet. PSK8
interacted with a number of proteins that are not very well characterised yet, but at least
some seem to be cell membrane or wall localized. Two proteins showed high homology to
the LLR-extensin family (LRX), which are localized in the cell wall. It has been suggested
that LRXs might control the development of the cell by regulating cell wall expansion, cell
polarization and the modification of localized cell wall domains during differentiation
(Baumberger et al., 2003). As BRs are involved in cell elongation, such an interaction is not
unlikely. PSK8 mediated phosphorylation of LRR-extensins might affect their stability,
activity or disrupt interaction with other proteins. PSK8 also interacted with a homolog of
AtREM6.3, a member of the REMORIN family. REMORINS are plant specific and are
located in the plasma membrane and in lipid rafts (Raffaele et al., 2007). The precise
biological roles of REMORINS are not clear, but according to the available expression data
they may be involved in various signal transduction pathways triggered by abiotic and
biotic stressors. The expression level of AtREM6.3 is the highest in the embryo and
interestingly, a BR treatment decreased the expression level of AtREM6.3 (Raffaele et al.,
2007).
Experimental procedures
Brassinolide experiment
24- epibrassinolide was purchased from Wako Biochemistry, dissolved in DMSO (2mM)
and stored at -20°C. Young plantlets were sprayed each tow or three days until runoff with
1 µM brassinolide or with a solvent control (0.01% ethanol and 0.1% Tween 20) for a
couple of weeks.
Genomic DNA isolation and PCR
Petunia DNA was isolated by grinding one young leaf in liquid nitrogen. To this 400 µl
DNA extraction buffer was added (0.1 M Tris pH 8.0; 0.5 M NaCl; 50 mM EDTA and 10
mM β mercapto-ethanol) and samples were incubated for 15-30 min. at 65°C. Next 250 µl
100
Brassinosteroid biosynthesis and signalling in petunia
phenol/chloroform was added, samples were shaken and subsequently centrifuged for 5
minutes at maximum speed in a microcentrifuge. The supernatant was transferred to a clean
tube and DNA was precipitated using 2-propanol and washed with 70% EtOH. The pellet
was dissolved in 100 µl H2O containing DNase free RNase (10 mg/ml) followed by an
incubation step for 15-30 min. at 65°C. PCR reactions were performed by using 2 µl
genomic DNA in a standard PCR reaction. The nucleotide sequences of the primers used
are listed in Supplemental Table 1. All PCR products were separated by electrophoresis in a
1% (w/v) agarose gel.
Isolation of 3’ gene fragments by RACE PCR
In order to isolate cDNA fragments, total RNA was isolated from leaves followed by first
strand cDNA synthesis using the RACE1 primer as described previously (Quattrocchio et
al., 2006). PCR amplification was carried out using gene-specific primers or, to isolate 3’
ends of cDNAs, by using one gene-specific primer in combination with the RACE2 primer
under standard PCR conditions (annealing temperature 55˚C). All used primer sequences
are listed in Supplemental Table 1. PCR fragments were directly sequenced or cloned in
pGEM-Teasy (Promega) and sequenced.
Isolation of 5’ gene fragments by SOTI PCR
To complete sequences at the 5´ end, for some genes a somatic insertion-mediated PCR was
performed (Rebocho et al., 2008). Here, we make use of somatic dTph1 insertions in the
target gene that may occur in some cells of petunia W138 plants. Multiple W138 genomic
DNAs from leaves of individual plants or leaves of pooled plants were subjected to nested
PCR using gene-specific primers and dTph1 transposon primers (see Supplemental Table 1
for primer sequences). In the first PCR the dTph1 transposon primers out11 or out12 were
used in a standard PCR reaction with annealing temperature 55˚C. In the second, nested
PCR, 1 µl of the out11 reaction was re-amplified with out15 and a nested gene-specific
primer while the out12 reaction was re-amplified with out6 and a nested gene-specific
primer. The second reaction was a touch-down PCR where annealing started at 70˚C and
decreased with one ˚C every cycle until reaching 55 ˚C, followed by 25 additional cycles at
55 ˚C. PCR fragments were directly sequenced or cloned in pGEM-Teasy (Promega) and
sequenced.
101
Chapter 4
Phylogenetic analysis
Phylogenetic trees were generated using the neighbour-joining method of ClustalX
(ftp://ftp.ebi.ac.uk/pub/software/clustalw2/). Bootstrap mode (1000 replications) was used
for estimating the level of confidence assigned to the particular nodes in the tree. The tree
was visualized with help of Treeviewer 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.
html) and rooted using ClustalW (http://www.bioinformatics.nl/tools/clustalw.html).
Alignments were coloured using Boxshade (http://www.ch.embnet.org/software/BOX_
form.html).
Yeast two-hybrid
The full length coding sequences of PhBEH2 was amplified by RT-PCR on total RNA
isolated from leaves using Phusion polymerase (Finnzymes) and primers M736 and M737.
The amplification product was cloned EcoRI/SalI in pBD-GAL4 and pAD-GAL4
(Stratagene). The PSK8-GAL4BD fusion was made by cutting PhBEH2 yeast two-hybrid
interactor 78 with EcoRI/SalI and ligation of the fragment in pBD-GAL4. The plasmids
were introduced into the yeast strain PJ69 (James et al., 1996), which carries HIS3, ADE2
and LACZ reporter genes driven by distinct GAL4-responsive promoters. For the yeast
two-hybrid screen, PhBEH2/PSK8 were used as bait against a cDNA library from young
petunia inflorescences (Souer et al., 2008). Library screen was performed as described
previously (Quattrocchio et al., 2006). A total of 100.000 and 300.000 transformed yeast
cells were screened for PhBEH2 and PSK8, respectively. Plasmid DNA was isolated from
positive (HIS, ADE) colonies, transformed to Escherichia coli, sequenced and subsequently
reintroduced in PJ69, either with the empty pBD-GAL4 vector or with the bait plasmid
expressing PhBEH2/PSK8. Only plasmids that grew on media lacking histidine and
adenine when co-transformed with the bait vector, but not when co-transformed with the
empty pBD-GAL4, were considered as positives and analyzed further.
For the yeast two-hybrid assay between members of the BES1/BZR1 family and
GSK3/SHAGGY-like family members from Arabidopsis and petunia were amplified by
RT-PCR using Phusion polymerase (Finnzymes) on Petunia leaf total RNA or from
existing plasmid clones. The amplification products were cloned EcoRI/SalI in pBD-GAL4
and/or pAD-GAL4 (Stratagene).
