Dheeraj Mittal1, Yasuaki Enoki2, Dhruv Lavania1, Amanjot Singh1, Hiroshi Sakurai2and Anil Grover1 1 Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, In
Trang 1shock element types and heat shock factors in rice
(Oryza sativa L.)
Dheeraj Mittal1, Yasuaki Enoki2, Dhruv Lavania1, Amanjot Singh1, Hiroshi Sakurai2and
Anil Grover1
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; heat shock element; heat shock
protein; heat shock 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 heat shock factors (Hsfs) to heat shock elements (HSEs) leads to transcriptional regulation of heat shock 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, 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, heat shock element; Hsf, heat shock transcription factor; Hsp, heat shock protein.
Trang 2The synthesis of heat shock 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 heat shock 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, rice and 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
Trang 3panel 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 and binding 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
37C In the case of OsHsfB4b, trimerization was noted at 22 and 32C 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 32P-labeled model 3P-type HSE (Fig 3A) as a function of four different incubation temperatures, i.e
12, 22, 32 and 37C (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 37C; 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.
Trang 4Hsf1 [19] OsHsfA9 showed binding to 4P- and
3P-type HSEs but not to G- and S-3P-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 mM 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 B4b B4c 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.
Trang 5BiFC 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
pBD-GAL4-OsHsfA2d
pBD-GAL4-OsHsfA7
pBD-GAL4-OsHsfA9
pBD-GAL4-OsHsfB4b
pBD-GAL4-OsHsfB4c
pBD-GAL4-OsHsfC1a
pBD-GAL4-OsHsfC1b
pBD-GAL4-OsHsfC2a
pBD-GAL4-OsHsf26
pBD-GAL4
1 mM 3-AT
SD-WH +
5 mM 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 m M 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
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 with32P-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.
Trang 6This 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 G2> A3> A4, 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] Usbind-ing in vitro crosslinkbind-ing 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
OsOsHsfA7 +
A2a OsHsfA7 + B4b OsHsfA7 + B4c OsHsfA7 + 26*OsHsfA7 + pAD
OsHsf
A2a + pAD
OsHsfB4b + pAD OsHsfB4c + B4c OsHsfB4c + 26*OsHsfB4c + pADOsHsf26* +pADOsHsf26* + 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 Interactions among 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.
Trang 7maximum 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 factors and 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 activaPhosphoryla-tion and inactivaPhosphoryla-tion 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 in rice 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 in rice 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
Trang 8In 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
polypeptides were normalized by the levels of incorporated
[35S] 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 with32P-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 mMNaCl
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 Phusion 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
URA3::UASGAL4_17mers_(x3)-TATACYC1-lacZ) (Stratagene)
Transformants were allowed to grow in SD medium
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
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-pAD-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
References
1 Sarkar NK, Kim YK & Grover A (2009) Rice sHsp genes: genomic organization and expression profiling under stress and development BMC Genomics 10, 393
2 Singh A, Singh U, Mittal D & Grover A (2010) Gen-ome-wide analysis of rice ClpB⁄ HSP100, ClpC and ClpD genes BMC Genomics 11, 95
