Bindingaffinitiesandinteractionsamongdifferent heat
shock elementtypesandheatshockfactorsin rice
(Oryza sativa L.)
Dheeraj Mittal
1
, Yasuaki Enoki
2
, Dhruv Lavania
1
, Amanjot Singh
1
, Hiroshi Sakurai
2
and
Anil Grover
1
1 Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India
2 Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Japan
Keywords
heat shock; heatshock element; heat shock
protein; heatshock transcription factor; rice
(Oryza sativa)
Correspondence
A. Grover, Department of Plant Molecular
Biology, University of Delhi South Campus,
New Delhi 110021, India
Fax: +91-11-24115270
Tel: +91-11-24117693/24115097
E-mail: anil.anilgrover@gmail.com
(Received 9 May 2011, revised 27 June
2011, accepted 29 June 2011)
doi:10.1111/j.1742-4658.2011.08229.x
Binding of heatshockfactors (Hsfs) to heatshock elements (HSEs) leads to
transcriptional regulation of heatshock genes. Genome-wide, 953 rice genes
contain perfect-type, 695 genes gap-type and 1584 genes step-type HSE
sequences in their 1-kb promoter region. The rice genome contains 13 class
A, eight class B and four class C Hsfs (OsHsfs) and has OsHsf26 (which is
of variant type) genes. Chemical cross-linking analysis of in vitro synthe-
sized OsHsf polypeptides showed formation of homotrimers of OsHsfA2c,
OsHsfA9 and OsHsfB4b proteins. Binding analysis of polypeptides with oli-
gonucleotide probes containing perfect-, gap-, and step-type HSE sequences
showed that OsHsfA2c, OsHsfA9 and OsHsfB4b differentially recognize
various model HSEs as a function of varying reaction temperatures. The
homomeric form of OsHsfA2c and OsHsfB4b proteins was further noted by
the bimolecular fluorescence complementation approach in onion epidermal
cells. In yeast two-hybrid assays, OsHsfB4b showed homomeric interaction
as well as distinct heteromeric interactions with OsHsfA2a, OsHsfA7, OsH-
sfB4c and OsHsf26. Transactivation activity was noted in OsHsfA2c, OsH-
sfA2d, OsHsfA9, OsHsfC1a and OsHsfC1b in yeast cells. These differential
patterns pertaining to binding with HSEs and protein–protein interactions
may have a bearing on the cellular functioning of OsHsfs under a range of
different physiological and environmental conditions.
Structured digital abstract
l
HSFA2C binds to HSFA2C by cross-linking study (View interaction)
l
HSFA2C physically interacts with HSFA2C by bimolecular fluorescence complementation (View
interaction)
l
HSFB4B physically interacts with HSFB4B by bimolecular fluorescence complementation
(View interaction)
l
HSFA2A physically interacts with HSFB4B by two hybrid (View interaction)
l
HSFB4B binds to HSFB4B by cross-linking study (View interaction)
l
HSFB4B physically interacts with HSF26 by two hybrid (View interaction)
l
HSFA9 binds to HSFA9 by cross-linking study (View interaction)
l
HSFA7 physically interacts with HSFB4B by two hybrid (View interaction)
l
HSFB4B physically interacts with HSFB4C by two hybrid (View interaction)
l
HSFB4B physically interacts with HSFB4B by two hybrid (View interaction)
Abbreviations
3-AT, 3-amino-1,2,4-triazole; BiFC, bimolecular fluorescence complementation; EGS, ethylglycol bis(succinimidylsuccinate); EMSA,
electrophoretic mobility shift assay; HS, heat shock; HSE, heatshock element; Hsf, heatshock transcription factor; Hsp, heatshock protein.
3076 FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS
Introduction
The synthesis of heatshock proteins (Hsps) represents
one of the most thoroughly studied induced gene
expression systems. Hsp genes are primarily regulated
by heat stress, metal stress and developmental cues
[1,2]. Hsp transcripts increase massively following heat
shock (HS), indicating that expression of Hsps is pro-
foundly regulated at the transcriptional level. The abil-
ity of HS promoters to sense and respond to heat is
mainly due to the presence of consensus sequences
called heatshock elements (HSEs). The eukaryotic
HSE consensus sequence has been defined by altering
units of 5¢-nGAAn-3¢. HSEs are separated into three
types: perfect (P), gap (G) and step (S) [3]. P-type
HSEs have three inverted repeats in a contiguous array
(nTTCnnGAAnnTTC). G-type HSEs have two consec-
utive inverted sequences, with the third sequence sepa-
rated by 5 bp (nTTCnnGAAn(5 bp)nGAAn). S-type
HSEs have 5-bp gaps separating all three modules
(nTTCn(5 bp)nTTCn(5 bp)nTTCn). In plants, the
importance of HSEs for heat-dependent transcriptional
regulation is reflected from experiments on promoter
deletions and by the capacity of a synthetic HSE
sequence integrated in a truncated CaMV35S promoter
to stimulate heat-inducible reporter gene expression in
transgenic tobacco plants [4].
Heat shock transcription factors (Hsfs) bind with
HSEs, eventually resulting in transcriptional activation
of HS genes. Hsfs have been characterized from various
plant species [5–15]. Plant Hsf circuitry appears more
complex than in yeast or animal systems, as Arabidop-
sis, riceand tomato contain over 20 Hsf genes [5,16].
Hsfs have a core structure comprising an N-terminal
DNA binding domain, an adjacent oligomerization
domain with heptad hydrophobic repeats (HR-A ⁄ B),
signal sequences for nuclear localization and export and
a C-terminal AHA type activation domain [5]. Based on
the sequence homology and domain structure, plant
Hsfs have been subdivided into three classes, A, B and C
[7]. Hsfs are differentially expressed in response to vari-
ous abiotic stresses, in a tissue- and stage-specific man-
ner [16,17]. It is suggested that the ‘early’ constitutively
expressed Hsf genes become activated immediately upon
HS and function as the primary regulators in the cell,
while the expression of ‘late’ Hsf genes is enhanced sig-
nificantly following HS by early Hsfs [18].
The activation of Hsfs occurs in two stages: (a) the
induction of high affinity binding to HS promoters
accomplished by trimerization and cooperative interac-
tions between Hsf trimers and (b) the exposure of one
or more dedicated activator domains [19]. Further,
divergence of HSE architecture influences gene- and
stress-specific responses: it is suggested that HSE archi-
tecture is an important determinant of which Hsf mem-
bers are recruited and provides enormous functional
diversity in transcriptional regulation of target genes
[19,20]. Plant Hsfs can potentially form homo- or het-
ero-oligomers resulting in altered nuclear localization
as well as enhanced or suppressed transcription [21]. In
tomato, constitutively expressed HsfA1 and HS-induc-
ible HsfA2 have been shown to form hetero-oligomers
for nuclear transport [22], and the hetero-oligomers
synergistically induce HS response of genes [23]. In
Arabidopsis, homomeric interactions for HsfA1a and
HsfA1b as well as heteromeric interaction between
HsfA1a and HsfA1b were noted [6]. Both HsfA1a and
HsfA1b also make heteromeric interactions with
HsfA2; however, synergistic transactivation ability is
not observed in the heteromers [13]. In contrast to class
A Hsfs, class B HsfB1 and HsfB2b proteins showed ho-
momeric interactions but did not interact with each
other [6]. The homo-oligomer of Arabidopsis HsfA4
acts as an activator of heat stress, whereas HsfA5
forms hetero-oligomers with HsfA4, thereby interfering
in the HsfA4 DNA binding capacity and thus acting as
a selective repressor [24]. In addition to Hsf–Hsf inter-
actions, a large body of information has been accumu-
lated on the importance of HSE structure for the
differential induction of Hsp genes [9,25–29].
Rice has 25 Hsf genes [30]. In addition, there is one
more entry for OsHsf, namely OsHsf26 (LOC_
Os06g22610), which is a predicted 190 amino acid pro-
tein and contains an oligomerization domain but lacks
the DNA binding domain, signal sequences for nuclear
localization and export and the AHA motif [16].
Considering this entry also as Hsf (though a variant
type), it is proposed that overall there are 26 genes
encoding for the OsHsf family. Taking cues from the
information available from Hsf–HSE systems of other
organisms, it is presumed that 26 rice Hsfs may have
different specificities for HSEs. In this study, we pro-
vide detailed genome-wide analysis of rice HSE types.
We further provide data on protein–protein interac-
tions, DNA binding characteristics and transactivation
activity of selected members of class A, B and C OsHsf
proteins.
Results
Genome-wide distribution of HSEs in rice
The rice genome database was searched for genes that
show HSE-like configurations (as shown in the upper
D. Mittal et al. Heatshockelementtypesandheatshockfactorsin rice
FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS 3077
panel in Fig. 1A) in their respective 1-kb upstream
promoter region (taking A of ATG as +1). These
genes were grouped on the basis of the presence of
the specific HSE types, irrespective of the number of
hits per sequence and the strand of a gene. In totality,
953 genes contained P-type, 695 genes G-type and
1584 genes S-type HSEs in the rice genome (Fig. 1A).
In total 711, 476 and 1368 genes contained exclusively
the P-, G- and S-type HSEs, respectively; 59 genes
showed both P- and G-type HSEs, 56 genes a combi-
nation of P- and S-type HSEs, while G- and S-type
HSEs were noted together in 33 genes. Also, 127
genes showed all three types of HSEs (P, G and S) in
their promoter region. Overall, a higher abundance of
S-type HSE was noted compared with P- and G-type
HSEs.
Employing HS-induced microarray analysis of rice
transcripts (unpublished data; also see [2]), we next
analyzed how many of the HS induced rice genes
from the microarray profiling contain the above HSE
configurations. A total of 880 genes showed HS
induced transcript profiling (Fig. 1B). Notably, 143 of
the 880 HS induced genes contained HSEs in their
1-kb upstream promoter sequences. Out of 143 HS
genes, only 22 genes represented the annotated
Hsf ⁄ Hsp types. Thus a subset of the HS induced genes
( 16%) contains the typical P-, G- or S-type HSE
configurations in their promoters.
In vitro analysis of homo-oligomerization
potential of OsHsfs andbinding of OsHsfs
with HSEs
OsHsfA2c, OsHsfA7, OsHsfA9, OsHsfB4b, OsHsfB4c
and OsHsfC1b polypeptides were synthesized by in vi-
tro transcription ⁄ translation reactions (Fig. 2A). When
analyzed for their capacity to undergo homo-oligomer-
ization by chemical crosslinking, OsHsfA2c, OsHsfA9
and OsHsfB4b showed homotrimer formation activity
(shown by black circles) from their respective mono-
meric forms (shown by white circles) (Fig. 2B). The
formation of trimers in these cases was noted at three
different reaction temperatures tested, i.e. 22, 32 and
37 °C. In the case of OsHsfB4b, trimerization was
noted at 22 and 32 °C but was barely visible at 37 °C.
In contrast, OsHsfA7, OsHsfB4c and OsHsfC1b did
not efficiently form homotrimers in the conditions
tested.
Electrophoretic mobility shift assay (EMSA) was
carried out to assess the DNA binding abilities of these
OsHsfs (Fig. 3). First, the EMSA was carried out
using
32
P-labeled model 3P-type HSE (Fig. 3A) as a
function of four different incubation temperatures, i.e.
12, 22, 32 and 37 °C (Fig. 3B). OsHsfA2c, OsHsfA9
and OsHsfB4b polypeptides formed protein–DNA
complexes. Binding activity was low at 12 °C and was
enhanced with increasing temperature; OsHsfA2c
showed maximum binding at 32 and 37 °C; OsHsfA9
showed efficient binding at 22, 32 and 37 °C; and
OsHsfB4b showed maximum binding at 22 °C. In con-
trast, the DNA affinity of OsHsfA7, OsHsfB4c and
OsHsfC1b polypeptides was not significant. Second,
the EMSA was carried out at 22 °C employing four
different HSE oligonucleotides, i.e. 4P-, 3P-, G- and
S-types (Fig. 3C). OsHsfA2c showed high affinity
binding to 4P- and 3P-type HSEs and low affinity
binding with G- and S-type HSEs. A slowly migrating
OsHsfA2c–HSE4P complex was noted: this complex
may contain two OsHsfA2c trimers, because coopera-
tive binding of two Hsf trimers to 4P-type HSE is
observed in yeast Hsf1, Drosophila Hsf and human
P-type: nTTCnnGAAnnTTC
G-type: nTTCnnGAAn(5 bp)nGAAn
S-type: nTTCn(5 bp)nTTCn(5 bp)nTTCn
A
0
400
800
1200
1600
B
0
200
400
600
800
1000
HS inducible genes Genes with HSEs
P
G
S
P + G
P + S
G + S
P + G + S
P*
G*
S*
Fig. 1. Genome-wide distribution of HSEs in rice. (A) Frequency
analysis of perfect-type (P-type), gap-type (G-type) and step-type
(S-type) HSEs in the 1-kb promoter region of rice genes. P, G and
S indicate classes of genes which contained P-type, G-type or
S-type in any combination, in totality. P*, G* and S* indicate clas-
ses of genes showing exclusively P-type, G-type and S-type HSEs,
respectively. (B) Analysis of genes showing HSEs out of the total
genes showing HS-inducible expression.
Heat shockelementtypesandheatshockfactorsinrice D. Mittal et al.
3078 FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS
Hsf1 [19]. OsHsfA9 showed binding to 4P- and 3P-
type HSEs but not to G- and S-type HSEs. OsHsfB4b
showed binding to 4P- and 3P-type HSEs with much
higher affinity than the binding for G-type HSE.
Therefore, three OsHsfs differentially recognize various
model HSEs in vitro.
Transactivation activity of OsHsfs in yeast cells
To test the activator potential of OsHsfs, fusions of
the Gal4 DNA binding domain and OsHsfs were
expressed in yeast cells containing a Gal4-regulated
His3 reporter construct [31]. Among five class A OsH-
sfs (OsHsfA2a, OsHsfA2c, OsHsfA2d, OsHsfA7 and
OsHsfA9) Gal4 fusions of OsHsfA2c, OsHsfA2d and
OsHsfA9 supported yeast cell growth on medium con-
taining 3-amino-1,2,4-triazole (3-AT), a competitive
inhibitor of His3 protein, which suggests that these
proteins function as activators in yeast cells (Fig. 4). In
class B and class C members, OsHsfC1a and OsH-
sfC1b showed transactivation capacity on medium
containing 5 m
M 3-AT. OsHsf26, a variant type of
OsHsf, lacked transactivation activity.
Interactions among OsHsfs in yeast and onion
epidermal cells
Selected OsHsfs were analyzed for their possible ho-
momeric and heteromeric interactions, using yeast
two-hybrid assays (Fig. 5). In this assay, OsHsfs fused
to the Gal4 DNA binding domain and activation
domain were expressed in yeast cells containing a
Gal4-regulated lacZ reporter construct, and the inter-
actions were scored based on b-galactosidase activity.
OsHsfA2a, OsHsfA7, OsHsfB4b, OsHsfB4c and OsH-
sf26 were tested for homomeric and heteromeric
interactions. Of these, OsHsfB4b showed a clear
homomeric interaction. OsHsfB4b also showed hetero-
meric interactions with OsHsfA7, OsHsfA2a, OsH-
sfB4c and OsHsf26 proteins.
The bimolecular fluorescence complementation
(BiFC) technique has provided support in indicating
that Hsfs show protein–protein interactions. This tech-
nique has also been used for visualization of the subcel-
lular locations of the interacting proteins in the cell
[6,32]. Subsequently, OsHsfA2c and OsHsfB4b were
analyzed for their potential to form homomers by the
Temp (
o
C)
EGS
HsfA9
22 32 37
HsfA7
22 32 37
HsfC1b
22 32 37
HsfB4c
22 32 37
HsfB4b
22 32 37
HsfA2c
22 32 37
70
20
35
50
25
A7
A2c
A9
B4c
B4b
C1b
100
70
140
35
50
25
100
240
A
B
Fig. 2. Homo-oligomerization activity of in vitro synthesized OsHsf polypeptides. (A)
35
S-labeled OsHsfA2c (A2c), OsHsfA7 (A7), OsHsfA9
(A9), OsHsfB4b (B4b), OsHsfB4c (B4c) and OsHsfC1b (C1b) polypeptides were subjected to SDS ⁄ PAGE electrophoresis and phosphor-imag-
ing. Equivalent amounts of polypeptides were electrophoresed, and the different band intensities were due to the different methionine con-
tents. The positions of molecular mass markers are shown on the left in kilodaltons. (B) Labeled polypeptides were incubated at the
indicated temperatures, treated (+) or untreated ()) with 1.0 m
M EGS for 12 min, and subjected to SDS ⁄ PAGE electrophoresis and phos-
phor-imaging. Positions of monomers are indicated by white circles, and lower and higher levels of homotrimers are indicated by gray and
black circles, respectively.
D. Mittal et al. Heatshockelementtypesandheatshockfactorsin rice
FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS 3079
BiFC experiment using onion epidermal cells (Fig. 6).
A positive BiFC reaction was noted for both OsHsfA2c
and OsHsfB4b proteins, indicating that these proteins
interact to produce active reporter yellow fluorescent
protein. As the BiFC reaction was clearly noted in
nuclei in both cases, it is apparent that homomeric
forms of these proteins are localized in nuclei.
Discussion
This study noted that in the rice genome with an esti-
mated size of 67 393 genes, 2830 genes contain at least
one of the three HSE-type configurations in their 1-kb
upstream promoter region. Overall, 953 genes contain
P-type, 695 genes G-type and 1584 genes S-type HSEs.
pBD-GAL4-OsHsfA2a
pBD-GAL4-OsHsfA2c
p
BD-GAL4-OsHsfA2d
pBD-GAL4-OsHsfA7
pBD-GAL4-OsHsfA9
pBD-GAL4-OsHsfB4b
pBD-GAL4-OsHsfB4c
pBD-GAL4-OsHsfC1a
p
BD-GAL4-OsHsfC1b
pBD-GAL4-OsHsfC2a
pBD-GAL4-OsHsf26
pBD-GAL4
SD-WH SD-WH +
1 m
M 3-AT
SD-WH +
5 m
M 3-AT
SD-W
Fig. 4. Transactivation activity of OsHsfs.
Auxotrophic growth assay on SD-W (syn-
thetically defined tryptophan dropout med-
ium), SD-WH (synthetically defined
tryptophan and histidine dropout medium)
and SD-WH + 1 m
M 3-AT and WH + 5 mM
3-AT (synthetically defined tryptophan and
histidine dropout media with 1 and 5 m
M of
3-AT). The lane with pBD-GAL4 represents a
negative control.
Temp (
o
C)
Effect of Temperature (probe, HSE3P)
HsfA9
12 22 32 37
HsfA7
12 22 32 37
HsfB4b
12 22 32 37
HsfA2c
12 22 32 37
HsfA9
4P 3P G S
HSE Specificity (at 22
o
C)
HsfA2c
4P 3P G S
HsfB4b
4P 3P G S
HsfA7
4P 3P G S
Probe
HsfC1b
12 22 32 37
HsfB4c
12 22 32 37
HsfB4c
4P 3P G S
HsfC1b
4P 3P G S
B
C
3P HSE-probe:
4P HSE-probe:
S HSE-probe:
G HSE-probe:
tcgacTTCtaGAAgcTTCcaGAAattagtgctactcga
A
tcgacTTCtaGAAgcTTCcactaattagtgctactcga
tcgacTTCtaGAAgctagcaGAAattagtgctactcga
tcgacTTCtactagcTTCcactaatTTCtgctactcga
Fig. 3. Binding assay of in vitro synthesized
OsHsf polypeptides with HSEs. (A) Nucleo-
tide sequences of the four HSE oligonucleo-
tides are shown. ‘GAA’ and inverted ‘TTC’
sequences are shown by bold uppercase
letters. (B) Binding of OsHsfs with 3P-type
HSE was analyzed at various temperatures.
Unlabeled polypeptides were incubated with
32
P-labeled 3P-type HSE at the indicated
temperatures and subjected to PAGE elec-
trophoresis and phosphor-imaging. White
and black arrowheads indicate positions of
unbound DNA fragments and protein–DNA
complexes, respectively. (C) Binding of
OsHsfs with various HSE types was ana-
lyzed at 22 °C. Unlabeled polypeptides were
incubated with
32
P-labeled 4P-, 3P-, G- and
S-type HSEs at 22 °C and subjected to
PAGE electrophoresis and phosphor-imag-
ing. Double arrowheads show a complex
containing two OsHsfA2c trimers and 4P-
type HSE.
Heat shockelementtypesandheatshockfactorsinrice D. Mittal et al.
3080 FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS
This study further shows that, as only 16% of HS
induced genes contain the canonical HSE types, a
major population of the HS induced genes are not
associated with canonical HSE types. The order of
importance of the bases in the nGAAn repeat of HSE
is G
2
>A
3
>A
4
, and some deviations from the
canonical HSE types are tolerated in functional HSEs
[19]. In yeast, some of the HS induced genes contain
HSE-like sequences slightly diverged from nGAAn
[33]. Nonetheless, it appears that cis-acting sequences
different from the typical HSEs may also be playing a
role in HS inducibility. HsfA1a of Arabidopsis has
been shown to bind TT-rich sequence and stress
responsive elements, in addition to P- and G-type
HSEs [34]. Recent observations showed that a novel
9-bp AZC (
L-azetidine-2-carboxylic acid) responsive
element works as an alternative Hsf binding sequence
in rice [35]. It is a possibility that rice genes that are HS
inducible and do not have canonical HSEs may harbor
such or other novel cis elements. There are reports
suggesting that HSEs are present on non-HS-inducible
genes as well [27,35,36]. Similar to these observations,
we also noted that 2687 rice genes, which are not HS
inducible as per our microarray results, have HSEs in
their promoter region. Put together, these observations
highlight the inadequacies in our current understanding
of the relevance of HSEs in HS response in rice.
The formation of the trimeric form of Hsfs is con-
sidered important for attaining their high affinity bind-
ing to HSEs [19]. Using in vitro crosslinking and yeast
two-hybrid assays, we noted that OsHsfA2c, OsHsfA9
and OsHsfB4b form homomers. However, homomer
formation activity was lacking for OsHsfA2a, OsH-
sfA7, OsHsfB4c, OsHsfC1b and OsHsf26 proteins.
BiFC analysis showed that OsHsfA2c and OsHsfB4b
form a homomeric state and further showed that
homomeric OsHsfA2c and OsHsfB4b forms are clearly
localized in the nucleus. In vitro, homotrimerized OsH-
sfA2c, OsHsfA9 and OsHsfB4b bound to 3P-type
HSE. OsHsfB4b showed maximum trimerization and
DNA binding activities at lower temperature than
0
0.5
1
1.5
2
2.5
3
3.5
4
Miller units
OsOsHsfA7 + A2a
OsHsfA7 + B4b
OsHsfA7 + B4c
OsHsfA7 + 26*
OsHsfA7 + pAD
OsHsf
A2a + pAD
OsHsfB4b + pAD
OsHsfB4c + B4c
OsHsfB4c + 26*
OsHsfB4c + pAD
OsHsf26* +pAD
OsHsf26* + 26*
PC
NC
OsHsfB4b + B4b
OsHsfB4b + B4c
OsHsfB4b + 26*
OsHsf A2a + A2a
OsHsf A2a + B4b
OsHsf A2a + B4c
OsHsf
A2a + 26*
OsOsHsfA7 + A7
Fig. 5. Interactionsamong OsHsfs in yeast
two-hybrid assays. Yeast two-hybrid assays
showing b-galactosidase activity in YRG2
yeast cells transformed with different con-
structs. PC, positive control (pSE1111-
ScSNF1 and pSE1112-ScSNF4 transformed
YRG2 strain to yield YRG2-pSE1111-
ScSNF1+pSE1112-ScSNF4 cells); NC, nega-
tive control (pAD + pBD vector transformed
YRG2 cells). Respective OsHsfs cloned in
pBD + pAD transformed in YRG2 cells were
also used as a negative control.
OsHsfA2c-YFPN
35S
OsHsfA2c
YFPN
NosT
OsHsfA2c-YFPC
35S
OsHsfA2c
YFPC
NosT
OsHsfB4b-YFPN
35S
OsHsfB4b
YFPN
NosT
OsHsfB4b-YFPC
35S
OsHsfB4b
YFPC
NosT
A
B
Fig. 6. Analysis for homomeric protein–protein interactions and
subcellular localization of OsHsfA2c and OsHsfB4b by the BiFC
approach. (A) Details of the OsHsfA2c–YFPN + OsHsfA2c–YFPC
fusion construct are shown in the upper panel. Onion epidermal
cells transformed with OsHsfA2c–YFPN+OsHsfA2c–YFPC fusion
construct are shown in the lower panel. (B) Details of the OsH-
sfB4b–YFPN + OsHsfB4b–YFPC fusion construct are shown in the
upper panel. Onion epidermal cells transformed with the OsH-
sfB4b–YFPN + OsHsfB4b–YFPC fusion construct are shown in the
lower panel. In both (A) and (B), panels in the middle display bright
field images while the right panel shows a merged image. NosT
refers to nopaline synthase transcription termination signal.
D. Mittal et al. Heatshockelementtypesandheatshockfactorsin rice
FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS 3081
maximum activities noted in OsHsfA2c and OsHsfA9.
This study reflects the first case of plant Hsfs showing
that trimerization and DNA binding activities in dif-
ferent members is temperature-dependent to differen-
tial extents. The trimerization and DNA binding
activities at permissive and cooler temperatures (Figs 2
and 3) indicate the possible role of OsHsfs in
unstressed control conditions. It remains to be seen
what relevance this temperature-dependent pattern
under in vitro conditions has in terms of in vivo physio-
logical conditions. When various model HSE types
were employed in the EMSA, OsHsfA2c showed low-
affinity binding to G- and S-type HSEs; however,
OsHsfA9 and OsHsfB4b showed no or little binding to
G- and S-type HSEs. Although S-type HSEs appear
most prevalent in the promoter region in the rice gen-
ome, binding with S-type HSE under in vitro condi-
tions is of low affinity with the select group of OsHsfs
tested herein. Because lower affinity sites contribute in
the binding of transcription factorsand gene regula-
tion [37], the low affinity binding noted in this study
may be relevant for the in vivo functioning of OsHsfs.
It was postulated that the property of transactivator
function resides in the AHA motifs present at the
C-terminus [31]. Class A OsHsfs have AHA while class
B and C Hsfs lack these motifs [7]. In rice, transactiva-
tion activity has previously been reported for
OsHsfA2e protein [10]. We noted that three class A
proteins (OsHsfA2c, OsHsfA2d and OsHsfA9) show
transactivation activity; however, two class A members
(OsHsfA2a and OsHsfA7) containing AHA motifs lack
activity. Class B OsHsfB4b and OsHsfB4c proteins
lacked transactivation activity. Of the OsHsfC1a,
OsHsfC1b and OsHsfC2a class C members tested,
OsHsfC1a and OsHsfC1b showed transactivation
activity that appeared comparable with class A mem-
bers in extent. Weak transactivation potential has also
been noted in Arabidopsis class C Hsf [31]. It thus
seems that elements apart from AHA motifs can make
a contribution to transactivation activity. Phosphoryla-
tion is implicated in the activation and inactivation of
transactivation potential of human HSF1 [38]. We note
multiple putative phosphorylation sites in OsHsfs
(Table S1). However, involvement of phosphorylation
reaction at these sites remains to be established in
response to HS conditions.
We have earlier shown that OsHsf26 has an oligo-
merization domain but lacks the domain for DNA
binding activity [16]. The OsHsf26 transcript is
expressed under complex stress combinations involving
high and low temperatures coupled with oxidative
stress inrice seedlings (D. Mittal and A. Grover,
unpublished data). In this study, we have shown that
OsHsf26 interacts with OsHsfB4b. It is possible that
OsHsf26 works as a competitive inhibitor of OsHsfB4b
oligomerization, which results in inhibition of DNA
binding of OsHsfB4b. Thus, the variant OsHsf26 form
may have regulatory roles controlling downstream
gene expression via non-functional hetero-oligomeriza-
tion with OsHsfs.
From the above account, we note that OsHsfB4b is
predominantly involved inrice HS response. OsHsfB4b
forms homomeric interactions to form a trimeric state
and makes heteromeric interactions with various OsH-
sfs. We have recently noted that OsHsfB4b binds to
OsHsfA2c and OsClpB-cyt ⁄ Hsp100 protein (A. Singh,
D. Mittal, D. Lavania, M. Agarwal, R. C. Mishra and
A. Grover, unpublished data). In addition, high tran-
script abundance of OsHsfB4b gene is noted following
heat stress and oxidative stress [16]. OsHsfB4b itself
lacks transactivation ability, implying that OsHsfB4b
homotrimer binds to P- and G-type HSEs and represses
transcription. It is also possible that HSE specificity of
OsHsfB4b changes via hetero-oligomerization with
OsHsfA2a, OsHsfA7 and OsHsfB4c. The homotrimer
of OsHsfA2c may be a potent HS-inducible activator of
genes containing various HSE types (the OsHsfA2c
transcripts increase upon HS [16]). OsHsfA9 homotri-
mer may be involved in activation of genes containing
P-type HSEs; however, its transcripts are relatively low
under normal and stress conditions [16]. In conclusion,
we suggest that these differential patterns may have a
bearing on cellular functioning of OsHsfs under a range
of different physiological and environmental conditions,
which influence synthesis of different target proteins
governed by HSE–Hsf interactions.
Materials and methods
Genome-wide analysis of HSE in the rice genome
The 1-kb upstream regions to the translation start site of
all the rice genes were downloaded from the RGA database
(http://rice.plantbiology.msu.edu/; release 6.1) and analyzed
for the presence of consensus P-, S- and G-type HSE ele-
ments (Fig. 1A). The motif search function of the CLC
Main Workbench 5 (http://www.clcbio.com) was employed
to execute the respective queries in default parameters.
Cloning of rice OsHsf genes
Rice OsHsf genes were cloned from respective KOME
clones (Rice Genome Resource Centre, National Institute
of Agrobiological Sciences, Tsukuba, Japan) using HS
responsive cDNA synthesized in the laboratory. Various
primers used in this study are shown in Table S2.
Heat shockelementtypesandheatshockfactorsinrice D. Mittal et al.
3082 FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS
In vitro polypeptide synthesis, chemical
crosslinking and EMSA
OsHsf cDNA fragments were cloned into the pTNT vector
(Promega, Madison, MA, USA). Using these plasmid
DNAs, OsHsf polypeptides were prepared by in vitro tran-
scription ⁄ translation (TNT coupled reticulocyte lysate sys-
tem with SP6 RNA polymerase; Promega) according to
the manufacturer’s protocol, except that the reaction was
carried out at 22 °C for 2.5 h. The relative amounts of
polypeptides were normalized by the levels of incorporated
[
35
S] methionine and by the methionine contents. Equal
amounts of OsHsf polypeptides were subjected to chemical
crosslinking analysis with ethylglycol bis(succinimidylsucci-
nate) (EGS) and to EMSA with
32
P-labeled oligonucleotide
probes containing 4P-, 3P-, G- and S-type HSE sequences
(see Fig. 3A) as described [39], except that the reaction
mixtures contained 50 m
M NaCl.
Transactivation assay in yeast cells
OsHsf ORFs PCR amplified using gene-specific primer sets
(Table S2) were cloned in the pBD-GAL4 vector (Stratagene
Agilent Technologies, La Jolla, CA, USA) in the EcoRI and
SmaI sites. All PCRs were done using PhusionÔ Hi-Fi DNA
polymerase in the presence of 3% dimethyl sulfoxide. pBD-
GAL4+OsHsfs were introduced into yeast strain YRG2
(MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3
112gal4-542 gal80-538 LYS2::UAS
GAL1
-TATA
GAL1
-HIS3
URA3::UAS
GAL4_17mers_(x3)
-TATA
CYC1
-lacZ) (Stratagene).
Transformants were allowed to grow in SD medium lack-
ing amino acid tryptophan at 28 °C. Different dilutions
were spotted on SD medium lacking amino acids trypto-
phan and histidine. To check leaky expression of HIS3
reporter gene, dilutions were also dotted on the respective
dropout media supplemented with 1 m
M and 5 mM of
3-AT. Each experiment was repeated three times.
Yeast two-hybrid assay
Yeast two-hybrid assays were carried out using pAD-
GAL4 (activation domain fusion, prey) and pBD-GAL4
(binding domain fusion, bait) vectors (Stratagene). For
cloning in pAD-GAL4 vector (Stratagene), OsHsf frag-
ments excised from pBD-GAL4+OsHsfs by EcoRI and
XbaI digestion were cloned in pAD-GAL4 vector in the
sites EcoRI and XbaI. OsHsf-bait and OsHsf-prey pairs
were co-transformed into YRG2 and transformants were
selected on medium lacking leucine and tryptophan.
b-galactosidase activity was measured by the quantitative
liquid culture method using O-nitrophenyl b-
D-galactopyr-
anoside as substrate and by filter lift assay (Yeast Proto-
cols Handbook; Clontech Laboratories Inc., Mountain
View, CA, USA). Each experiment was repeated three
times.
BiFC assays
PCR amplified OsHsfA2c and OsHsfB4b genes were cloned
into BiFC vectors pUC-SPYCE and pUC-SPYNE [32]. For
transient expression in onion epidermal cells, the fusion
proteins with N- or C-terminal parts of yellow fluorescent
protein in pUC-SPYCE and pUC-SPYNE vectors were
introduced into onion epidermal cells by particle bombard-
ment as described previously [2]. After incubation for 16 h,
the cells were visualized by confocal laser scanning micro-
scope (Leica TCS SP5). Yellow fluorescent protein was
excited with an argon laser at 514 nm.
Acknowledgements
DM, DL and AS acknowledge the Council of Scien-
tific and Industrial Research (CSIR), New Delhi, for
the Fellowship award. BiFC vectors pUC-SPYCE and
pUC-SPYNE were kindly provided by F. Schoffl and
C. Oecking, University of Tubingen, Germany. This
work was supported in part by the Indo-Finland pro-
ject grant from the Department of Biotechnology
(DBT), Government of India, to AG and Grants-in-
Aid for Scientific Research from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of
Japan to HS.
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Supporting information
The following supplementary material is available:
Table S1. In silico analysis for the putative phosphory-
lation sites inrice Hsfs.
Table S2. List of primers used in this study.
This supplementary material can be found in the
online version of this article.
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D. Mittal et al. Heatshockelementtypesandheatshockfactorsin rice
FEBS Journal 278 (2011) 3076–3085 ª 2011 The Authors Journal compilation ª 2011 FEBS 3085
. Binding affinities and interactions among different heat
shock element types and heat shock factors in rice
(Oryza sativa L. )
Dheeraj Mittal
1
, Yasuaki. indicated by gray and
black circles, respectively.
D. Mittal et al. Heat shock element types and heat shock factors in rice
FEBS Journal 278 (201 1) 3076–3085