102
Brassinosteroid biosynthesis and signalling in petunia
Acknowledgements
We thank Allan Awanijan and Longinus Igbojionu for their assistance during some of the
cloning experiments and Maartje Kuijpers for plant care. This work was supported by a
grant of the Netherlands Organisation for Scientific Research (NWO).
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107
Chapter 4
Supplemental data
Supplemental Table 1. Primers used for PCR
Gene
Primer
Sequence 5´-3´
Gene
Primer
Sequence 5´-3´
RACE1
P007
GACTCGAGTCGACATCGATTTTTTTTTTTTTTT
PhCYP85A1
M673
GGACTGGTCCCAGGATATCCACAGTC
RACE2
P008
GACTCGAGTCGACATCGA
M674
CATAAGGTAACCAAGCATATCATGTTG
dTph1 OUT6
P016
CCTTTGCACCAAGGAGCTCCGCC
M698
TGCTGCTGTCCATGGTTCTGCTCACA
dTph1 OUT11
P423
CGAAGGGGTGTCAATGCTG
M699
TCAATTAGTGTCCTTAGCAAGCTGAC
dTph1 OUT12
P630
MCAGCATTGACACCCCTTC
M810
GGCATGAATTCTTGAGCTAAGGAG
dTph1 OUT15
P1114
TATTTGAACRGTTGTCCTCTTGAA
M811
CTTCCCAATACTAACTATCGTTG
PhBEH2
M711
GCTCTTTGTACTGAAGCTGGTTGG
M815
CAATCCCAGCAATTTGTTTCAATAGTG
M712
GATGGTACCACTTATCGCAAGG
PhBRI1
PSK8
PSK9
PhCPD
108
M656
CATTTCCCACCTTCATCTTATGTC
M717
GATGTATGACGACGGATTTCCGTCACAG
M657
GCTCGAGGAACCAAATTAGCACA
M718
CTGGCATGGTAAGAAGGTACAGGA
M679
CTTACGGCAAACACTACACTTCAGCTG
M736
GGGAATTCACAGCTGCCGGCGGTGGAGGTG
M703
AGCCAATTCGAGACCTCTGCCTATG
M737
GGGGTCGACGAAGCTCCTTCAAGCATGTGCC
M704
CAGCAAAAGACCATGTCACTAACCATTC
M631
GTTGAAGGATGGCAGTGTCGTAG
M662
GAATAATGGCTTCCAGGTATTGGGT
M632
GAGTGTCCATAGCACTCATGAG
M663
CGAGAATGGAACGTACTTTCTTCTC
M694
TCAGGTCAGCGGACAGGGTGATCG
M680
CATTGAAGGGGTCTGCTCCAAGACAC
M695
TGTAGGAAAGCCAACCCTCTAGCA
M706
TGTTACAGTATAGAGAGCATACTC
M709
GGCTCGAGGGATGGACATTCTCATTCTGCAAC
M658
GACCTTCTTGTCCAGTTCAGATG
M714
CAACCCAAAGATGCAGAAGAGGGAG
M659
GATCAATCTTCAGGCTCATCAAC
M719
CCTGCTCCATGGCACTCCTTACTCC
M678
GACGCCAACATCAGTATACATTTGAG
M723
GGCAATTGGATGGTCATTCTCATTC
M689
GCTCAAATGTATACTGATGTTGGCGTC
M741
TCCTTCAGCTTTGACAATGACC
M705
GGGAGCTTGATGATCTGACCGTTG
M743
CACCAAGTTGGCTTGCCAACACACC
M747
CTCTTTGAGGCGCTTCACGACCTC
M746
GAGGACACCAATGTAAAGTCAACAC
M753
GAACGAGCACGCTTATAGTCAC
M746B
TATTTGAACRGTTGTCCTCTTGAA
M775
TTGAACGGTTGTCCTCTTGAAC
M826
CTTTTCTACGACTAGTAGAGATGAGC
M814
GACGCAGTTGGTGGACTTTCTTGTC
M834
CAAGGTCCTTGGTACTCCTACTCG
M654
CTCCAACTGGACATAGGCCTGTCT
M738
GGGAATTCCTGTTAAGGGATGTGAATGATG
M655
CTCTTGCCTCTGCCTGTCACAATC
M744
CTGAACGCGTAGTGGGAACTGGATC
M696
GGGATGGAGTTATATCCTCGTATTG
M745
GGGTCGACCTTCACGTCATGGCATCGTGTG
M697
GTGCAATATCCATTGTGTACCAGTAC
PhDET2
PhDET3
PhDIM
PhDWARF5
PhDWARF7
M660
CTTGAAGCTATTCGTGGAGGAGAC
M781
GCTTCGGCTGGCCATTCTTCCCTC
M782
GGTTGTGGAAATTATATGACCAGTGAC
M661
GAACAACAAAAGAGATGAACGTTAC
M809
GGGAATTCTCTGCTCCTGTTATGGATGTGAATG
M677
CTAAGTACATGATTGCAAATGGATGGAC
M664
GAAAATCCTGAACCCTTCATCGATGA
M701
GGCAATGAGTTATCACAGCCTTAGC
M665
CTCAAACAAGCTTCGATGTTGGAC
M675
M671
CTGATAATGTTCCTGCTTTTGTTGACAC
CACCACCCATATATTTGGTGAACCGAC
M672
GAAAGATTGACCCCAACCCCATTC
M676
GGTTTCATAGCCAGCCACAAGTAATGC
M702
CTTGTTACTGATATTTATTGAATGG
M700
GGACCATTTGTTGGTCGATATAGAC
M720
CTCTTCAGGTTTTCTGCAGAGAATC
M748
AGCGAACCAGGATAACTAGATTC
M797
CAACAGATGCAGAGGTGAACAAAGTGGT
M752
CACACACTAGTGGAGTATATAAGAGG
M798
CCTTCACCTCAGTTCACATGGATGAAG
M774
CAAGTCCATGCCCTTCACTCAATG
M799
CAAGCCTTGAAAGTTCTAAACCAGGAC
M812
CTTCCCACTGGTAAACACTCAACTC
M866
CTTCTGATACTTTTTATGTGTCTTC
M867
GTTATACCATTCCAAAAGGGTGGAAG
M868
CTCAACGCATCTGCCACCTTCTTTCTC
M869
CTCATAACCAGGACATCGCCGTGGTC
PhSMT2
PhROT3
Brassinosteroid biosynthesis and signalling in petunia
Supplemental Table 2. Genbank accession numbers
Gene
Genbank accession number
BES1/BZR1 family
AtBES1
AtBZR1
AtBEH1
AtBEH2
AtBEH3
AtBEH4
NP_973863
NP_565099
NP_190644
Q94A43
NP_193624
NP_565187
BRI1
AtBRI1
LeBRI1
PsBRI1
NbBRI1
NtBRI1
OsBRI1
HvBRI1
Gene
Genbank accession number
GSK3/SHAGGY kinases
NP_195650
AAN85409
BAC99050
ABO27628
ABR18799
NP_001044077
BAD01654
AtSK11
ASK12
ASK13
ASK31
ASK32
ASK41
ASK42
AtBIN2
AtGSK1
AtASKζ
PSK4
PSK6
PSK7
NP_568486
NP_187235
NP_196968
NP_974471
NP_191981
NP_172455
NP_176096
NP_193606
NP_172127
NP_180655
CAA58594
CAA11861
CAA11862
Other
AtCLV1
AtMPK1
D83257
Q39021
109
Chapter 4
putative leucine
PhBRI1
NbBRI1
LeBRI1
AtBRI1
1
1
1
1
PhBRI1
NbBRI1
LeBRI1
AtBRI1
46
60
51
43
MNSQNNVIYN-------LFLLSFCLH-------LIFFLPPASPAS-INGLFKDTQQLLSF
MKPHKSALYQHHCSLKKIFLLSFYLQPLFILLLIIFFLPPASPAS-VNGLLKDSQQLLSF
MKAHKTVFNQHPLS---LNKLFFVLL-------LIFFLPPASPAASVNGLYKDSQQLLSF
MKTFSSFFLS--------VTTLFFFS----------FFSLSFQASPSQSLYREIHQLISF
24
698
715
707
698
LGGLKNVAILDLSYNRLNGSIPNSLTSLTLLGEIDLSNNNLSGLIPESAPFDTFPDYRFA
LGGLKNVAILDLSYNRLNGSIPNSLTSLTLLGELDLSNNNLTGPIPESAPFDTFPDYRFA
LGGLKNVAILDLSYNRFNGTIPNSLTSLTLLGEIDLSNNNLSGMIPESAPFDTFPDYRFA
VGDLRGLNILDLSSNKLDGRIPQAMSALTMLTEIDLSNNNLSGPIPEMGQFETFPPAKFL
PhBRI1
NbBRI1
LeBRI1
AtBRI1
758
775
767
758
*
*
NN-SLCGYPL-TPCN-SGASNANLHQKSH-RKQASWQG-VAMGLLFSLFCIFGLIIVAVE
NT-SLCGYPL-QPCGSVGNSNSSQHQKSH-RKQASLAGSVAMGLLFSLFCIFGLIIVAIE
NN-SLCGYPLPIPCSSGPKSDANQHQKSH-RRQASLAGSVAMGLLFSLFCIFGLIIVAIE
NNPGLCGYPL-PRCDPSNADGYAHHQRSHGRRPASLAGSVAMGLLFSFVCIFGLILVGRE
PhBRI1
NbBRI1
LeBRI1
AtBRI1
813
832
825
817
MKKRRKKKEAALEAYMDGHSHS---ATANSAWKFTSAREALSINLAAFEXPLRKLTFADL
TKKRRKKKEAALEAYMDGHSNS---ATANSAWKFTSAREALSINLAAFEKPLRKLTFADL
TKKRRRKKEAALEAYMDGHSHS---ATANSAWKFTSAREALSINLAAFEKPLRKLTFADL
MRKRRRKKEAELEMYAEGHGNSGDRTANNTNWKLTGVKEALSINLAAFEKPLRKLTFADL
PhBRI1
NbBRI1
LeBRI1
AtBRI1
870
889
882
877
LEATNGFHNDSLIGSGGFGDVYRAQLKDGSVVAIKKLIQVSGQGDREFTAEMETIGKIKH
LEATNGFHNDSLIGSGGFGDVYKAQLKDGSVVAIKKLIHVSGQGDREFTAEMETIGKIKH
LEATNGFHNDSLVGSGGFGDVYKAQLKDGSVVAIKKLIHVSGQGDREFTAEMETIGKIKH
LQATNGFHNDSLIGSGGFGDVYKAILKDGSAVAIKKLIHVSGQGDREFMAEMETIGKIKH
PhBRI1
NbBRI1
LeBRI1
AtBRI1
930
949
942
937
RNLVPLLXYCKVGEERLLVYEYMKYGSLEDVLHDRKKNGIKLNWAARRKIAIGAARGLAF
RNLVPLLGYCKVGEERLLVYEYMKYGSLEDVLHDRKKNGIKLNWHARRKIAIGAARGLAF
RNLVPLLGYCKVGEERLLVYEYMKYGSLEDVLHDRKKIGIKLNWPARRKIAIGAARGLAF
RNLVPLLGYCKVGDERLLVYEFMKYGSLEDVLHDPKKAGVKLNWSTRRKIAIGSARGLAF
PhBRI1
NbBRI1
LeBRI1
AtBRI1
990
1009
1002
997
LHHNCIPHIIHRDMKSSNVLLDENLEARVSDFGMARLMSAMDTHLSVSTLAGTPGYVPPE
LHHNCIPHIIHRDMKSSNVLLDENLEARVSDFGMARLMSAMDTHLSVSTLAGTPGYVPPE
LHHNCIPHIIHRDMKSSNVLLDENLEARVSDFGMARLMSAMDTHLSVSTLAGTPGYVPPE
LHHNCSPHIIHRDMKSSNVLLDENLEARVSDFGMARLMSAMDTHLSVSTLAGTPGYVPPE
PhBRI1
NbBRI1
LeBRI1
AtBRI1
1050
1069
1062
1057
YYQSFRCSTKGDVYSYGVVLLELLTGRQPTDSADFGDNNLVGWVKQQ-KMKISDVFDREL
YYQSFRCSTKGDVYSYGVVLLELLTGRTPTDSADFGDNNIVGWVRQHAKLKISDVFDREL
YYQSFRCSTKGDVYSYGVVLLELLTGKQPTDSADFGDNNLVGWVKLHAKGKITDVFDREL
YYQSFRCSTKGDVYSYGVVLLELLTGKRPTDSPDFGDNNLVGWVKQHAKLRISDVFDPEL
PhBRI1
NbBRI1
LeBRI1
AtBRI1
1109
1129
1122
1117
LKEDPTIEIELLQHLKVARACLDDRHWKRPTMIQVMAMFKEIQAGSGIDSSSTIA-TDDC
LKEDPSIEIELLQHLKVACACLDDRHWKRPTMIQVMAMFKEIQAGSGIDSSSTIA-ADDV
LKEDASIEIELLQHLKVACACLDDRHWKRPTMIQVMAMFKEIQAGSGMDSTSTIG-ADDV
MKEDPALEIELLQHLKVAVACLDDRAWRRPTMVQVMAMFKEIQAGSGIDSQSTIRSIEDG
PhBRI1
NbBRI1
LeBRI1
AtBRI1
1168
1188
1181
1177
NFNAVEGGIEMGINESIKEGNELSKHL
NFSAVEGGIEMGISESIKEGNELSKHL
NFSGVEGGIEMGINGSIKEGNELSKHL
GFSTIEM-----VDMSIKEVPEGKL--
transmembrane domain
zipper motif
1
2
*
*
KSSLP--STTLQGLAASTDPCSYTGVSCKNSRVVSIDLSNTLLSVDFTLVSSYLLTLSNL
KSSLPNTQAQLQNWLSSTDPCSFTGVSCKNSRVSSIDLTNTFLSVDFTLVSSYLLGLSNL
KAALPPTPTLLQNWLSSTGPCSFTGVSCKNSRVSSIDLSNTFLSVDFSLVTSYLLPLSNL
KDVLPD-KNLLPDWSSNKNPCTFDGVTCRDDKVTSIDLSSKPLNVGFSAVSSSLLSLTGL
3
25
PhBRI1
NbBRI1
LeBRI1
AtBRI1
4
PhBRI1 104 ETLVLKNANLSGSLTSASKSQCGVSLNSLDLSENTISGPVNDVSSLGSCSNLKSLNLSRN
NbBRI1 120 ESLVLKNANLSGSLTSAAKSQCGVSLNSIDLAENTISGSVSDISSFGPCSNLKSLNLSKN
LeBRI1 111 ESLVLKNANLSGSLTSAAKSQCGVTLDSIDLAENTISGPISDISSFGVCSNLKSLNLSKN
AtBRI1 102 ESLFLSNSHING---SVSGFKCSASLTSLDLSRNSLSGPVTTLTSLGSCSGLKFLNVSSN
kinase domain
5
6
PhBRI1 164 LMDSPLKEAKFQSFSLSLQVLDLSYNNISGQNLFPWLFFLRFYELEYFSVKGNKLAGTIP
*
NbBRI1 180 LMDPPSKEIKAS--TLSLQVLDLSFNNISGQNLFPWLSSMRFVELEYFSLKGNKLAGNIP
LeBRI1 171 FLDPPGKEMLKAA-TFSLQVLDLSYNNISGFNLFPWVSSMGFVELEFFSLKGNKLAGSIP
AtBRI1 159 TLDFPGKVSGGLK-LNSLEVLDLSANSISGANVVGWVLSDGCGELKHLAISGNKISGDVD
7
8
9
PhBRI1 224 ELDFKNLSYLDLSANNFSTGFPLFKDCGNLQHLDLSSNKFVGDIGGSLAACVKLSFVNLT
NbBRI1 238 ELDYKNLSYLDLSANNFSTGFPSFKDCSNLEHLDLSSNKFYGDIGASLSSCGRLSFLNLT
LeBRI1 230 ELDFKNLSYLDLSANNFSTVFPSFKDCSNLQHLDLSSNKFYGDIGSSLSSCGKLSFLNLT
AtBRI1 218 VSRCVNLEFLDVSSNNFSTGIPFLGDCSALQHLDISGNKLSGDFSRAISTCTELKLLNIS
10
11
12
PhBRI1 284 NNMFVGFVPKLQSESLEFLYLRGNDFQGVLASQLGDLCKSLVELDLSFNNFSGFVPETLG
NbBRI1 298 SNQFVGLVPKLPSESLQFMYLRGNNFQGVFPSQLADLCKTLVELDLSFNNFSGLVPENLG
LeBRI1 290 NNQFVGLVPKLPSESLQYLYLRGNDFQGVYPNQLADLCKTVVELDLSYNNFSGMVPESLG
AtBRI1 278 SNQFVGPIPPLPLKSLQYLSLAENKFTGEIPDFLSGACDTLTGLDLSGNHFYGAVPPFFG
13
14
PhBRI1 344 ACSKLELLDVSNNNFSGKLPVDTLLKLSNLKTLVLSFNNFIGGLPESLSSLVK-LETLDV
NbBRI1 358 ACSSLELLDISNNNFSGKLPVDTLLKLSNLKTMVLSFNNFIGGLPESFSNLLK-LETLDV
LeBRI1 350 ECSSLELVDISYNNFSGKLPVDTLSKLSNIKTMVLSFNKFVGGLPDSFSNLLK-LETLDM
AtBRI1 338 SCSLLESLALSSNNFSGELPMDTLLKMRGLKVLDLSFNEFSGELPESLTNLSASLLTLDL
15
16
PhBRI1 403 SSNNLTGLIPSGICKDPLNSLKVLYLQNNLFTGPIPDSLGNCSRLVSLDLSFNYLTERIP
NbBRI1 417 SSNNITGVIPSGICKDPMSSLKVLYLQNNWLTGPIPDSLSNCSQLVSLDLSFNYLTGKIP
LeBRI1 409 SSNNLTGVIPSGICKDPMNNLKVLYLQNNLFKGPIPDSLSNCSQLVSLDLSFNYLTGSIP
AtBRI1 398 SSNNFSGPILPNLCQNPKNTLQELYLQNNGFTGKIPPTLSNCSELVSLHLSFNYLSGTIP
17
18
19
PhBRI1 463 SSLGSLSKLKDLVLWLNQLSGEIPQELMYLKSLENLILDFNDLSGSIPASLSNCTNLNWI
NbBRI1 477 SSLGSLSKLKDLILWLNQLSGEIPQELMYLKSLENLILDFNDLTGSIPASLSNCTNLNWI
LeBRI1 469 SSLGSLSKLKDLILWLNQLSGEIPQELMYLQALENLILDFNDLTGPIPASLSNCTKLNWI
AtBRI1 458 SSLGSLSKLRDLKLWLNMLEGEIPQELMYVKTLETLILDFNDLTGEIPSGLSNCTNLNWI
21
20
PhBRI1 523 SLSNNMLSGEIPASLGRLVNLAILKLK-ITQSQEYPAEWG-CQSLIWLDLNNNFLNGSIR
NbBRI1 537 SMSNNLLSGEIPASLGGLPNLAILKLGNNSISGNIPAELGNCQSLIWLDLNTNLLNGSIP
LeBRI1 529 SLSNNQLSGEIPASLGRLSNLAILKLGNNSISGNIPAELGNCQSLIWLDLNTNFLNGSIP
AtBRI1 518 SLSNNRLTGEIPKWIGRLENLAILKLSNNSFSGNIPAELGDCRSLIWLDLNTNLFNGTIP
68 amino acid non-repetitive island
PhBRI1 581 -RHVKQSGKIAVAFLTGKRYVYIKNDGSK-ECHGAGNLLEFGGIRQEQLDRISTRHPCNF
NbBRI1 597 GPLFKQSGNIAVALLTGKRYVYIKNDGSK-ECHGAGNLLEFGGIRQEQLDRISTRHPCNF
LeBRI1 589 PPLFKQSGNIAVALLTGKRYVYIKNDGSK-ECHGAGNLLEFGGIRQEQLDRISTRHPCNF
AtBRI1 578 AAMFKQSGKIAANFIAGKRYVYIKNDGMKKECHGAGNLLEFQGIRSEQLNRLSTRNPCNI
22
23
PhBRI1 639 T-RVYRGITQPTFNHNGSMIFLDLSYNKLEGSIPKELGSMFYLSILNLGHNDLSSAIPQE
NbBRI1 656 T-RVYRGITQPTFNHNGSMIFLDLSYNKLEGSIPKELGSMYYLSILNLGHNDLSGVIPQE
LeBRI1 648 T-RVYRGITQPTFNHNGSMIFLDLSYNKLEGSIPKELGAMYYLSILNLGHNDLSGMIPQQ
AtBRI1 638 TSRVYGGHTSPTFDNNGSMMFLDMSYNMLSGYIPKEIGSMPYLFILNLGHNDISGSIPDE
Supplemental Figure 1. Alignment of the deduced amino acid sequences of BRI1 and homologs. Ph, petunia;
Nb, Nicotiana benthamiana; Le, tomato and At, Arabidopsis. Identical residues in three out of four sequences are
indicated in black and similar amino acids in gray. Dashed lines represent gaps to facilitate alignment. A putative
leucine zipper motif, 68 amino acid non-repetitive island, N- and C- terminal LRR-flanking cysteine pairs (*),