3 Yamamoto A, Mizukami Y & Sakurai H (2005) Identi-fication of a novel class of target genes and a novel type
of binding sequence of heat shock transcription factor
in Saccharomyces cerevisiae J Biol Chem 280, 11911– 11919
4 Scho¨ffl F, Rieping M, Baumann G, Bevan M & Angermu¨ller S (1989) The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco Mol Gen Genet
217, 246–253
5 von Koskull-Do¨ring P, Scharf KD & Nover L (2007) The diversity of plant heat stress transcription factors Trends Plant Sci 12, 452–457
6 Li M, Doll J, Weckermann K, Oecking C, Berendzen
KW & Scho¨ffl F (2010) Detection of in vivo inter-actions between Arabiopsis class A-HSFs, using a novel BiFc fragment, and identification of novel class B-HSF interacting proteins Eur J Cell Biol 89, 126–132
Trang 97 Nover L, Bharti K, Do¨ring P, Mishra SK, Ganguli A
& Scharf KD (2001) Arabidopsis and the heat stress
transcription factor world: how many heat stress
tran-scription factors do we need? Cell Stress Chaperones 6,
177–189
8 Baniwal SK, Bharti K, Chan KY, Fauth M, Ganguli
A, Kotak S, Mishra SK, Nover L, Port M, Scharf KD
et al.(2004) Heat stress response in plants: a complex
game with chaperones and more than twenty heat stress
transcription factors J Biosci 29, 471–487
9 Kotak S, Vierling E, Ba¨umlein H & von
Koskull-Do¨r-ing P (2007) A novel transcriptional cascade regulatKoskull-Do¨r-ing
expression of heat stress proteins during seed
develop-ment of Arabidopsis Plant Cell 19, 182–195
10 Yokotani N, Ichikawa T, Kondou Y, Matsui M,
Hirochika H, Iwabuchi M & Oda K (2008) Expression
of rice heat stress transcription factor OsHsfA2e
enhances tolerance to environmental stresses in
trans-genic Arabidopsis Planta 227, 957–967
11 Yoshida T, Sakuma Y, Todaka D, Maruyama K, Qin
F, Mizoi J, Kidokoro S, Fujita Y, Shinozaki K &
Yamaguchi-Shinozaki K (2008) Functional analysis of
an Arabidopsis heat-shock transcription factor HsfA3
in the transcriptional cascade downstream of the
DREB2A stress-regulatory system Biochem Biophys
Res Commun 11, 515–521
12 Liu JG, Qin QL, Zhang Z, Peng RH, Xiong AS &
Chen JM (2009) OsHSF7 gene in rice, Oryza sativa L.,
encodes a transcription factor that functions as a high
temperature receptive and responsive factor BMB Rep
42, 16–21
13 Li M, Berendzen KW & Scho¨ffl F (2010) Promoter
specificity and interactions between early and late
Ara-bidopsis heat shock factors Plant Mol Biol 73,
559–567
14 Nishizawa A, Tainaka H, Yoshida E, Tamoi M,
Yabu-ta Y & Shigeoka S (2010) The 26S Proteasome function
and Hsp90 activity involved in the regulation of HsfA2
expression in response to oxidative stress Plant Cell
Physiol 51, 486–496
15 Xin H, Zhang H, Chen L, Li X, Lian Q, Yuan X, Hu
X, Cao L, He X & Yi M (2010) Cloning and
character-ization of HsfA2 from Lily (Lilium longiflorum) Plant
Cell Rep 29, 875–885
16 Mittal D, Chakrabarti S, Sarkar A, Singh A &
Gro-ver A (2009) Heat shock factor gene family in rice:
genomic organization and transcript expression
profil-ing in response to high temperature, low temperature
and oxidative stresses Plant Physiol Biochem 47,
785–795
17 Swindell WR, Huebner M & Weber AP (2007)
Tran-scriptional profiling of Arabidopsis heat shock proteins
and transcription factors reveals extensive overlap
between heat and non-heat stress response pathways
BMC Genomics 8, 125
18 Wunderlich M, Doll J, Busch W, Kleindt CK, Loh-mann C & Scho¨ffl F (2007) Heat shock factors: regula-tors of early and late functions in plant stress response Plant Stress 1, 16–22
19 Sakurai H & Enoki Y (2010) Novel aspects of heat shock factors: DNA recognition, chromatin modulation and gene expression FEBS J 277, 4140–4149
20 Yamamoto N, Takemori Y, Sakurai M, Sugiyama K & Sakurai H (2009) Differential recognition of heat shock elements by members of the heat shock transcription factor family FEBS J 276, 1962–1974
21 Miller G & Mittler R (2006) Could heat shock tran-scription factors function as hydrogen peroxide sensors
in plants? Ann Bot 98, 279–288