transmembrane and kinase domain are shown. Numbers 1-25 depict the first amino acid of each Leucine Rich
Repeat (LRR).
110
Brassinosteroid biosynthesis and signalling in petunia
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
1
1
1
1
1
MASLPLAPQHHHVPPDNHHPLQHDQPPAAVKHAVAGARPEMESDKEMSAAVVEGNDAVTG
----------------------------------------MADDKEMPAAVVDGHDQVTG
----------------------------------------------MSAPVMDVNDAVTG
MASLPLGP---------QPHALAP-PLQLHDGDALKRRPELDSDKEMSAAVIEGNDAVTG
MTSIPLGPP--------QPPSLAPQPPHLHGGDSLKRRPDIDNDKEMSAAVIEGNDAVTG
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
241
201
195
231
233
ICSRYYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGENAVDQLVEIIKVLGTP
ICSRFYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGENAVDQLVEIIKVLGTP
ICSRFYRAPELIFGATEYTDSIDIWSAGCVLAELLLGQPLFPGENAVDQLVEIIKVLGTP
ICSRYYRAPELIFGATEYTASIDIWSAGCVLAELLLGQPLFPGENSVDQLVEIIKVLGTP
ICSRYYRAPELIFGATEYTSSIDIWSAGCVLAELLLGQPLFPGENSVDQLVEIIKVLGTP
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
61
21
15
51
53
HIISTTIGGKNGEPKRTISYMAERVVGTGSFGIVFQAKCLETGETVAIKKVLQDKRYKNR
HIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGETVAIKKVLQDRRYKNR
HIISTTIGGKNGQPKQTVSYMAERVVGTGSFGVVFQAKCLENGETVAIKKVLQDRRYKNR
HIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGESVAIKKVLQDRRYKNR
HIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGESVAIKKVLQDRRYKNR
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
301
261
255
291
293
TREEIRCMNPNYTDFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALEACA
TREEIRCMNPHYTDFRFPQIKAHPWHKIFHKRMPPEAIDFASRLLQYSPSLRCTALEACA
TREEIRCMNPNYTDFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALDACA
TREEIRCMNPNYTDFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALEACA
TREEIRCMNPNYTDFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALEACA
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
121
81
75
111
113
ELQLMRLMDHPNVICLKHCFFSTTSRDELFLNLVMDYVPESLYKVLKHYSNSNQRMPLIY
ELQLMRVMDHPNVVCLKHCFFSTTSKDELFLNLVMEYVPESLYRVLKHYSSANQRMPLVY
ELQLMRTMDHPNVVSLKHCFYSTTSKNELFLNLVMEYVPETMYRMLKHYSNMNQRMPLIY
ELQLMRPMDHPNVISLKHCFFSTTSRDELFLNLVMEYVPETLYRVLRHYTSSNQRMPIFY
ELQLMRLMDHPNVVSLKHCFFSTTTRDELFLNLVMEYVPETLYRVLKHYTSSNQRMPIFY
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
361
321
315
351
353
HSFFDELREPNARLPNGRPFPPLFNFKQELTGASPELVNKLIPEHVWRQIGLNFPYPGAT
HPFFDELREPNARLPNGRPFPPLFNFKQEVAGSSPELVNKLIPDHIKRQLGLSFLNQSGT
HPFFDELREPNARLPNGRPLPPLFNFKQELSGSSPDLINRLIPEHIKRQMGLQFFTSHDA
HPFFNELREPNARLPNGRPLPPLFNFKQELGGASMELINRLIPEHVRRQMSTGLQNS--HPFFNELREPNARLPNGRPLPPLFNFKQELSGASPELINRLIPEHVRRQMNGGFPFQAGP
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
181
141
135
171
173
VKLYMYQIFRGLAYIHNVPRVCHRDVKPQNLLVDPLTHQVKLCDFGSAKVLVNGEANISY
VKLYMYQIFRGLAYIHNVAGVCHRDLKPQNLLVDPLTHQVKICDFGSAKQLVKGEANISY
VKLYIYQVFRGLAYMHTVAGVCHRDLKPQNILVDPVTHQVKICDFGSAKLLVKGEANISY
VKLYTYQIFRGLAYIHTVPGVCHRDVKPQNLLVDPLTHQVKLCDFGSAKVLVKGEPNISY
VKLYTYQIFRGLAYIHTAPGVCHRDVKPQNLLVDPLTHQCKLCDFGSAKVLVKGEANISY
PSK8
AtBIN2
PSK9
AtGSK1
AtASKdzeta
-381 -375 MT
-413 --
*
PS H2
K9
Ph
14
BD -33ε
BE
Ph
Ph
BR
I1
cy
t
Supplemental Figure 2. Alignment of the deduced amino acid sequences of class II GSK3/SHAGGY-like
kinases from Arabidopsis and petunia. Ph, petunia; At, Arabidopsis. Identical residues in three out of five
sequences are indicated in black and similar amino acids in gray. Dashed lines represent gaps to facilitate
alignment. The red asterisk marks the phospho-tyrosine residue (pTyr200, Kim et al., 2009).
- TX-gal
- TH
- THA
Supplemental Figure 3. Yeast one-hybrid to test for auto-activation of baits. The cytoplasmic domain of
PhBRI1 and the full-length sequences of PhBEH2, PSK9 and Ph14-3-3ε were fused to the GAL4-binding domain.
Auto-activation was determined by growth on medium lacking histidine (-TH), histidine and adenine (-THA) and
by blue colouring using 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (-TX-gal).
111
Summary
As the world population is growing continuously, food production needs to keep pace.
Apart from improved fertilization and cultivation techniques, plant breeding greatly
improved productivity.
One of the major abiotic stresses that affects productivity in the agricultural sector is soil
salinity. Desertification, floods and unsuitable irrigation methods increase the amount of
salt all over the world. Removal of salt from soil is costly and therefore it is economically
important to find solutions that allow crops to grow in salty habitats. Currently two genetic
approaches are used to improve salt stress tolerance in crops: 1) direct selection of existing
plant varieties that are salt tolerant and cross them into cultivars and 2) the generation of
transgenic plants to introduce novel genes or alter the level of expression of existing genes
that play a role in salt stress tolerance.
The initial goal of my PhD research project was to investigate the role of the GSK3-like
kinase GSK1 in salt stress signalling by using the model plant Arabidopsis thaliana. The
Arabidopsis genome contains ten GSK3-like kinases that based on phylogenetic analysis
can be classified into four subgroups. BIN2, a group II member, is the best-studied GSK3like kinase and plays a negative role in the brassinosteroid (BR) signalling pathway.
Based on previous research by another group we expected that mutation of the GSK1 gene
would result in a plant more susceptible to salt stress. We considered this might be the same
for two other GSK3-like kinases (ASKζ and BIN2) as they are very similar to GSK1, all
belonging to the group II GSK3-like kinases. In Chapter 2, we checked whether mutation
of these genes altered the response of Arabidopsis towards salt. In contrast to prior
research, we could not detect a role for GSK1 in tolerance against Na+ stress, neither for the
other group II members ASKζ and BIN2. However, based on research by others, the
involvement of not only group II GSK3-like kinases but also GSK3-like kinases of other
subgroups in salt tolerance seems likely. Instead of playing a positive role in salt stress
resistance, GSK3-like kinases seem to play a negative role. The potential role of GSK3-like
kinases in salt stress signalling might be an indirect effect that originates from the role that
we and others discovered for GSK1 as well as ASKζ in BR signalling.
Brassinosteroids (BR) are steroid hormones that are essential for plant growth and
development. Chapter 1 reviews the current knowledge about BR biosynthesis, BR
signalling and its involvement in various growth- and developmental processes such as cell
113
Summary
division and elongation, vascular differentiation, abiotic- and biotic stresses, photomorphogenesis, germination, senescence and fertility.
BIN2 negatively regulates the BR signalling pathway by phosphorylating the transcription
factors BES1 and BZR1. In Chapter 2 we show that GSK1 as well as ASKζ interact with
the BES1/BZR1 transcription factor family. The fact that group II GSK3-like kinases have
redundant roles in the BR signalling pathway was confirmed by studying several kinds of
mutants.
In order to learn more about the mode of actions of GSK1 and the group II GSK3-like
kinases in BR signalling and possibly in other signal transduction pathways we tried to
purify protein complexes containing group II GSK3-like kinases via various Tandem
Affinity Purification (TAP) methods which are described in Chapter 3. Till date, TAP
purification was unsuccessful due to technical complications and maybe also because the
TAP tagged versions of GSK3-like kinases seem to be biologically inactive.
BR biosynthesis and signalling has been extensively studied in Arabidopsis and to some
extent in other plant species such as tomato, rice and Catharanthus roseus. Within our
petunia collection we found several mutant families that resemble sterol/BR biosynthesis
and signalling mutants and this tempted us to research whether the BR biosynthesis and
signalling pathways are also conserved in petunia (Chapter 4). Cross-pollination between
the mutant families revealed five different complementation groups. Four mutant groups
clearly responded to spraying with the most biologically active BR, 24-epibrassinolide, by
elongation of their internodes, petioles and leaves. Complementation of the mutant
phenotype in these plants indicated that these are sterol/BR biosynthesis mutants. For one
mutant group we could reveal that this mutant phenotype was caused by the insertion of a
transposable dTph1 element in the BR biosynthesis gene CPD. One mutant group did not
respond to exogenous application of 24-epibrassinolide and therefore it was considered to
be a BR signalling mutant. Interestingly, this family turned out to have a dTph1 transposon
insertion in a gene homologous to the BR receptor BRI1 from Arabidopsis.
With help of a PCR-based approach we succeeded to isolate a member of the BES1/BZR1
transcription factor family (PhBEH2) and a group II GSK3-like kinase (PSK9) from
petunia. In a yeast two-hybrid experiment PhBEH2 interacted with another group II GSK3like kinase (PSK8) and 14-3-3 proteins, supporting the idea that the BR signalling pathway
is conserved among the plant kingdom. This was further supported by the fact that PSK8
could interact with members of the BES1/BZR1 family from Arabidopsis and BIN2 from
Arabidopsis with PhBEH2 from petunia.
Interestingly PhBEH2 also interacted with proteins involved in gibberellin (GA), auxin
(IAA) and abscisic acid (ABA) signalling and a protein that plays a role in the autonomous
flowering pathway. These interactions reveal new mechanisms, not recognised from
114
Summary
Arabidopsis research that suggest extensive cross-talk between BR signalling and other
(hormone) signalling pathways.
This thesis provides some knowledge about BR biosynthesis and signalling in Arabidopsis
and petunia. For a long time BRs are applied in the agriculture and horticulture to increase
crop yield and quality. With help of the acquired knowledge about the BR pathway,
superior crops could be developed in the future with a desirable stature, high yield and good
quality.
115
Samenvatting
Brassinosteroïden biosynthese en signalering in
Arabidopsis thaliana en Petunia hybrida
Aangezien de wereldbevolking alsmaar toeneemt, is het van belang om de voedselproductie
op peil te houden. Naast verbeterde bemestingstechnieken en teeltmethoden, speelt ook
plantenbiotechnologie een grote rol in de vergroting van de gewassenopbrengst.
Door slechte irrigatiemethoden, overstromingen en woestijnvorming wordt de
landbouwgrond steeds zouter en dit is erg nadelig voor het verbouwen van gewassen. Het
verwijderen van zout uit de bodem is erg duur en daarom is het van economisch belang om
oplossingen te vinden die er voor kunnen zorgen dat gewassen in verzilte gebieden kunnen
groeien. Op dit moment worden twee technieken gebruikt om zouttolerantie in gewassen te
vergroten: 1) directe selectie van bestaande variëteiten die al zouttolerant zijn en deze
tolerantie door middel van kruisen over te brengen in landbouwgewassen en 2) de generatie
van transgene planten door nieuwe genen te introduceren of het expressieniveau te wijzigen
van genen die een rol spelen in zoutstress tolerantie.
Het aanvankelijke doel van dit PhD onderzoeksproject was het onderzoeken van de rol van
de GSK3 kinase GSK1 in zoutstress signalering in de modelplant Arabidopsis thaliana. In
het genoom van Arabidopsis zijn 10 GSK3 kinasen geïdentificeerd die op basis van hun
fylogenie in 4 subgroepen kunnen worden verdeeld. BIN2 is de best bestudeerde GSK3
kinase en speelt een negatieve rol in de brassinosteroïden (BR) signaal transductie.
Op basis van voorafgaand onderzoek door een Koreaanse onderzoeksgroep kregen wij het
idee dat het uitschakelen van het GSK1 gen zou resulteren in een zoutgevoelige plant.
Aangezien BIN2 en een andere GSK3 kinase ASKζ heel erg veel op GSK1 lijken, zou dit
ook voor hun kunnen gelden. In hoofdstuk 2 hebben we bekeken of mutaties van deze
genen invloed hadden op de zouttolerantie in Arabidopsis. In tegenstelling tot het
Koreaanse onderzoek vonden wij geen rol voor GSK1 in zoutstress tolerantie en ook niet
voor de andere GSK3 subgroep II leden BIN2 en ASKζ. Recente onderzoeksresultaten van
andere onderzoekers suggereren dat niet alleen subgroep II betrokken is in zouttolerantie,
maar ook andere subgroepen. Daarnaast lijkt het er ook sterk op dat GSK3 kinasen in plaats
van een positieve rol juist een negatieve rol spelen in zoutstress signalering. Een vraag die
nog beantwoord dient te worden is of GSK3 kinasen direct de zout tolerantie reguleren of
indirect via de BR signaal transductie cascade waar GSK3 kinases ook een belangrijke rol
spelen.
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Samenvatting
BRs zijn steroïde hormonen die van essentieel belang zijn voor de groei en ontwikkeling
van een plant. Hoofdstuk 1 geeft een samenvatting weer over de huidige kennis omtrent
BR biosynthese, BR signalering en de rol van BRs in verschillende groei en ontwikkelingsprocessen zoals celdeling en celstrekking, vaatbundel ontwikkeling, abiotische en biotische
stress, fotomorfogenese, kieming, veroudering en vruchtbaarheid.
BIN2 reguleert op een negatieve wijze de BR signaal transductie cascade door de
transcriptiefactoren BES1 en BZR1 te fosforyleren. In hoofdstuk 2 laten wij zien dat GSK1
zowel als ASKζ ook een interactie kunnen aangaan met leden van de BES1/BZR1
transcriptiefactor familie. Dat BIN2, GSK1 en ASKζ een mutuele rol spelen in BR
signalering wordt verder bevestigd door een onderzoek aan verschillende soorten mutanten.
Om meer te weten te komen over de functie van GSK1 en de andere groep II GSK3 kinasen
in BR signalering en mogelijk in andere signaal transductie ketens hebben we groep II
GSK3 kinasen gemerkt met verschillende Tandem Affinity Purification (TAP) tags in
Arabidopsis en getracht bindingspartners te isoleren (hoofdstuk 3). Het is ons niet gelukt
om bindingspartners te identificeren, dit vanwege technische complicaties en mogelijk ook
doordat de GSK3 kinasen inactief lijken te zijn door de TAP tag die er aan vast zit.
BR biosynthese en signalering is uitgebreid bestudeerd in Arabidopsis en in enigermate in
plantensoorten zoals tomaat, rijst en roze maagdenpalm. In onze petunia collectie vonden
wij verschillende mutanten families die defect lijken te zijn in de sterol/BR biosynthese of
in de BR signaal transductie keten en dit stimuleerde ons om te onderzoeken of in petunia
de BR biosynthese en BR signaal transductie keten geconserveerd zijn (hoofdstuk 4).
Kruisbestuiving tussen de verschillende mutanten families leverde vijf verschillende
complementatie groepen op. Vier van die groepen bleken sterol/BR biosynthese mutanten
te zijn, omdat ze complementeerden na gesprayd te zijn met de meest actieve BR, 24epibrassinolide. Voor één groep mutanten konden we achterhalen dat het dwerg fenotype
was ontstaan door een dTph1 transposon insertie in het BR biosynthese gen CPD. Eén
groep mutanten herstelde niet na gesprayd te zijn met 24-epibrassinolide en zou dus een BR
signaleringsmutant moeten zijn. Inderdaad, deze groep mutanten bleek een transposon
insertie te hebben in een gen dat homoloog is aan de BR receptor BRI1 uit Arabidopsis.
Met behulp van PCR gebaseerde methoden hebben wij een lid van de BES1/BZR1
transcriptiefactor familie (PhBEH2) en een groep II GSK3 kinase (PSK9) uit petunia weten
te isoleren. In een gist ´two-hybrid´ systeem, een techniek om eiwit-eiwit interacties te
bepalen, vertoonde PhBEH2 interactie met een andere GSK3 kinase (PSK8) en 14-3-3
eiwitten. De vondst van deze eiwitten insinueert dat de BR signaal transductie route
geconserveerd is in het plantenrijk en dit wordt verder bekrachtigd door het feit dat PSK8
van petunia in gist een eiwit-eiwit interactie kan aangaan met leden van de BES1/BZR1
familie uit Arabidopsis en BIN2 van Arabidopsis met PhBEH2 van petunia.
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Samenvatting
PhBEH2 ging ook interacties aan met eiwitten die betrokken zijn in bijvoorbeeld
gibberelline, auxine en abscisinezuur signalering. Deze interacties zijn nog niet eerder
gevonden en mogelijk dat de BR signaleringsroute en andere (hormoon) signaal transductie
ketens met elkaar kunnen communiceren via deze eiwit-eiwit interacties.
Dit proefschrift levert informatie op over BR biosynthese en signalering in Arabidopsis en
petunia. BR bevattende oplossingen worden al een tijd gebruikt in de horticultuur en
agricultuur om de opbrengst en kwaliteit van gewassen te verhogen. Met behulp van alle
kennis die is en wordt opgedaan omtrent de BR biosynthese- en signaleringsroute, kan in de
toekomst de BR route zodanig genetisch gemanipuleerd worden dat dit kan leiden tot de
komst van superieure gewassen met een juist formaat, een hoge productie en kwaliteit.
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Dankbetuiging
Vanzelfsprekend zou dit proefschrift niet tot stand zijn gekomen zonder de bijdrage en
steun van velen en hiervoor wil ik iedereen van harte bedanken, maar een aantal mensen
toch nog even in het speciaal.
Mijn dank gaat allereerst uit naar mijn co-promotor Erik en promotor Ronald die mij in de
gelegenheid hebben gesteld om het onderzoek te verrichten dat tot dit proefschrift leidde.
Erik, omdat we samen aan het project werkten waren we echt op elkaar aangewezen.
Gedurende die periode heb ik veel van je geleerd. Je hebt me in mijn werk veel vrijheid en
vertrouwen gegeven, waardoor ik de gelegenheid heb gehad om me goed te ontplooien.
Daarnaast heb ik veel waardering voor de opbouwende kritiek die je me gaf tijdens het
schrijven van mijn proefschrift. Zonder jou was dit proefschrift nooit zo geworden zoals het
nu is.
Alhoewel mijn naaste collega´s niet direct betrokken waren bij het onderzoek, hebben zij
altijd voor me klaar gestaan om me bijvoorbeeld nieuwe technieken aan te leren of me raad
te geven. Dus Jan, Toon, Francesca, Tarcies , Xana, Walter, Elske, Rob, Tijs, Kees ,Ilja etc.
heel erg bedankt voor alles! Voor de goede zorg van mijn plantjes in de kas wil ik Maartje,
Daisy, Pieter en Martina bedanken. En Joke, ik kwam maar al te graag bij je op het
kantoortje langs om effe lekker te beppen. Bedankt voor alle administratieve rompslomp
waar je me bij hebt geholpen en bij het voorbereiden van mijn promotiefeest. Natuurlijk wil
ik ook nog alle MolMic en H0 mensen bedanken voor de hulp en gezelligheid. Henk, wil ik
in het bijzonder bedanken voor zijn hulp met betrekking tot de zoutstressexperimenten en
voor het gebruik maken van je klimaatkamers. Ik ben zeer vereerd dat je deel uitmaakt van
mijn promotiecommissie.
Tijdens mijn VU tijd heb ik ook studenten mogen begeleiden. Over het algemeen pakte dit
positief uit. Annegret, many thanks for your good work. I wish you all the luck with your
PhD. Allan, bedankt voor het deelnemen aan het petuniaonderzoek. Jammer, dat je niet
door bent gegaan voor je Master. Ik wens je heel veel succes toe met je opleiding tot piloot.
Katja, destijds de stagiaire van Jan, wil ik bedanken voor de gezellige tijd op het lab. Heel
veel succes met je AIO!
Mies, ik had nooit gedacht dat ik zo’n goede vriend zou tegenkomen op mijn werk. Ik kan
niet alleen verschrikkelijk met je lachen, maar ook zoveel met je delen. Ook al spreek en
zie ik je nog regelmatig, ik mis ons snackie van de week, de foute plaatjes die we draaiden
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Dankbetuiging
en onze dagelijkse onderonsjes. Als ik het even niet meer zag zitten, wist je me altijd op te
beuren. Ik ben ingenomen met het feit, dat je als paranimf aan mijn zijde zult staan.
Een andere collega moet ik ook niet vergeten. Lieve Bets, ik heb me van begin af aan altijd
op mijn gemak gevoeld bij jou. Ik vond het heerlijk om met jou op het lab te werken en
lekker tussendoor te babbelen over onze poezenbeesten en natuurlijk niet te vergeten
Expeditie Robinson. Ik vind het een hele grote eer dat jij mijn paranimf wilt zijn.
Als ik terugdenk aan de vier jaren op de VU roept dit warme herinneringen op. Ik denk dan
terug aan de sinterklaasavondjes, kerstdiners, het heerlijke eten van Francesca tijdens de
vele meetings, het labtuinieren van Tarcies, de fietspuzzeltocht en niet te vergeten alle fijne
kletspraatjes die ik op het lab, in de wandelgangen, koffiekamer, kas tot in de kelders van
de VU heb gehad.
Ook buiten de VU om heb ik veel steun gehad tijdens het schrijven van mijn proefschrift.
Sylvia, ik wil jou in het bijzonder bedanken voor jouw hulp bij het werken met Photoshop
en Illustrator. Als ik jou niet had gehad, was ik nu nog bezig geweest! Vrienden en
collega´s van NSure, bedankt voor jullie belangstelling en luisterend oor, maar vooral ook
voor jullie gezelligheid waar ik mijn energie uit kon putten.
Roland, ik wil je bedanken voor je steun, geduld en begrip. Je interesse lag niet zo zeer bij
de aard van mijn werk, maar wel hoe het met je grote zus ging. Dit was heel belangrijk voor
mij.
Pap en mam, jullie onvoorwaardelijke steun en liefde heeft er voor een heel groot deel aan
bijgedragen dat dit proefschrift nu af is. Dankzij jullie bleef ik met beide benen op de grond
staan en heb ik me kunnen ontwikkelen tot de persoon die ik nu ben. Ik draag dit
proefschrift dan ook graag aan jullie op.
Ten slotte, wil ik mijn opa en oma bedanken voor hun morele en financiële steun. Wat fijn
zou het zijn geweest als ze mijn promotie nog hadden mogen meemaken.
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