22 Scharf KD, Heider H, Ho¨hfeld I, Lyck R, Schmidt E & Nover L (1998) The tomato Hsf System: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules Mol Cell Biol 18, 2240–2251
23 Chan-Schaminet KY, Baniwal SK, Bublak D, Nover L
& Scharf KD (2009) Specific interaction between tomato HsfA1 and HsfA2 creates hetero-oligomeric su-peractivator complexes for synergistic activation of heat stress gene expression J Biol Chem 284, 20848–20857
24 Baniwal SK, Chan KY, Scharf KD & Nover L (2007) Role of heat stress transcription factor HsfA5 as specific repressor of HsfA4 J Biol Chem 282, 3605–3613
25 Carranco R, Almoguera C & Jordano J (1999) An imperfect heat shock element and different upstream sequences are required for the seed-specific expression
of a small heat shock protein gene Plant Physiol 121, 723–730
26 Almoguera C, Prieto-Dapena P & Jordano J (1998) Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock ele-ments Plant J 13, 437–446
27 Diaz-Martin J, Almoguera C, Prieto-Dapena P, Espin-osa JM & Jordano J (2005) Functional interaction between two transcription factors involved in the devel-opmental regulation of a small heat stress protein gene promoter Plant Physiol 139, 1483–1494
28 Nishiwaza A, Yabuta Y, Yoshida E, Maruta T, Yoshim-ura K & Shigeoka S (2006) Arabidopsis heat shock tran-scription factor as a key regulator in response to several types of environmental stress Plant J 48, 535–547
29 Nishizawa A, Yoshida E, Yabuta Y & Shigeoka S (2009) Analysis of the regulation of target genes by an Arabidopsis heat shock transcription factor, HsfA2 Biosci Biotechnol Biochem 73, 890–895
30 Guo J, Wu J, Ji Q, Wang C, Luo L, Yuan Y, Wang Y
& Wang J (2008) Genome-wide analysis of heat shock transcription factor families in rice and Arabidopsis J Genet Genomics 35, 105–118
31 Kotak S, Port M, Ganguli A, Bicker F & von Koskull-Do¨ring P (2004) Characterization of C-terminal
Trang 10domains of Arabidopsis heat stress transcription factors
(Hsfs) and identification of a new signature
combina-tion of plant class A Hsfs with AHA and NES motifs
essential for activator function and intracellular
locali-zation Plant J 39, 98–112
32 Walter M, Chaban C, Schu¨tze K, Batistic O,
Wecker-mann K, Na¨ke C, Blazevic D, Grefen C, Schumacher
K, Oecking C et al (2004) Visualization of protein
interactions in living plant cells using bimolecular
fluo-rescence complementation Plant J 40, 428–438
33 Sakurai H & Takemori Y (2007) Interaction between
heat shock transcription factors (HSFs) and divergent
binding sequences: binding specificities of yeast HSFs
and human HSF1 J Biol Chem 282, 13334–13341
34 Guo L, Chen S, Liu K, Liu Y, Ni L, Zhang K & Zhang
L (2008) Isolation of heat shock factor HsfA1a-binding
sites in vivo revealed variations of heat shock elements
in Arabidopsis thaliana Plant Cell Physiol 49, 1306–
1315
35 Guan JC, Yeh CH, Lin YP, Ke YT, Chen MT, You
JW, Liu YH, Lu CA, Wu SJ & Lin CY (2010) A 9 bp
cis-element in the promoters of class I small heat shock
protein genes on chromosome 3 in rice mediates
L-azetidine-2-carboxylic acid and heat shock responses
J Exp Bot 61, 4249–4261
36 Hahn JS, Hu Z, Thiele DJ & Iyer VR (2004) Genome
wide analysis of the biology of stress responses through
heat shock transcription factor Mol Cell Biol 24, 5249–
5256
37 Pan Y, Tsai CJ, Ma B & Nussinov R (2010) Mecha-nisms of transcription factor selectivity Trends Genet
26, 75–83
38 Guettouche T, Boellmann F, Lane WS & Voellmy R (2005) Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress BMC Bio-chem 6, 4
39 Enoki Y & Sakurai H (2011) Diversity in DNA recogni-tion by heat shock transcriprecogni-tion factors (HSFs) from model organisms FEBS Lett 585, 1293–1298
Supporting information
The following supplementary material is available: Table S1 In silico analysis for the putative phosphory-lation sites in rice Hsfs
Table S2 List of primers used in this study
This supplementary material can be found in the online version of this article
Please note: As a service to our authors and readers, this journal provides supporting information supplied
by the authors Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors