doi:10.1093/plcell/koaa026
THE PLANT CELL 2021: 33: 338–357
Research Article
Jianyan Huang
,1,2,* Xiaobo Zhao
,3 Marco Bürger
,2 Yurong Wang
2,4
and Joanne Chory
1,2,
*
1 Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
2 Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA
3 Institute of Nuclear Agricultural Sciences, Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Zhejiang Province,
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
4 Division of Biological Sciences, University of California, Davis, CA 95616, USA
*Authors for correspondence: jyhuang@salk.edu, chory@salk.edu
J.H., X.Z., and J.C designed the research. J.H., X.Z., M.B., and Y.W performed the research. J.H analyzed the data. J.H., X.Z., M.B. and J.C. wrote the
manuscript.
The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is (are): Joanne Chory (chory@salk.edu).
Abstract
The ethylene response factor (ERF) transcription factors are integral components of environmental stress signaling
cascades, regulating a wide variety of downstream genes related to stress responses and plant development. However, the
mechanisms by which ERF genes regulate the heat stress response are not well understood. Here, we uncover the positive
role of ethylene signaling, ERF95 and ERF97 in basal thermotolerance of Arabidopsis thaliana. We demonstrate that ethylene signaling-defective mutants exhibit compromised basal thermotolerance, whereas plants with constitutively activated
ethylene response show enhanced basal thermotolerance. EIN3 physically binds to the promoters of ERF95 and ERF97.
Ectopic constitutive expression of ERF95 or ERF97 increases the basal thermotolerance of plants. In contrast, erf95 erf96
erf97 erf98 quadruple mutants exhibit decreased basal thermotolerance. ERF95 and ERF97 genetically function downstream
of EIN3. ERF95 can physically interact with ERF97, and this interaction is heat inducible. ERF95 and ERF97 regulate a
common set of target genes, including known heat-responsive genes and directly bind to the promoter of HSFA2. Thus,
our study reveals that the EIN3-ERF95/ERF97-HSFA2 transcriptional cascade may play an important role in the heat stress
response, thereby establishing a connection between ethylene and its downstream regulation in basal thermotolerance
of plants.
Introduction
Temperature is an environmental factor that dramatically
affects seasonal growth and geographical distribution
of plants. During the past 200 years, human activity has
increased the release of greenhouse gases, resulting in global
warming that is predicted to reach 1.0 C above preindustrial levels (likely range of 0.8–1.2 C; https://www.ipcc.
ch/sr15/). However, global warming could reach 4.0 C at the
end of the century if increases continue at the current rate
(Betts et al., 2011). One side effect of global warming is a
decrease in agricultural yield (Lobell et al., 2011; Zhao et al.,
2017). Each Celsius-degree increase in global mean temperature would, on average, reduce the global yields of wheat
(Triticum) by 6.0%, rice (Oryza sativa) by 3.2%, maize (Zea
mays) by 7.4%, and soybean (Glycine max) by 3.1% (Zhao
et al., 2017). Therefore, studying the mechanisms by which
Received October 15, 2020. Accepted November 6, 2020. Advance access publication December 16, 2020
C American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com
V
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Two interacting ethylene response factors regulate
heat stress response
The Plant Cell, 2021 Vol. 33, No. 2
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| 339
IN A NUTSHELL
Background: Temperature is an environmental factor that has one of the most dramatic effects on seasonal growth
and geographic distribution of plants. During the past 200 years, human activity has increased the release of
greenhouse gases, resulting in global warming. One side effect of global warming is a decrease in agricultural yield.
Therefore, studying how plants respond to heat stress is important for maximizing agricultural production and
promoting world food security in the coming years. Ethylene response factors (ERFs) display a great degree of
functional plasticity and participate in the regulation of many biotic and abiotic stress responses, making them good
candidates for engineering stress-tolerant crops.
Findings: We found that ethylene, ERF95 and ERF97 are positive regulators in the heat stress response of
Arabidopsis. Ectopic constitutive expression of ERF95 or ERF97 increases the thermotolerance of plants. These two
ERFs act downstream of ethylene signaling component EIN3 and regulate a common set of downstream target
genes in heat stress response. ERF95 and ERF97 can form protein dimers binding to the promoter of heatresponsive marker gene HSFA2 to regulate the expression of HSFA2. Together, our results establish a connection
for ethylene, ethylene response factors and HSFA2 in the heat stress response of plants.
Next steps: Ectopic and constitutive expression of ERF95 and ERF97 led to the growth arrest of Arabidopsis, which
would limit their biotechnological application. To overcome the growth-stress tradeoff will be the next step. One
promising strategy for breeding thermotolerant crops is to use host-specific stress-inducible promoters to drive
endogenous ERF95 and ERF97 in crops.
plants respond to high temperature-mediated stress is
important for maximizing agricultural production and
promoting world food security in the coming years.
Being sessile, plants must cope with environmental
stresses such as heat, drought, cold, and high light, and
adapt to these stresses to ensure survival and reproductive
success. Key players in the ability of plants to respond to
heat stress are the heat shock transcription factors (HSFs),
which are rapidly activated under heat stress to enhance the
expression of many genes encoding heat shock proteins
(HSPs; Busch et al., 2005; Charng et al., 2007). HSPs act as
molecular chaperones, protecting cellular proteins by preventing their denaturation and aggregation, and facilitating
the refolding of proteins damaged by heat (Wang et al.,
2004). In addition, phytohormones are involved in the heat
stress response of plants (Verma et al., 2016).
Ethylene is a gaseous hormone that controls many physiological processes from seed germination to tissue senescence
(Johnson and Ecker, 1998). The ethylene signaling cascade is
initiated by binding of ethylene to its receptors ETR1 (ethylene response 1), ERS1 (ethylene resistant 1), ETR2 (ethylene
response 2), ERS2 (ethylene resistant 2), and EIN4 (ethylene
insensitive 4; Chang et al., 1993; Hua et al., 1995, 1998;
Bleecker et al., 1988; Sakai et al., 1998). CTR1 (constitutive
triple response 1) functions downstream of these receptors
to negatively regulate ethylene signaling in the absence of
ethylene (Kieber et al., 1993), and EIN2 acts downstream of
CTR1 (Roman et al., 1995). ethylene insensitive 3 (EIN3) and
its closest homolog EIN3-LIKE 1 (EIL1) are two primary transcription factors downstream of EIN2 (Chao et al., 1997;
Solano et al., 1998). In addition to its role in the regulation
of plant growth and development, ethylene has also been
implicated in responses to various stresses, including
heat (Wang et al., 2013; Müller and Munné-Bosch, 2015;
Tao et al., 2015). For example, ethylene production is induced in a heat susceptible wheat cultivar after heat exposure (Hays et al., 2007) and ethylene is required to protect
against heat stress-induced oxidative damage in Arabidopsis
(Larkindale and Knight, 2002). However, the molecular
mechanism by which ethylene participates in the heat stress
response has not been established.
The APETALA2/ethylene responsive element binding protein (AP2/EREBP) gene family includes 147 members and is
one of the plant-specific transcription factor families in
Arabidopsis (Dietz et al., 2010). Sixty-five ethylene response
factors (ERFs) constitute the largest subfamily of the AP2/
EREBP family (Feng et al., 2005; Dietz et al., 2010). ERFs were
initially identified as binding factors that mediate ethylene
response (Fujimoto et al., 2000). ERFs participate in many
developmental processes and play pivotal roles in adaptation to biotic or abiotic stresses, such as pathogen attack,
UV irradiation, cold, heat, drought, and salinity (Mizoi et al.,
2012; Licausi et al., 2013; Xie et al., 2019a, 2019b). Subgroup
IXc of the ERF subfamily contains eight ERFs named from
ERF91 to ERF98. ERF95–ERF98 are four small ERFs containing 128–139 amino acids (Nakano et al., 2006). Among
them, ERF95 (also named ESE1) is a direct target of EIN3
and plays a role in salt stress response of Arabidopsis
(Zhang et al., 2011). ERF96 and ERF97 (also named AtERF14)
have functions in plant defense response (O~
nate-Sánchez
et al., 2007; Catinot et al., 2015; Wang et al., 2015a). ERF98 is
involved in salt tolerance through transcriptional activation
of genes that synthesize ascorbic acid (Zhang et al., 2012).
Interestingly, ectopic expression of a number of ERF genes
under the control of constitutive expression promoters
such as the Cauliflower mosaic virus (CaMV) 35S promoter
can enhance the tolerance of plants to various stresses, including heat, indicating that ERFs also participate in heat
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Question: We wanted to know how ERF-mediated transcriptional regulation worked in plant heat stress response.
We chose two ERF genes, ERF95 and ERF97, and studied their role in thermotolerance.
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Results
Ethylene plays a positive role in plant basal
thermotolerance
To elucidate the role of ethylene in heat stress response of
Arabidopsis, we employed a basal thermotolerance assay
(Silva-Correia et al., 2014; Li et al., 2014): 7-day-old plants
were submerged in a 43 C water bath in darkness for 22
min (Basal heat stress) and then allowed to recover in a
growth chamber at 22 C for 7 days before the survival
rate of plants was assessed. We first investigated the phenotype of plants lacking EIN2 and EIN3, two key positive
regulators of the ethylene signaling pathway. Following 22
min of 43 C basal heat treatment, the loss-of-function
mutants ein2-5 and ein3 eil1 each exhibited a survival rate
of about 8%, compared with about 20% for wild-type
plants (Figure 1, A–C). Conversely, ctr1-1 mutants or EIN3
overexpression (EIN3ox) lines, which have a constitutive
ethylene response, each exhibited a survival rate 480%
(Figure 1, A–C). The relative electrolyte leakage serves as
an indicator of plant cell membrane integrity damage
caused by stress. Therefore, we measured the electrolyte
leakage of wild-type Col-0, ein2-5, ein3 eil1, ctr1-1, and
EIN3ox lines before and after basal heat treatment. The
ein2-5 and ein3 eil1 mutants had higher electrolyte leakage,
whereas the ctr1-1 and EIN3ox plants had lower electrolyte
leakage than wild-type after basal heat treatment
(Figure 1D). The electrolyte leakage levels before basal
heat treatment were comparable among all these lines
(Figure 1D). Taken together, these observations suggest
that the ethylene signaling pathway plays a positive role in
basal thermotolerance of plants.
Ectopic and constitutive expression of ERF95
enhances the basal thermotolerance of plants
ERFs are downstream targets of the ethylene signaling pathway, and it has been reported that ERF95 is a direct target
of EIN3 (Zhang et al., 2011). A number of studies have
shown that ectopic and constitutive expression of AP2/ERF
genes can increase the tolerance of plants to various biotic
or abiotic stresses (Wang et al., 2016). To investigate the
role of ERF95 in basal thermotolerance, we generated transgenic lines ectopically and constitutively expressing ERF95
under the control of the CaMV 35S promoter with a C-
terminal 3HA-3FLAG tag (p35S:ERF95). p35S:ERF95
plants were smaller under normal growth conditions compared with wild-type, and this phenotype associated with
the expression level of ERF95 proteins (Supplemental Figure
1, A and B). We then examined the response of p35S:ERF95
and erf95 T-DNA insertion mutants to basal heat treatment.
After 22 min of 43 C basal heat treatment, 480% of
p35S:ERF95 plants survived, compared with 20% for wildtype, suggesting that p35S:ERF95 plants had enhanced basal
thermotolerance. However, the survival rate of erf95 mutants
was similar to wild-type (Figure 2, A–C). Consistent with
this result, significantly reduced electrolyte leakage was
detected in the 35S:ERF95 lines compared with wild-type after basal heat treatment (Figure 2D).
In plants, an enhanced stress response often compromises
growth and development, which is regarded as a trade-off
between growth and stress responses. To assess the contribution of plant size to the basal thermotolerance phenotype
of p35S:ERF95 lines, we used another previous studygenerated ERF95 ectopic expression line called ESE1ox, which
also under the control of the CaMV 35S promoter but does
not affect the size of plants (Zhang et al., 2011). ESE1ox also
showed enhanced basal thermotolerance (Supplemental
Figure 1, C–F). Moreover, we performed basal thermotolerance on some mutants with smaller plant size than the
wild-type, e.g. the cop1, pifq, and bzr1-1D mutants. The
results showed that cop1, pifq, and bzr1-1D mutants were
more sensitive rather than more resistant to basal heat
treatment than wild-type. This result indicates that the
smaller size of plants is not always associated with the enhanced basal thermotolerance phenotype (Supplemental
Figure 2).
ERF95 physically interacts with another ERF: ERF97
To identify partners of ERF95 in basal heat stress response,
we subjected ERF95 to immunoprecipitation-mass spectrometry (IP-MS) analysis. In addition to the known ERF95interacting protein mediator subunit 25 (MED25; Ou et al.,
2011), another ERF, ERF97, was co-precipitated with ERF95
(Figure 3A). We validated the interaction between ERF95
and ERF97 using a firefly luciferase complementation imaging (LCI) assay (Chen et al., 2008). ERF97-NLuc (N-terminal
luciferase fusion) co-expressed with ERF95-CLuc (C-terminal
luciferase fusion) in Arabidopsis protoplasts resulted in the
complementation of luciferase. Similar results were observed
with the co-transformation of ERF97-CLuc with ERF95-NLuc
(Figure 3B). As negative controls, ERF97-NLuc was coexpressed with CLuc, or NLuc was co-expressed with ERF95CLuc. Both resulted in relatively weak luciferase activity
(Figure 3B). The LCI assay also showed that both ERF95 and
ERF97 can form homodimers (Figure 3B). Moreover, we
used bimolecular fluorescence complementation (BiFC;
Waadt et al., 2008) to confirm and localize the interactions.
Co-expression of ERF97-nYFP (N-terminal yellow fluorescent
protein fusion) and ERF95-cYFP (C-terminal yellow fluorescent protein fusion) in protoplasts resulted in YFP signals in
the nucleus and cytosol, demonstrating that ERF95
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stress response (Cheng et al., 2013; Hsieh et al., 2013; Yao
et al., 2017). However, the role of ERF-mediated transcriptional regulation in heat stress response remains unclear.
In this study, we demonstrate a positive role for ethylene and two interacting ERFs, ERF95 and ERF97, in plant
basal thermotolerance. These two ERFs act downstream of
EIN3 and share many common downstream regulatory
genes. Furthermore, ERF95 and ERF97 can directly bind to
the promoter of HSFA2 and regulate the expression of
HSFA2. Together, our study establishes a connection
between ethylene, ERFs, and HSFA2 in the heat stress response of plants.
J. Huang et al.
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ein2-5
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EIN3ox
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*
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43°C
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ct il1
r1
EI -1
N
3o
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Survival rate (%)
100
100
Electrolyte leakage (%)
D
120
Figure 1 Ethylene is a positive regulator in basal thermotolerance of plants. (A) Phenotype of wild-type (Col-0), ein2, ein3 eil1, ctr1-1, and EIN3ox
seedlings grown under long day (16 h light and 8 h dark) conditions at 22 C for 7 days. Scale bar is 1 cm. (B) Phenotype of wild-type (Col-0), ein2,
ein3 eil1, ctr1-1, and EIN3ox seedlings in basal thermotolerance assay (43 C for 22 min, and recovered at 22 C for 7 days). Scale bar is 1 cm. (C)
Survival rates of Col-0, ein2, ein3 eil1, ctr1-1, and EIN3ox seedlings in basal thermotolerance assay. Values are mean ± SEM of three biological replicates. Asterisks indicate statistically significant differences (*P 5 0.05, ***P 5 0.001) compared with the wild-type as determined by one-way
analysis of variance (ANOVA) followed by Fisher’s LSD test. (D) Electrolyte leakage assay of Col-0, ein2, ein3 eil1, ctr1-1, and EIN3ox seedlings. Data
are mean ± SEM (three biological replicates). Asterisks indicate statistically significant differences (*P 5 0.05, **P 5 0.01, ***P 5 0.001) compared
with wild-type under the same condition, as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. NS, not significant.
interacted with ERF97 in vivo (Figure 3C). We also observed
interactions of ERF95 with itself and ERF97 with itself
(Figure 3D). The interaction between ERF95 and ERF97 was
further confirmed by co-immunoprecipitation (Co-IP) assays.
FLAG-tagged ERF95, HA-tagged ERF97 or HA-tagged ERF014
(negative control) were co-infiltrated into the Nicotiana benthamiana leaves. ERF97 protein was co-immunoprecipitated
with ERF95 from FLAG beads, while ERF014 proteins were
not, indicating that ERF95 was able to interact with ERF97
(Figure 3E). To assess whether the interaction between
ERF95 and ERF97 is heat inducible or not, we performed a
split luciferase assay using N. benthamiana leaves. As the activity of luciferase will be abolished under 43 C (Pozzo et al.,
2018), we used a lower temperature heat treatment of 37 C.
A 15-min 37 C heat treatment significantly increased the luciferase activity in leaves co-transformed with ERF95-NLuc
and ERF97-CLuc versus before heat treatment, while 37 C
heat treatment slightly decreased the activity of luciferase in
leaves transformed with whole luciferase driven by the
CaMV 35S promoter (Supplemental Figure 3). Together,
these results demonstrate that ERF95 can interact with
ERF97 to form a heterodimer, and heat stress enhances the
interaction between ERF95 and ERF97, implying that ERF95
and ERF97 may function as a protein complex to regulate
plant growth and heat stress response.
Ectopic and constitutive expression of ERF97
enhances basal thermotolerance of plants and
quadruple ERF mutants exhibit decreased basal
thermotolerance
Given the interaction between ERF95 and ERF97, we tested
whether ERF97 was also involved in basal heat stress response. We generated ERF97 ectopic and constitutive expression lines under the control of the CaMV 35S promoter
(p35S:ERF97) and found that p35S:ERF97 plants showed a
similar but stronger phenotype compared to p35S:ERF95
plants. p35S:ERF97 plants had dark green leaves, and were
smaller, growth retarded, and late flowering (Supplemental
Figure 4, A–D). We then subjected wild-type, p35S:ERF97,
and erf97 mutant plants to the basal thermotolerance assay.
After 22-min basal heat treatment at 43 C, 490% of
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C
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Col-0
J. Huang et al.
p35S:ERF95#5
erf95
p35S:ERF95#6
80
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Electrolyte leakage (%)
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Survival rate (%)
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5S
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p3
5#
5S
5
:E
R
F9
5#
6
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Figure 2 Ectopic and constitutive expression of ERF95 confers a tolerant phenotype to basal heat stress. (A) Phenotype of wild-type (Col-0),
p35S:ERF95 and erf95 seedlings grown under long day condition at 22 C for 7 days. Two independent p35S:ERF95 lines are shown. Scale bar is 1
cm. (B) Phenotype of wild-type (Col-0), p35S:ERF95, and erf95 seedlings in basal thermotolerance assay. Scale bar is 1 cm. (C) Survival rates of Col0, p35S:ERF95, and erf95 seedlings in basal thermotolerance assay. Values are mean ± SEM of three biological replicates. Asterisks indicate statistically significant differences (***P 5 0.001) compared with the wild-type as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. NS, not significant. (D) Electrolyte leakage assay of Col-0, p35S:ERF95, and erf95 seedlings. Data are mean ± SEM (three biological
replicates). Asterisks indicate statistically significant differences (*P 5 0.05, **P 5 0.01) compared with wild-type under the same condition, as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. NS, not significant.
the p35S:ERF97 plants survived, compared with 20% for
wild-type (Figure 4, A–C), indicating that ectopic and constitutive expression of ERF97 also enhanced basal thermotolerance. Similar to erf95, erf97 mutants exhibited wild-type
levels of basal thermotolerance (Figure 4, A–C). Consistent
with this result, significantly reduced electrolyte leakage was
detected in p35S:ERF97 lines compared with wild-type after
basal heat treatment (Figure 4D).
ERF95 and ERF97, along with two close homologs, ERF96
and ERF98, belong to the small IXc subgroup of AP2/ERF
family. Since ERF95 and ERF97 single mutants resembled
wild-type plants in basal thermotolerance assays, we created
ERF95 and ERF97 double mutants (erf95 erf97). The survival
rate of erf95 erf97 was comparable to that of the wild-type
(Figure 4, E and G). ERF95, ERF96, ERF97, and ERF98 have
very similar protein sequences (Wang et al., 2015a), and it is
impossible to generate the ERF95/ERF98 double mutant by
hybridization, as the physical distance between these two
genes on the chromosome is too small. We therefore turned
to the CRISPR/Cas9 system. We chose two guide RNAs
(gRNA) each for ERF96 and ERF98, and transformed them
into the erf95 erf97 double mutant to generate the quadruple mutant of these four ERFs (erfq; Supplemental Figure
4E). The two gRNAs caused a 246-bp deletion in ERF96
(Supplemental Figure 4F). For ERF98, gRNA1 did not work,
but gRNA2 caused a 1-bp deletion in line #3 and a 50-bp
deletion in line #23 (Supplemental Figure 4F). We selected
two homozygous erfq lines (erfq#1 from line #3 and erfq#2
from line #23) to perform the basal thermotolerance assay.
Survival rates of 10% for erfq mutants compared with
27% for wild-type plants indicated that erfq mutants were
sensitive to basal heat treatment (Figure 4, E–G). The
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B
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A
C
Peptide counts
Locus
YFP
Chlorophyll
Merged
Brightfield
ERF97-nYFP+
ERF95-cYFP
Col-0#1 Col-0#2 p35S:ERF95#5 p35S:ERF95#6
ERF95
AT3G23220
0
0
20
20
MED25
AT1G25540
0
0
7
3
ERF97
AT1G04370
0
0
2
2
ERF97-nYFP+
cYFP
NLuc+ERF95-CLuc
D
ERF95-NLuc+CLuc
ERF95-nYFP+
ERF95-cYFP
NLuc+ERF97-CLuc
ERF97-NLuc+CLuc
ERF95-nYFP+
cYFP
ERF95-NLuc+ERF97-CLuc
ERF95-NLuc+ERF95-CLuc
ERF97-nYFP+
ERF97-cYFP
ERF97-NLuc+ERF95-CLuc
ERF97-NLuc+ERF97-CLuc
E
ERF95-FLAG
ERF014-HA
ERF97-HA
+
+
_
+
_
100
200
300
400
500
F
+
22°C
IB:α-FLAG
NLuc
+CLuc
Input
IB:α-HA
IB:α-FLAG
nYFP+
ERF97-cYFP
600 700 800 900 1000 1100
ERF95-NLuc
+CLuc
ERF95-NLuc
+
ERF97-CLuc
NLuc+
ERF97-CLuc
IP:
α-FLAG
IB:α-HA
3.5
37°C
Relative LUC activity
0
***
3
2.5
2
1.5
1
0.5
0
22°C
37°C
Figure 3 ERF95 and ERF97 can Form Hetero- and Homo-Dimers. (A) Peptide counts for ERF95, MED25, and ERF97 proteins from ERF95 IP-MS experiment. (B) LCI assay showing the interactions between ERF95 and ERF97, and ERF95, ERF97 with itself. Luciferase activity upon co-transformation
of ERF95-NLuc and ERF97-CLuc, ERF95-NLuc and ERF95-CLuc, ERF97-NLuc and ERF95-CLuc, ERF97-NLuc and ERF97-CLuc compared to controls.
The X-axis is the relative luciferase (LUC) activity. Y-axis shows the different co-transformations. Data shown are the mean ± SEM of three biological
replicates. (C) BiFC assay of ERF95 interaction ERF97. Co-transformation of ERF97-nYFP and ERF95-cYFP leads to the reconstitution of YFP signal,
whereas no signal is detected when ERF97-nYFP and cYFP or ERF95-cYFP and nYFP are co-expressed. Scale bar is 10 lm. (D) BiFC assay of ERF95
and ERF97 in vivo homodimer formation. Co-transformation of ERF95-nYFP and ERF95-cYFP or ERF97-nYFP and ERF97-cYFP results in the YFP signal, whereas no signal is detected when ERF97-cYFP and nYFP or ERF95-nYFP and cYFP are co-expressed. Scale bar is 10 lm. (E) ERF95 interaction
with ERF97 in Co-IP assay. ERF95-FLAG and ERF97-HA, or ERF014-HA were co-infiltrated into N. benthamiana leaves. After 3-d infiltration, proteins
were extracted from N. benthamiana leaves and FLAG dyna beads were used to immunoprecipitate ERF97-HA and ERF014-HA proteins. Gel blots
were probed with anti-FLAG or anti-HA antibody. (F) Heat stress enhanced the interaction between ERF95 and ERF97 as showing by split luciferase
assay using N. benthamiana leaves. Five replications were used for relative luciferase activity quantification. For relative luciferase (LUC) activity, the
LUC activity of each sample under 22 C was set as one. Asterisks indicate the Student’s t-test significant differences (***P 5 0.001).
electrolyte leakage assay showed that erfq had higher electrolyte leakage than the wild-type in response to basal heat
treatment (Figure 4H). This difference suggests that ERF95,
ERF96, ERF97, and ERF98 have functional redundancy in
basal heat stress response.
ERF97 acts downstream of EIN3
ERF95 is a direct binding target of EIN3 (Zhang et al., 2011).
Promoter sequence analysis shows that the EIN3 binding ciselement ATGTA (Solano et al., 1998) is also present in the
ERF97 promoter (Figure 5A). Both electrophoretic mobility
shift assay (EMSA) and chromatin immunoprecipitation
(ChIP)-qPCR assay confirmed that EIN3 binds directly to the
promoter of ERF97 (Figure 5, B and C). To explore the role
of ERF97 in ethylene response, we examined the expression
level of ERF97 in 7-day-old wild-type seedlings (Col-0)
treated with ethylene. Consistent with the previous study
(O~
nate-Sánchez et al., 2007), ERF97 transcript levels were elevated by ethylene (Figure 5D). We further analyzed the expression pattern of ERF97 in ethylene signaling mutants
ein2-5, ein3 eil1, ctr1-1, and EIN3ox. Reverse transcriptionqPCR (RT-qPCR) analysis showed that before ethylene treatment, ERF97 had a lower expression level in ein2-5 and ein3
eil1 mutants but had a higher expression level in ethylene
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nYFP+
ERF95-cYFP
Relative LUC activity
B
| 343
p35S:ERF97#7
p35S:ERF97#9
erf97
C
B
D
120
100
***
***
80
60
40
ns
20
100
Electrolyte leakage (%)
Col-0
J. Huang et al.
80
60
20
5
p3 S:E Co
5S R F l-0
:E 97
R #
F9 7
7#
er 9
f9
p3
7
5
p3 S:E Co
5S R F l-0
:E 97
R #7
F9
7#
er 9
f9
7
er
f9
7
F
p3
40
G
H
30
ns
20
**
***
10
0
100
Electrolyte leakage (%)
F9
p3
7#
5S
7
:E
R
F9
7#
9
C
ol
-0
:E
R
5S
erfq#2
ns ns ns
0
p3
erfq#1
Survival rate (%)
erf95 erf97
*** ***
80
22°C
43°C
ns **
*
60
40
20
ns ns ns
#1
#2
fq
er
fq
er
er
f9
7
er
f9
5
C
ol
-0
0
Figure 4 Ectopic and constitutive expression of ERF97 confers a tolerant phenotype to basal heat stress and quadruple mutation of erf95 erf96
erf97 erf98 (erfq) decreases the tolerance of plants to basal heat stress. (A) Phenotype of wild-type (Col-0), p35S:ERF97, and erf97 seedlings grown
under long day conditions at 22 C for 7 days. Two independent p35S:ERF97 lines are shown. Scale bar is 1 cm. (B) Phenotype of wild-type (Col-0),
p35S:ERF97, and erf97 seedlings in basal thermotolerance assay. Scale bar is 1 cm. (C) Survival rates of Col-0, p35S:ERF97 and erf97 seedlings in basal
thermotolerance assay. Values are mean ± SEM from three biological replicates. Asterisks indicate statistically significant differences (***P 5
0.001) compared with the wild-type as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. NS, not significant. (D)
Electrolyte leakage assay of Col-0, p35S:ERF97 and erf97 seedlings. Data are mean ± SEM (three biological replicates). Asterisks indicate statistically
significant differences (***P 5 0.001) compared with wild-type under the same condition, as determined by one-way ANOVA followed by
Dunnett’s multiple comparisons. NS, not significant. (E) Phenotype of wild-type (Col-0), erf95 erf97 and erfq seedlings grown under long day condition at 22 C for 7 days. Scale bar is 1 cm. (F) Phenotype of wild-type (Col-0), erf95 erf97, and erfq seedlings in basal thermotolerance assay. Scale
bar is 1 cm. (G) Survival rates of Col-0, erf95 erf97, and erfq seedlings in basal thermotolerance assay. Values are mean ± SEM from three biological
replicates. Asterisks indicate statistically significant differences (**P 5 0.01, ***P 5 0.001) compared with the wild-type as determined by one-way
ANOVA followed by Dunnett’s multiple comparison. NS, not significant. (H) Electrolyte leakage assay of Col-0, erf95 erf97, and erfq seedlings. Data
are mean ± SEM (three biological replicates). Asterisks indicate statistically significant differences (*P 5 0.05, **P 5 0.01) compared with wildtype under the same condition as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. NS, not significant.
constitutive-response mutants: ctr1 and EIN3ox (Figure 5D).
The ethylene induction of ERF97 was abolished in ein2-5 and
ein3 eil1 mutants (Figure 5D). These results suggest that the
ethylene-induced expression of ERF97 depends on the ethylene signaling pathway. Then, ERF95 or ERF97 was expressed
under the control of the CaMV 35S promoter in the ein3
eil1 mutant background (p35S:ERF95/ein3 eil1 and
p35S:ERF97/ein3 eil1, respectively) and we examined the phenotypes of the resulting plants in basal thermotolerance
assays. Both p35S:ERF95 and p35S:ERF97 rescued the basalheat-sensitive phenotype of ein3 eil1 mutants (Figure 5, E–
H). These results genetically confirm that ERF95 and ERF97
function downstream of EIN3 in basal heat stress response.
ERF95 and ERF97 modulate basal heat stress
response through many common downstream
genes, including known heat stress-responsive genes
To explore how ectopic and constitutive expression of
ERF95 and ERF97 enhanced the basal thermotolerance of
plants, we performed mRNA sequencing (RNA-seq) analysis.
Seven-day-old Col-0, p35S:ERF95#6, and p35S:ERF97#9 seedlings were subjected to basal heat stress treatment (HT),
whereas controls were not (NT). In NT, we identified 2,094
differentially expressed genes (DEGs) in p35S:ERF95 and 1941
DEGs in p35S:ERF97 compared with the wild-type
(Supplemental Data Sets 1 and 2). In wild-type plants, 4,476
genes, which included 1,564 upregulated and 2,912 downregulated genes, were differentially expressed in HT
(Supplemental Data Set 3). Since ERF95 and ERF97 positively
regulated heat stress response, we hypothesized that ERF95and ERF97-regulated genes were regulated by heat stress in
the same direction. To test this idea, we examined the overlaps between heat-regulated genes and genes affected in
p35S:ERF95 or p35S:ERF97 plants by performing clustering
analysis with specific gene groups. The comparisons showed
that ERF95 or ERF97-regulated genes were largely regulated
by heat in the same way (Figure 6, A and B). Specifically,
18.2% (211 out of 1,161, Group 1) of ERF95-induced genes
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Col-0
ns
40
0
E
22°C
43°C
er C
f9 ol5 0
er
f9
er 7
fq
er #1
fq
#2
er C
f9 ol5 0
er
f9
er 7
fq
er #1
fq
#2
A
| THE PLANT CELL 2021: 33: 338–357
Survival rate (%)
344
The Plant Cell, 2021 Vol. 33, No. 2
0.025
10
0.015
p35S:ERF97/
ein3 eil1
*
*
6
**
4
ns
2
ns
***
ein2-5
ein3 eil1
100
22°C
0
Actin
Col-0
G 100
H
***
***
80
60
40
20
ctr1-1
EIN3ox
43°C
80
*
***
60
40
ns
20
ns
0
ei
ei
ei
R
5S
p3
:E
5S
p3
:E
R
R
F9
F9
7/
5/
ei
ei
n3
n3
ei
n3
ei
:E
5S
p3
l1
l1
l1
0
F9 n3
5/
ei
l1
e
F9 in3
ei
7/
l1
ei
n3
p3
ei
l1
5S
:
p3 ER e i n
5S F9
3
:E 5/e e i l 1
R
F9 in3
e
7/
ei il1
n3
ei
l1
p35S:ERF95/
ein3 eil1
ERF97P
Survival rate (%)
F
ein3 eil1
0
***
*
8
Figure 5 ERF97 is a direct binding target of ethylene signaling component EIN3 and the response of ERF97 to ethylene depends on ethylene signaling pathway. (A) Positions of cis-element ATGTA, which can be bound by EIN3 in the promoter of ERF97. The red arrows indicate the positions of
probes used for EMSA. (B) EMSA of the binding of EIN3 to the promoter of ERF97. Competitor or mutated competitor fragments were added in
100 or 300 excess to analyze the specificity of binding. (C) ChIP-qPCR assay confirms the binding of EIN3 to the promoter of ERF97. The
amounts of the indicated DNA in the immune complex were tested by qPCR. ACTIN2 was used as the negative control. (D) Relative expression
level of ERF97 in ethylene signaling related ein2-5, ein3 eil1, ctr1-1, and EIN3ox seedlings. Seeds were germinated on 1/2 LS medium for 7 day under
long day conditions and then treated with air or 10 ppm ethylene gas for 4 h. Values are mean ± SEM of three biological replicates. Asterisks indicate the Student’s t-test significant differences (*P 5 0.05, **P 5 0.01, ***P 5 0.001). NS, not significant. (E) Phenotype of ein3 eil1, p35S:ERF95/
ein3 eil1, and p35S:ERF97/ein3 eil1 seedlings grown under long day conditions at 22 C for 7 days. (F) Phenotype of ein3 eil1, p35S:ERF95/ein3 eil1
and p35S:ERF97/ein3 eil1 seedlings in basal thermotolerance assay. Scale bar is 1 cm. (G) Survival rates of ein3 eil1, p35S:ERF95/ein3 eil1, and
p35S:ERF97/ein3 eil1 seedlings in basal thermotolerance assay. Values are mean ± SEM of three biological replicates. Asterisks indicate statistically
significant differences (***P 5 0.001) as determined by one-way ANOVA followed by Dunnett’s multiple comparisons. (H) Electrolyte leakage assay of ein3 eil1, p35S:ERF95/ein3 eil1, and p35S:ERF97/ein3 eil1 seedlings. Data are mean ± SEM (three biological replicates). Asterisks indicate statistically significant differences (*P 5 0.05, ***P 5 0.001) compared with wild-type under the same condition, as determined by one-way ANOVA
followed by Dunnett’s multiple comparisons. NS, not significant.
were upregulated by heat in wild-type (Figure 6, A and C)
and 25.4% (237 out of 934, Group 2) of ERF95-repressed
genes were downregulated by heat in wild-type (Figure 6, A
and D). In contrast, a smaller proportion of ERF95-induced
genes were repressed by heat in wild-type (93 out of 1,161
genes, 8%) or repressed by ERF95 were induced by heat (105
out of 934 genes, 11.2%; Figure 6A). Similar results were
obtained for ERF97. Specifically, 23.6% (272 out of 1,154,
Group 3) of ERF97-induced genes were upregulated by heat
(Figure 6, B and E) and 36.1% (284 out of 787, Group 4) of
ERF97-repressed genes were downregulated by heat
(Figure 6, B and F). In contrast, a smaller proportion of
ERF97-induced genes were repressed by heat in wild-type
(76 out of 1,154 genes, 6.6%) or repressed by ERF97 were induced by heat (51 out of 787 genes, 6.5%; Figure 6B).
Since ERF95 and ERF97 can interact with each other, we
compared the DEGs between ERF95 and ERF97 to determine
the common targets shared between ERF95 and ERF97.
Before heat stress, a total of 1,246 DEGs were common between p35S:ERF95 (1,246 out of 2,095, 59%) and p35S:ERF97
(1,246 out of 1,941, 64%; Figure 7A) with similar expression
patterns (Figure 7B). Under basal heat stress treatment,
2,140 and 1,778 genes were differentially expressed in
p35S:ERF95 and p35S:ERF97 compared with the wild-type,
respectively (Supplemental Data Set 4). In total, 1,150
DEGs were common between p35S:ERF95-HT (1,150 out of
2,140, 54%) and p35S:ERF97-HT (1,150 out of 1,778, 65%;
Figure 7C). These common genes also showed a similar
expression pattern in p35S:ERF95 and p35S:ERF97
(Figure 7D). Transcriptome data also revealed that some
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0.005
E
*
ET
12
0.02
0.01
Free probe
air
14
EIN3-GFP
R
+
+
+
+
+
+
100x 300x m(300x)
16
ein3 eil1
:E
+
_
D
0.03
5S
Biotin-labeled probe
Unlabeled probe
+
+
_
C
+1
Electrolyte leakage (%)
-377
| 345
p3
ATGTA
-413
EIN3 _
ERF97 relative expression
B
ATGTA
% of input
A
THE PLANT CELL 2021: 33: 338–357
| THE PLANT CELL 2021: 33: 338–357
A
J. Huang et al.
Down-regulated genes
in p35S:ERF95
857
2582
93
Heat-induced
genes
0
284
P=3.19e-122
C
Group 4
P=9.73e-114
51
E
Group 3: Heat-induced and Up-regulated in p35S:ERF97
0
−0.5
−0.5
−1
−1
Row
Z-score
Row
Z-score
p3
5S
:E
R
p3
p3
5S
5S
:E
R
F9
-0
ol
F9
7NT
-H
T
-N
-0
ol
C
R
:E
R
:E
5S
p3
7HT
0.5
0
T
1
0.5
5HT
1
F9
5NT
F9
-0
C
C
ol
ol
-0
-H
-N
T
T
Group 1: Heat-induced and Up-regulated in p35S:ERF95
D
F
Group 2: Heat-repressed and Down-regulated in p35S:ERF95
Group 4: Heat-repressed and Down-regulated in p35S:ERF97
1
1
0.5
0.5
0
0
−0.5
−0.5
−1
−1
Row
Z-score
F9
:E
5S
p3
p3
5S
:E
R
R
C
ol
F9
-0
7HT
-H
7NT
T
T
-N
-0
ol
C
R
S:
E
p3
5
p3
5S
:E
R
F9
F9
5HT
5NT
T
-H
ol
C
-0
ol
C
-0
-N
T
Row
Z-score
Figure 6 ERF95 and ERF97 positively regulate heat-responsive genes. (A) Venn diagram of the overlapping heat-regulated genes and DEGs in
p35S:ERF95 without basal heat stress treatment (NT). Red number indicates genes differentially regulated by ERF95 and heat in the same direction.
(B) Venn diagram of the overlapping heat-regulated genes and DEGs in p35S:ERF97 without basal heat stress treatment (NT). Red number
indicates genes differentially regulated by ERF97 and heat in the same direction. P-values in A and B show the statistical significance of the overlap
between two groups of genes in Venn diagrams. (C) Clustering analysis of Group 1 genes. In all, 211 heat-induced genes are up-regulated
in p35S:ERF95 plants. (D) Clustering analysis of Group 2 genes. In all, 237 heat-repressed genes are down-regulated in p35S:ERF95 plants.
(E) Clustering analysis of Group 3 genes. In all, 272 heat-induced genes are up-regulated in p35S:ERF97 plants. (F) Clustering analysis of Group 4
genes. In all, 284 heat-repressed genes are down-regulated in p35S:ERF97 plants.
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0
0
Group 3
Group 2
P=1.15e-60
105
452
0
0
272
0
Group 1
P=8.12e-72
0
0
1248
237
0
Down-regulated
genes in p35S:ERF97
806
76
0
211
Up-regulated genes
in p35S:ERF97
2552
592
0
0
Heat-repressed
genes
Heat-induced
genes
0
0
1248
B
Up-regulated genes
in p35S:ERF95
Heat-repressed
genes
C
346
The Plant Cell, 2021 Vol. 33, No. 2
THE PLANT CELL 2021: 33: 338–357
A p35S:ERF95-NT vs B
NT common
10
Col-0-NT DEGs
(2095)
5
849
0
1246
P=0
−5
**
30
**
20
0
ns
Col-0 erfq Col-0 erfq
22°C
43°C
10
ns
0
Col-0 erfq Col-0 erfq
22°C
43°C
25 HSP20-like AT5G37670
20
***
*
15
40 PDF1.2c AT5G44430
30
*
**
10
5
0
Relative expression
20
Relative expression
Relative expression
HSFA7a AT3G51910
*
***
vs Col-0-HT
25 HSP90.1 AT5G52640
20
***
**
15
10
5
0
ns
Col-0 erfq Col-0 erfq
22°C
43°C
300 HSP17.6A AT5G12030
*
***
200
100
ns
Col-0 erfq Col-0 erfq
22°C
43°C
20
10
*
0
Col-0 erfq Col-0 erfq
22°C
43°C
Relative expression
40 PDF1.2b AT2G26020
30
Relative expression
Col-0 erfq Col-0 erfq
22°C
43°C
vs Col-0-HT
vs Col-0-NT
Relative expression
Relative expression
Relative expression
Relative expression
200 HSP20-like AT1G53540
150
*
***
100
50
ns
2
p35S:ERF97-HT vs
Col-0-HT DEGs
p35S:ERF95-HT p35S:ERF97-HT
(1778)
2
0
*
Col-0 erfq Col-0 erfq
22°C
43°C
20 PDF1.3 AT2G26010
15
*
*
10
5
0
ns
Col-0 erfq Col-0 erfq
22°C
43°C
Figure 7 ERF95 and ERF97 have a common set of regulated genes and defective expression of HSFs, HSPs, and PDFs in erfq mutants. (A) Venn diagram of the common DEGs between p35S:ERF95 and p35S:ERF97 without basal heat stress treatment (NT). P-values show the statistical significance of the overlap between two groups of genes in Venn diagrams. (B) Heatmap of expression patterns of common DEGs as shown in Figure
7A. (C) Venn diagram of the common DEGs of p35S:ERF95 and p35S:ERF97 with basal heat stress treatment (HT). P-values show the statistical significance of the overlap between two groups of genes in Venn diagrams. (D) Heatmap of expression pattern of common DEGs as shown in Figure
7C. (E) Expression levels of HSFs and HSPs in erfq mutants (erfq#1 is used). The X-axis is the different samples with or without basal heat stress
treatment. The Y-axis represents the relative expression level, and the expression level of each gene in Col-0 under 22 C is set to one. Data are
mean ± SEM (three biological replicates). Asterisks indicate the Student’s t-test significant differences (*P 5 0.05, **P 5 0.01, ***P 5 0.001). NS,
not significant. (F) Expression levels of PDF genes in erfq mutant. The X-axis is the different samples with or without basal heat stress treatment.
The Y-axis represents the relative expression level, and the expression level of each gene in Col-0 under 22 C is set to one. Data are mean ± SEM
(three biological replicates). Asterisks indicate the Student’s t-test significant differences (*P 5 0.05, **P 5 0.01). NS, not significant.
heat stress-responsive genes, including HSFA2, HSFA7a,
HSP90, HSP20-like, and HSP17.6A, were upregulated under
basal heat stress treatment in p35S:ERF95 and p35S:ERF97
compared with the wild-type (Supplemental Figure 5A).
ERF95 and ERF97 expressed at very low levels [reads per kilobase of transcript per million fRPKMg 51], and RNA-seq
data indicated that these two genes were not significantly
upregulated by basal heat stress treatment (Supplemental
Data Set 1). Therefore, we examined the responsiveness of
ERF95 and ERF97 to basal heat stress by RT-qPCR and immunoblot assays. ERF95 and ERF97 were not significantly induced by basal heat stress treatment at the transcript level
(Supplemental Figure 5B) or the protein level (Supplemental
Figure 5C).
As erfq quadruple mutants were hypersensitive to basal
heat stress, we examined expression of those heat stressresponsive HSF and HSP genes in erfq mutants. Expression
levels of HSFA2, HSFA7a, HSP90, HSP20-like, and HSP17.6A
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600 HSFA2 AT2G26150
500
***
*
400
300
200
ns
2
0
Col-0 erfq Col-0 erfq
22°C
43°C
10
1150
P=0
628
vs Col-0-NT
F
8
6
4
2
0
−2
−4
990
p35S:ERF97-NT vs
Col-0-NT DEGs
p35S:ERF95-NT p35S:ERF97-NT
(1941)
0
HT common
Col-0-HT DEGs
(
(2140)
695
E
Cp35S:ERF95-HT vs D
| 347
348
| THE PLANT CELL 2021: 33: 338–357
ERF95 and ERF97 modulate basal heat stress
response through HSFA2 and other common
downstream targets
To better understand how ERF95 and ERF97 regulate basal
heat stress response, due to undetectable protein abundance
in native promoter-driven ERF95 or ERF97 lines, we performed ChIP-seq on p35S:ERF95 and p35S:ERF97 seedlings
before and after basal heat stress treatment. Before treatment, 3,318 peaks linked to 3,993 neighbor genes and 1,642
peaks linked to 2,193 neighbor genes were identified in
p35S:ERF95 and p35S:ERF97 compared with IgG control, respectively (Supplemental Data Set 6). Similar enrichment
patterns were observed for both ERF95 and ERF97 datasets
before and after basal heat stress treatment (Figure 8A). In
total, 1,844 targets were common between p35S:ERF95
(1,844 out of 3,993, 46%) and p35S:ERF97 (1,844 out of
2,193, 84%; Figure 8B) and these targets were enriched for
ethylene-related GO terms and terms related to other hormones (Supplemental Figure 7A). Among these 1,844 genes,
227 genes were also the common DEGs of ERF95 and ERF97
(Figure 8C). De novo discovery of enriched motifs within
ERF95 and ERF97 top 1,000 binding peaks identified the
GCC-box as a top-scoring motif (Figure 8G), which is consistent with previous studies (Zhang et al., 2011; Cheng et al.,
2013). A majority of ERF95 and ERF97 binding peaks were in
the promoter regions (Supplemental Figure 7B), consistent
with that ERF95 and ERF97 function as transcription factors.
After basal heat treatment, 3,139 peaks were assigned to
3,542 neighbor genes and 7,122 peaks were assigned to
7,321 neighbor genes were identified in p35S:ERF95 and
p35S:ERF97 compared with IgG control, respectively
(Supplemental Data Set 6). These binding peaks were also
mainly in promoter regions (Supplemental Figure 7D). The
GCC-box was the top-scoring motif found in ERF95 and
ERF97 binding sites after treatment as well (Figure 8G). A total of 3,218 targets were common between p35S:ERF95
(3,218 out of 3,542, 91%) and p35S:ERF97 (3,218 out of
7,321, 44%; Figure 8D) and were considered as a highconfidence set of ERF95 and ERF97 targets. These targets
were enriched for stress-related GO terms and terms related
to hormones (Supplemental Figure 7C). To assess how many
ERF95 and ERF97 targets were also regulated by heat, we
performed comparison between heat-regulated genes and
ERF95 or ERF97 binding targets after basal heat treatment.
The results of these comparisons showed 644 targets of
ERF95 were heat-regulated genes, 1,353 targets of ERF97
were heat-regulated genes, and 592 genes were common
targets of ERF95 and ERF97 that were regulated by heat
(Figure 8E). To narrow down the genes that may be related
to the ERF95 and ERF97 basal heat stress response phenotype, we compared the ERF95 and ERF97 high-confidence
targets with ERF95 and ERF97-regulated genes and found
that 320 common binding targets were regulated by ERF95
and ERF97 after basal heat stress treatment (Figure 8F), of
which 86 genes were regulated by basal heat stress treatment in wild-type and the majority had higher expression in
p35S:ERF95 and p35S:ERF97 (Figure 8H). This finding suggests
that these genes may contribute to the function of ERF95
and ERF97 in basal heat stress response. Notably, HSFA2,
HSP20-like (AT1G53540) and AtERF#011 were among of
these 86 genes (Figure 8I–K). As HSFA2 is a known heat
stress-responsive gene, we examined the phenotype of hsfa2
mutant and found that hsfa2 seedlings were sensitive to
basal heat stress treatment (Figure 8L), which is consistent
with a previous study (Charng et al., 2007).
To further determine the physiological function of the interaction between ERF95 and ERF97, an EMSA experiment
was performed with recombinant ERF95 and ERF97 proteins
using a DNA probe containing GCC-box (from HSFA2 promoter; Supplemental Figure 8A). ERF95 and ERF97 each can
bind to GCC-box cis-element separately (Supplemental
Figure 8B). The binding of each was abolished with an unlabeled probe in 300 excess of the biotin-labeled probe.
When the competing probe was mutated, binding of both
proteins appeared again, which suggests the specific binding
of ERF95 and ERF97 to the GCC-box. Similar binding results
were obtained with ERF95 and ERF97 proteins together
(Supplemental Figure 8B). Since ERF95 (139 amino acids)
and ERF97 (133 amino acids) have almost the same predicted protein sizes, the binding bands when both proteins
are present more likely represent heterodimer of the two
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were low before treatment and induced by basal heat stress
treatment. All these genes were significantly downregulated
following basal heat stress treatment in the erfq mutants
compared with wild-type (Figure 7E). In addition to these
known heat stress-responsive genes, we observed that several PLANT DEFENSIN (PDF) genes, such as PDF1.2b, PDF1.2c,
and PDF1.3, were induced by basal heat stress and were
highly induced in p35S:ERF95 and p35S:ERF97 lines
(Supplemental Data Sets 3 and 4). Therefore, we examined
the expression of these PDF genes in erfq mutants and
found that the heat induction of these three PDF genes was
abolished in the erfq mutants (Figure 7F). This suggests that
the heat induction of these PDF genes depended on ERF95,
ERF96, ERF97, and ERF98. Together, the upregulation of
HSFA2, HSFA7a, HSPs, and PDFs in p35S:ERF95 and
p35S:ERF97 lines, as well as the downregulation of these
genes in erfq mutants, indicated that these genes may contribute to basal heat stress response mediated by ERF95 and
ERF97.
The top gene ontology (GO) terms enriched among DEGs
from p35S:ERF95-NT and p35S:ERF97-NT were similar to one
another, with main terms related to stress response
(Supplemental Figure 6, A and B; Supplemental Data Set 5).
Under basal heat stress treatment, the GO terms for
p35S:ERF95 and p35S:ERF97 DEGs also had similar enrichments (Supplemental Figure 6, C and D; Supplemental Data
Set 5). The extensive common DEGs and their similar expression profiles, as well as the similar enrichment of GO
terms in DEGs between p35S:ERF95 and p35S:ERF97, reveal
that these two ERF transcription factors regulate highly overlapping subsets of genes.
J. Huang et al.
The Plant Cell, 2021 Vol. 33, No. 2
p35S:ERF97-NT
(2193)
p35S:ERF95-NT p35S:ERF97-NT
(3993)
(2193)
310
1830
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Figure 8 ERF95 and ERF97 modulate basal heat stress response through HSFA2 and other common downstream targets. (A) Heatmap of the enrichment [log2(ChIP/control)] of all peaks (±2 kb) identified from ChIP assays. ERF95-NT and ERF97-NT represent ChIP experiments using
p35S:ERF95 and p35S:ERF97 lines without basal heat stress treatment (NT), respectively. p35S:ERF95-HT and p35S:ERF97-HT are ChIP experiments
using p35S:ERF95 and p35S:ERF97 lines with basal heat stress treatment (HT), respectively. (B) Overlap between p35S:ERF95-NT and p35S:ERF97NT ChIP targets. (C) Overlap among p35S:ERF95-NT ChIP targets, p35S:ERF97-NT ChIP targets, p35S:ERF95-NT DEGs, and p35S:ERF97-NT DEGs
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4102
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3
A
| 349
THE PLANT CELL 2021: 33: 338–357
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| THE PLANT CELL 2021: 33: 338–357
proteins, while each of them likely bind to the probe as a
homodimer. This result, together with the regulation of
HSFA2 expression by ERF95 and ERF97 under basal heat
stress treatment, as well as the direct binding of ERF95 and
ERF97 to the promoter of HSFA2, indicates that ERF95 and
ERF97 may modulate the basal heat stress response through
HSFA2.
Although many studies have shown that ethylene and ethylene signaling components play important roles in responses
to different stresses (Achard et al., 2006; Cao et al., 2007; Lei
et al., 2011; Zhang et al., 2011; Shi et al., 2012; Peng et al.,
2014), less attention has been paid to the role of ethylene in
heat stress. In this study, we established that two interacting
ERFs, ERF95 and ERF97, both of which act downstream of
EIN3 and directly bind to the promoter of HSFA2, are involved in plant basal thermotolerance, elucidating a potential critical role for the EIN3-ERF95/ERF97-HSFA2
transcriptional cascade in the heat stress response (Figure 9).
The p35S:ERF95 and p35S:ERF97 plants as well as the plants
with constitutively activated ethylene responses all show a
resistant phenotype to basal heat stress, indicating that amplifying the ethylene signaling may play a positive role in
basal thermotolerance of plants.
ERF95 and ERF97 play a major role in basal thermotolerance, even though the expression of these genes was barely
induced by heat stress. Our results do not rule out ERF95
and ERF97 being regulated by heat via transcriptional or
posttranscriptional regulation in a specific cell type or being
transiently regulated by heat. How heat regulates ERF95 and
ERF97 expression, at the level of transcription, translation, or
posttranslation, needs further investigation. However, we
have shown that the interaction between ERF95 and ERF97
is heat-inducible (Figure 3F), ERF95 and ERF97 can form a
dimer to bind targets (Supplemental Figure 8), and the binding of ERF95 or ERF97 to some downstream heat-responsive
genes is also enhanced under heat treatment (Figure 8). We
speculate that ERF95 and ERF97 respond to heat stress by
strengthening the interaction between themselves to enhance the binding to downstream targets. Moreover, many
heat stress-responsive genes, such as HSFA2, HSFA7a, and
HSPs, were upregulated by ERF95 and ERF97 ectopic and
constitutive expression but were downregulated in erfq
mutants (Supplemental Figure 5 and Figure 7E;
Supplemental Data Sets 1 and 4). Consistent with this pattern, p35S:ERF95 and p35S:ERF97 plants displayed enhanced
basal thermotolerance, whereas erfq mutants showed reduced basal thermotolerance. A similar phenomenon was
reported for ERF1, the expression of which was slightly induced by heat stress treatment, whereas 35S:ERF1 plants
showed enhanced thermotolerance (Cheng et al., 2013). It
has been speculated that MED25 may help recruit ERF1 to
stress-specific promoters (Cheng et al., 2013). MED25, previously known as phytochrome and flowering time1 (Cerdán
and Chory, 2003), interacts with ERF1, ERF95, ERF98, ERF15,
ORA59, and ERF91 (Ou et al., 2011; Çevik et al., 2012). Our
IP-MS results also revealed an interaction between ERF95
and MED25 (Figure 3A), the latter of which is required for
ERF1, ORA59, and MYC2 to activate transcription (Çevik
et al., 2012) and to play a role in abiotic and biotic stress
responses (Kidd et al., 2009; Elfving et al., 2011).
Furthermore, the binding activity of ERF95 and ERF97 to
heat stress-responsive genes, such as HSFA2 and HSP20-like
appear to increase under heat treatment (Figure 8I and J).
Whether MED25 (or another part of the transcriptional regulatory complex present at stress gene promoters) functions
as a signal integrator to control stress-specific recruitment of
ERFs to promoters deserves further investigation.
Although it has been reported that ERF97 plays a nonredundant role in plant defense responses, as erf97 mutants
are more susceptible to infection by Fusarium oxysporum
but not by Rhizoctonia solani (O~
nate-Sánchez et al., 2007),
no difference was observed between erf97 mutants and
wild-type in basal thermotolerance. ERF95 and ERF97 were
functionally redundant to ERF96 and ERF98, as the basal
heat stress sensitivity increased in higher-order mutants
(Figure 4). ERF1 has been shown to activate specific sets of
stress-responsive genes by targeting specific cis-elements
(Cheng et al., 2013). We infer that ERF97 may also regulate
downstream target genes in a precise stress-specific manner.
The ERF95 and ERF97 ectopic and constitutive expression
lines generated in this study showed different phenotypes
compared with those from previous studies (O~
nate-Sánchez
et al., 2007; Zhang et al., 2011). Zhang et al. (2011) reported
that ERF95 ectopic and constitutive expression results in no
obvious phenotypes, but our ERF95 ectopic and constitutive
expression lines had very obvious phenotypes, with stunted
growth and dark green leaves, especially at the young
Figure 8 (Continued)
identified from RNA-seq. (D) Overlap between p35S:ERF95-HT and p35S:ERF97-HT ChIP targets. (E) Overlap among p35S:ERF95-HT ChIP targets,
p35S:ERF97-HT ChIP targets, and heat-regulated genes in wild-type identified from RNA-seq. P-values in B, D, and E show the statistical significance of the overlap between two groups of genes in Venn diagrams. (F) Overlap among p35S:ERF95-HT ChIP targets, p35S:ERF97-HT ChIP targets,
p35S:ERF95-HT DEGs, and p35S:ERF97-HT DEGs identified from RNA-seq. (G) De novo binding site motif enrichment from p35S:ERF95-NT,
p35S:ERF97-NT, p35S:ERF95-HT and p35S:ERF97-HT ChIP-seq. (H) Heatmap of the expression pattern of 86 basal heat stress treatment-responsive
genes, which are regulated both in p35S:ERF95-HT, p35S:ERF97-HT, and which also the common binding targets of ERF95 and ERF97. The heatmap
shows the Z-score value of log2-transformed [(average RPKM) + 0.001] of each gene. (I–K) Visualization of the binding of ERF95 and ERF97 to
the promoter of HSFA2, HSP20-like (AT1G53540), and AtERF#011, respectively. (L) Phenotype and survival rates of Col-0 and hsfa2 seedlings in
basal thermotolerance assay. Scale bar is 1 cm. Values are mean ± SEM of three biological replicates. Asterisks indicate statistically significant differences (*P 5 0.05) as determined by Student’s t-test.
Downloaded from https://academic.oup.com/plcell/article/33/2/338/6039183 by National Taiwan University user on 08 September 2023
Discussion
J. Huang et al.
The Plant Cell, 2021 Vol. 33, No. 2
THE PLANT CELL 2021: 33: 338–357
EIN3
ERF95
HSFA2
| 351
other heat stressresponsive genes
HSFs
ethylene
HSFA2
HSPs
EIN3
ERF97
HSFA2
PDFs
Heat
Figure 9 Model for the role of ERF95 and ERF97 in plant heat stress response. The ethylene signaling pathway is involved in heat stress responses.
Ethylene signaling component EIN3 directly binds to the promoter of ERF95 and ERF97. ERF95 and ERF97 form hetero- and homo-dimers. ERF95
and ERF97 regulate HSFA2 expression through directly binding to the HSFA2 promoter and also regulate a large number of common downstream
targets including HSFs, HSPs, and PDFs to regulate plant heat stress responses.
seedling stage under normal growth conditions
(Supplemental Figure 1A). Phenotypic inconsistency between these ectopic and constitutive expression lines may
be caused by different expression levels. The weaker line,
p35S:ERF95#6, which had lower levels of ERF95 protein, had
a distinctive phenotype at the young seedling stage, whereas
line p35S:ERF95#5, which had higher levels of ERF95 protein,
exhibited a strong mutant phenotype across the plant’s entire life cycle (Supplemental Figure 1, A and B). O~
nateSánchez et al. (2007) reported that transgenic plants overexpressing ERF97 showed a stunted phenotype from an early
developmental stage, kept producing rosette leaves, never
bolted, and never produced seeds. Our p35S:ERF97 lines had
a similar growth retardation phenotype, with late flowering,
but were able to produce seeds (Supplemental Figure 4, A–
D). However, the stronger line p35S:ERF97#9 produced fewer
seeds than the weaker line p35S:ERF97#7. These observations
suggest that the levels of ERF95 and ERF97 expression are
strongly associated with developmental phenotypes of
plants.
A number of ERF genes from different plant species are
able to confer tolerance to multiple stresses when ectopically expressed (Wang et al., 2016). ERFs display a great degree of functional plasticity and are involved in the
regulation of responses to both biotic and abiotic stresses,
making them prominent candidates for engineering stresstolerant crops. However, as observed for the ectopic constitutive expression of transcriptional regulators, ERF95 and
ERF97 ectopic and constitutive expression led to growth arrest, which would limit biotechnological applications.
Therefore, it is clear that the balance between growth and
stress defense must be optimized to maximize crop yield
and to meet the rising global demand for food and biofuel.
One of the most promising strategies for breeding stressresistant crops is to use host-specific stress-inducible promoters. For example, one approach would be to engineer
the stress-inducible heterologous expression of Arabidopsis
ERF95 and ERF97 in other crops, such as wheat, brassica,
rice, and soybean. Another approach would be to generate
transgenic plants using endogenous ERF95 and ERF97 paralogs. Indeed, some prominent applications of ERFs in crop
breeding have already been reported (Quan et al., 2010;
Rong et al., 2014; Lee et al., 2016; Jung et al., 2017).
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used as
the wild-type in this study. The T-DNA insertion mutants of
erf95 (SALK_128736; Zhang et al., 2011), erf97
(SALK_118494C; O~
nate-Sánchez et al., 2007), and hsfa2
(SALK_008978) were obtained from ABRC and the erf95
erf97 double mutant was generated by crossing the single
mutants. All genotypes were confirmed by PCR. Primer
sequences used for genotyping were listed in Supplemental
Data Set 7. ctr1-1 (Kieber et al., 1993), EIN3ox (Chao et al.,
1997), 35S:EIN3-GFP/ein3 eil1 (He et al., 2011), ein2-5 (Alonso
et al., 1999), ein3 eil1 (Alonso et al., 2003), ESE1ox (Zhang
et al., 2011), cop1-4 (McNellis et al., 1994), pifq (Leivar et al.,
2008), and bzr1-1D (Wang et al., 2002) were described previously. To generate ERF95 (AT3G23220) and ERF97
(AT1G04370) ectopic and constitutive expression lines, the
coding sequences of ERF95 and ERF97 fused with sequences
for 3HA and 3FLAG tags were amplified by PCR and
cloned into binary vector pEarleyGate101 (Earley et al.,
2006).
The
resulting
constructs
(p35S:ERF953HA + 3Flag or p35S:ERF97-3HA + 3FLAG) were
transferred into Agrobacterium tumefaciens GV3101 strain.
Transgenic lines in the background of wild-type (Col-0) were
transformed by the floral dip method (Clough and Bent,
1998). p35S:ERF95/ein3 eil1 and p35S:ERF97/ein3 eil1 lines
were generated in the ein3 eil1 background using the same
plasmids and methods as above. For generating the erfq mutant lines, we employed the CRISPR/Cas9 method (Xing
et al., 2014; Wang et al., 2015b) in the erf95 erf97 double
mutant background. The guide RNAs are listed in
Supplemental Data Set 7. The T1 generation plants were
genotyped by PCR on ERF96 locus and confirmed by
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Heat stress response
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| THE PLANT CELL 2021: 33: 338–357
Basal thermotolerance assay
For basal thermotolerance assay, seeds were sown on 1/2 LS
plates with 0.75% agar and stratified at 4 C for 4 day in the
dark. Then the plates were moved to long day growth
chamber (100 lmol.m–2.s–1, 16-h light and 8-h dark, coolwhite fluorescent bulb) at 22 C. After 7 days, the plates
were sealed with waterproof tape and submerged in 43 C
water bath (Model 4100C, Fisherbrand isotemp heated immersion circulators) under dark for 22 min (basal heat stress
treatment), and then allowed to recover at 22 C for 7 days.
At the end of recovery, images were taken and the survival
rates were recorded. Seedlings that were still green and had
generated new leaves were scored as survivors.
Electrolyte leakage assay
Electrolyte leakage assays were performed as previously described with modification (Li et al., 2017). Seedlings were
harvested before and after basal heat stress treatment,
rinsed twice with Milli-Q water (Millipore-Sigma), placed
into 15-mL tubes containing 8 mL of Milli-Q water and
shaken for 24 h at room temperature before their electrical
conductivity was measured (FiveEasy Cond meter F30,
Mettler Toledo), giving S1. After detecting S1, the samples
were boiled for 30 min and shaken at room temperature for
another 1 h before their electrical conductivity were measured again, giving S2. The electrolyte leakage was calculated
as: S1/S2.
Protein–protein interaction experiments
For Firefly LCI assay, Arabidopsis protoplasts and N. benthamiana leaves were used. Full-length coding regions of
ERF95 and ERF97 were amplified by PCR and cloned to target plasmids. The plasmids used for protoplast transfection
were prepared using the QIAGEN Plasmid Maxi Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s instruction. The different construct combinations were cotransformed into protoplasts isolated from wild-type Col-0
mesophyll as previously described (Yoo et al., 2007). About
5 lg of each plasmid containing N-(pUC19-NLuc) or C-terminal (modified pUC19-CLuc; Zhao et al., 2019) fusions of
Luciferase (LUC) were co-transformed into protoplasts and
the transfected protoplasts were incubated in dark for 16–
20 h before LUC activity was measured using 1 mM luciferin
as substrate (Chen et al., 2008). The relative LUC activity
was recorded using the Tecan Safire2 multi-mode plate
reader.
For testing whether ERF95–ERF97 interaction is heatinducible, LCI assays were performed on N. benthamiana as
previously described with modification (Li et al., 2019).
Briefly, EcoRI and HindIII were used to introduce the NLuc,
CLuc, ERF95-NLuc and ERF97-CLuc from pUC19-NLuc,
pUC19-CLuc, pUC19-ERF95-NLuc, and pUC19-ERF97-CLuc
vectors to binary vector pEarleyGate101. The whole sequence for luciferase was amplified by PCR using primers
Luciferase-attB1 and Luciferase-attB2, then cloned into binary vector pEarleyGate101. The plasmids were transferred
to Agrobacterium tumefaciens GV3101 and co-infiltrated
into leaf epidermal cells of 4-week-old N. benthamiana.
Three days after infiltration, the leaves were sprayed with
firefly luciferase substrate and incubated in darkness for 10
min. Luciferase imaging was performed using a CCD camera
(Andor, iKon-M 934) and PlantLab software (BioImaging
Solutions). After recording the bioluminescence signal from
a leaf, the leaf was surface dried with Kimwipes (KimberlyClark) and the leaf was placed in a plate sealed with waterproof tape and submerged into a 37 C water bath for
15 min. The heat-treated leaves were sprayed with new
luciferase substrate and incubated in dark for 10 min.
The images were recorded using the same settings as before
heat treatment. ImageJ software was used to quantify the
bioluminescence signal.
For BiFC assays, 5 lg of each plasmid containing N- (pUCpSPYNE173) or C-terminal (pUC-pSPYCE (M)) fusions of
YFP (Waadt et al., 2008) were co-transformed into protoplasts and then incubated in dark for 16–20 h. The fluorescence signal was obtained using a Zeiss LSM 710 confocal
laser scanning microscope.
For Co-IP and mass spectrometric experiments,
p35S:ERF95 lines fused with HA and FLAG tag were used.
Generally, 8 g total each Col-0, p35S:ERF95#5, and
p35S:ERF95#6 seedlings grown at 22 C (100 mmol.m–2.s–1,
16-h light and 8-h dark, cool-white fluorescent bulb) for
7 days were collected. Then, 12mL IP buffer (100mM phosphate buffer (pH 8), 150mM NaCl, 5mM EDTA, 5mM EGTA,
0.1% Triton X-100, 1 complete protease inhibitor mixture
(Roche), 75lM MG132, 10mM NaF) was added to the
ground samples and rotated at 4 C for 10 min. The supernatants were separated by centrifugation for 30 min at 4 C,
25,107 g and incubated with 100lL Anti-FLAGV M2
Magnetic Beads (M8823, Millipore-Sigma) for 1 h at 4 C
with rotation. Beads were washed with 1mL IP buffer five
times for 5 min each. Then, bound proteins were eluted
twice using 100 lL of the 3FLAG peptide (MilliporeSigma; 150 ng/lL) at 25 C with rotation for 15 min each.
The mass spectrometric analysis was performed at the Mass
Spectrometry Core of the Salk Institute for Biological Studies
as previous described (Zhao et al., 2019).
For Co-IP and immunoblotting, the Agrobacterium tumefaciens GV3101 cells harboring p35S:ERF95-FLAG and
p35S:ERF014-HA or p35S:ERF95-FLAG, and p35S:ERF97-HA expression vector were mixed and co-infiltrated into leaf epidermal cells of 4-week-old N. benthamiana. Three days after
infiltration, two N. benthamiana leaves were homogenized
R
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sequencing on ERF98 locus. The homozygous T3 lines were
used.
Seeds were surface sterilized using chlorine gas for 4 h and
plated on half strength LS (1/2 LS) medium pH 5.7 (Caisson
Laboratories, North Logan, UT) with 0.75% agar. After 4-day
stratification under dark at 4 C, plates were moved to
growth chamber (Long day, 100 lmol.m–2.s–1, 16-h light
and 8-h dark, cool-white fluorescent bulb) at 22 C for the
indicated number of days.
J. Huang et al.
The Plant Cell, 2021 Vol. 33, No. 2
THE PLANT CELL 2021: 33: 338–357
R
Whole-genome RNA-seq and analysis
For RNA-seq experiment, plants (Col-0, p35S:ERF95#6,
p35S:ERF97#9) were grown under long day conditions (100
lmol.m–2.s–1, 16-h light and 8-h dark, cool-white fluorescent
bulb) for 7 days at 22 C. We treated half plates with 43 C
water bath for 22 min and then collected the heat-treated
RNA samples and control RNA samples from 22 C as well.
Two biological replicates were used for RNA-seq analysis.
Total RNA was extracted using the RNeasy Plant Mini Kit
(Qiagen) following the manufacturer’s instructions and
digested with DNase I. Single-end sequencing was performed
on Illumina HiSeq 2500 platform at the Next-Generation
Sequencing Core of the Salk Institute for Biological Studies.
BRB-SeqTools (https://brb.nci.nih.gov/seqtools/) was used
for RNA-seq data analysis. Briefly, RNA-seq reads were
aligned to the Arabidopsis reference genome (TAIR10) using
TopHat version 2.1.1 (Kim et al., 2013) and gene-level raw
count data files were generated using HTSeq version 0.6.0
(Anders et al., 2015). The raw count data was imported into
Bioconductor package edgeR (Robinson et al., 2010) in R
language to identify the DEGs, and to calculate the RPKM
mapped reads of each gene. When determining the DEGs, a
gene was retained only if it was expressed at a count-permillion 40.5 in at least two samples. Those genes had a
fold change 1.5 or more or –1.5 or less with an false discovery rate 40.05 were considered as DEGs. GO-enrichment
analysis was determined using agriGO (Du et al., 2010). The
Venn diagrams were generated using interactivenn (http://
www.interactivenn.net/; Heberle et al., 2015).
Protein expression and purification
The sequence encoding an EIN3 fragment (68–352 aa) was
amplified by PCR using primers EIN3N-PGEX4T-F and
EIN3N-PGEX4T-R, then cloned into pGEX-4T-GW vector.
The BL21-CodonPlus (DE3)-RIL competent cells (Agilent
Technologies, Santa Clara, CA) were transformed with the
corresponding vector. Cells were grown at 37 C in 1L terrific
broth (TB) medium with 50 lg/mL ampicillin. Protein expression was induced when the cell culture reached an
OD600 of 0.6 by adding 0.1 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG) for 18 h at 20 C. The cell culture was harvested by centrifugation for 10 min at 4,000g. The pellet was
resuspended in 50 mL of cold buffer 1 (10 mM Tris, 150
mM NaCl, 5% glycerol, and 1 mM TCEP, pH7.7, and 1
Protease inhibitor cocktail), sonicated with a Sonic
Dismembrator (Fisher Scientific, Hampton, USA), and then
centrifuged for 30 min at 75,600g. The supernatant was
loaded onto a pre-equilibrated glutathione–Sepharose 4B
column (GE Healthcare, Little Chalfont, UK) and washed
twice with buffer 1, once with 0.5% Tween buffer (250 lL
Tween to 50 mL buffer 1), once with buffer 2 (50 mM Tris,
50 mM KCl, 20 mM MgCl2, 5 mM ATP, 1 mM TCEP, pH
7.7), once with buffer 3 (50 mM Tris, 2 M NaCl, 1 mM
TCEP, pH7.7), and once with buffer 1. The GST tag was removed by adding human rhinovirus (3C) protease on the
column at 4 C overnight. EIN3 protein was eluted in two 5
mL steps using buffer 1. The eluate was concentrated using
a centrifugal filter (Amicon Ultra-10K, Millipore-Sigma, USA)
for buffer exchange according to the manufacturer’s
protocol.
The coding sequence of ERF95 was amplified by PCR using
primers ERF95-pGEX4T-F and ERF95-pGEX4T-R, then cloned
into pGEX-4T-GW vector. The coding sequence of ERF97
was amplified by PCR using primers ERF97-attB1 and ERF97attB2, then cloned into pDEST-HISMBP vector. The BL21CodonPlus (DE3)-RIL competent cells (Agilent Technologies)
were transformed with the corresponding vector. Cells were
grown at 37 C in 1L TB medium with 50 lg/mL ampicillin.
Protein expression was induced when the cell culture
reached an OD600 of 0.6–0.8 by adding 1 mM IPTG for 1 h
at 37 C. The recombinant proteins were purified using either glutathione beads (GE Healthcare) or amylose resin
(NEB).
Electrophoretic mobility shift assay
EMSA assays were performed using the LightShift
Chemiluminescent EMSA Kit (Thermo Scientific, Waltham,
MA). Briefly, EIN3, ERF95 and ERF97 proteins were purified
as above. EIN3 protein-binding reaction was performed in a
total volume of 20 lL by incubation of an appropriate 2 lg
purified EIN3 protein with 200 fmol biotin-labeled DNA
probe, in binding buffer [10 mM Tris, pH 7.5, 50 mM KCl, 1
mM DTT, 3 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 lg
poly fdI-dCg] at room temperature for 20 min. ERF95 and
ERF97 protein binding reactions were performed in a total
volume of 20 lL by incubation of purified ERF95 and ERF97
proteins with 200-fmol biotin-labeled DNA probe, in binding
buffer [10 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 10%
glycerol, 1 lg poly fdI-dCg] at room temperature for 20
min. The binding products were resolved in 6% polyacrylamide gel running in 0.5 Tris-borate-EDTA buffer. The
specimens were electrotransferred onto a 0.45-lm HybondN nylon membrane (Amersham Pharmacia biotech, Little
Chalfont, UK) at 380 mA for 30 min at 4 C, and crosslinked to the membrane using a UV Stratalinker 1800
(Stratagene, San Diego, CA). The blots were developed using
Downloaded from https://academic.oup.com/plcell/article/33/2/338/6039183 by National Taiwan University user on 08 September 2023
in 3mL protein lysis buffer (50 mM Tris, pH 7.6; 150 mM
NaCl; 5 mM MgCl2; 10% glycerol; 0.1% NP40, 1 complete
cocktail protease inhibitors, 50 lM MG132, and 1 mM
PMSF). The lysate was cleared by centrifugation. The supernatants were incubated with 30 lL Anti-FLAGV M2
Magnetic Beads (M8823, Millipore-Sigma) for 2 h at 4 C
with rotation. The beads were then washed eight times for
5 min with 1 mL of lysis buffer and resuspended in 40 lL
2SDS–PAGE loading buffer. The proteins were detected
by immunoblotting using either anti-FLAG M2 monoclonal
peroxidase-conjugated antibody (A8592, Millipore-Sigma) at
a dilution of 1:5,000 or anti-HA monoclonal peroxidaseconjugated antibody (12013819001, Millipore-Sigma) at a dilution of 1:1,000. All immunoblots were developed using the
SuperSignalTM West Pico Plus Chemilunescent Substrate
(34580, Thermo scientific) or SuperSignalTM West Femto
Maximum Sensitivity Substrate (34094, Thermo scientific).
| 353
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| THE PLANT CELL 2021: 33: 338–357
the Chemiluminescent Nucleic Acid Detection Module Kit
(Thermo Scientific).
J. Huang et al.
– a 2000 – b 2000 – bs = 10). Finally, the data were plotted
using the plotHeatmap tool as previously described (Zhou
et al., 2018).
ChIP, ChIP-Seq, and analysis
Reverse transcription-qPCR analysis
RNA samples were isolated from plants treated as in basal
thermotolerance assay using the RNeasy Plant Mini Kit
(Qiagen) following the manufacturer’s instruction and
treated with DNase I (Qiagen). The first-strand cDNA was
synthesized using Maxima First Strand cDNA Synthesis Kit
for RT-qPCR (Thermo). qPCR was performed on a
TM
CFX384
Real-Time PCR Detection System (Bio-rad) using
TM
iTaq Universal SYBRV Green Supermix (Bio-rad). The following thermal conditions were used: 95 C for 3 min, 45
cycles of 95 C for 10 s and 60 C for 30 s. Expression levels
for all assayed genes were normalized using PP2AA3
(AT1G13320) as the internal control. Primers used for RTqPCR are listed in Supplemental Data Set 7.
R
Accession numbers
Sequence data from this article can be found in the
GenBank/EMBL data libraries under the following accession
numbers: ERF95, AT3G23220; ERF96, AT5G43410; ERF97,
AT1G04370; ERF98, AT3G23230; ERF014, AT1G44830;
HSFA2, AT2G26150; HSFA7a, AT3G51910; HSP90.1,
AT5G52640; HSP17.6A, AT5G12030; PDF1.2b, AT2G26020;
PDF1.2c, AT5G44430; and PDF1.3, AT2G26010. The raw
RNA-seq data and ChIP-seq data have been deposited into
the National Center for Biotechnology Information (NCBI)
Gene Expression Omnibus database under the accession
number GSE148745.
Supplemental Data
Supplemental Figure 1. Phenotype of p35S:ERF95 lines under normal growth condition and tolerant phenotype of
ESE1ox plants in basal thermotolerance assay.
Supplemental Figure 2. Some mutants with smaller plant
size than wild-type are sensitive to basal heat stress
treatment.
Supplemental Figure 3. Heat stress decreases luciferase
activity in N. benthamiana leaves.
Supplemental Figure 4. Phenotype of p35S:ERF97 lines
and generation of erfq quadruple mutants using CRISPR/
Cas9 system.
Supplemental Figure 5. Expression level of some heatresponsive HSFs, HSPs in p35S:ERF95 and p35S:ERF97 lines
and the expression pattern of ERF95, ERF97 under basal
heat stress treatment.
Supplemental Figure 6. Heatmaps showing the top 30
GO term enrichment of p35S:ERF95 and p35S:ERF97 DEGs.
Supplemental Figure 7. Heatmaps showing the top 30
GO term enrichment of p35S:ERF95 and p35S:ERF97 common targets before and after heat stress, and the genomic
distribution of ERF95 and ERF97 binding peaks.
Supplemental Figure 8. ERF95 and ERF97 can bind to
the downstream target as a homodimer and a heterodimer.
Downloaded from https://academic.oup.com/plcell/article/33/2/338/6039183 by National Taiwan University user on 08 September 2023
ChIP assay for EIN3 was performed as described previously
(Song et al., 2016). In brief, 4 g of 7-day old seedlings grown
under long day condition were harvested and cross-linked
within 1% formaldehyde solution for 20 min by vacuum.
Cross-linked was quenched by adding 2 M glycine (final concentration to 125 mM) under vacuum for 5 min. The chromatin was then isolated and sonicated with Bioruptor
(Diagenode). Chromatin from 35S:EIN3-GFP/ein3 eil1 and
ein3 eil1 seedlings was immunoprecipitated with anti-GFP
antibody (11814460001, Millipore-Sigma). Quantitative PCR
reactions were performed using the CFX384 Real-Time PCR
Detection System.
ChIP-seq experiments using 7-day-old seedlings treated
with and without basal heat stress treatment were performed as previously described (Kaufmann et al., 2010;
Zander et al., 2019). Two biological replicates were used
for ChIP-seq analysis. In brief, plants (Col-0, p35S:ERF95#6,
p35S:ERF97#9) were grown under long day condition (100
lmol.m–2.s–1, 16-h light and 8-h dark, cool-white fluorescent bulb) for 7 days at 22 C, then half of the plants were
treated in 43 C water bath for 22 min for basal heat stress
treated samples and the rest plants were kept under 22 C
for nonheat-treated samples. Monoclonal anti-HA antibody (11867423001, Millipore-Sigma) was used for IP and
mouse IgG (015-000-003, Jackson Immuno Research) was
served as the negative control. Multiplexed libraries were
sequenced on an Illumina HiSeq 2500 at the NextGeneration Sequencing Core of the Salk Institute for
Biological Studies.
Sequencing reads were aligned to TAIR10 genome using
Bowtie2 version 2.2.7 with default parameters (Langmead
and Salzberg, 2012). SAMtools (Li et al., 2009) was used to
merge biological ChIP-seq replicates. MACS2 version 2.2.5
(Zhang et al., 2008) was used to call peaks using q value
0.001 with IP from p35S:ERF97 by anti-IgG antibody as a
control. Peaks were then associated to TAIR10 annotated
genes using R BioConductor package ChIPseeker with TSS
region = ± 1 kb, addFlankGeneInfo = TRUE, and
flankDistance = 1,000 (Yu et al., 2015). Peaks were visualized
using IGV browser (Thorvaldsdóttir et al., 2013).
AnnotatePeak within ChIPseeker was used to annotate the location of a given peak in terms of genomic features. Bedtools
(Quinlan and Hall, 2010) was used to get the total 100-bp
flanking the binding summits of 1,000 top-ranking peaks for
each experiment and MEME-ChIP suite (http://meme-suite.
org/tools/meme-chip; Machanick and Bailey, 2011) was used
for identification of each DNA binding motif. The ChIP enrichment relative to IgG controls was visualized using
deepTools (Ramı́rez et al., 2016). Briefly, the sorted bam files
derived from Bowtie2 mapping were compared to IgG control
using the bamCompare tools, and the resulting bigwig files
were used to generate a data matrix using the
computeMatrix tool (reference-point – referencePoint center
The Plant Cell, 2021 Vol. 33, No. 2
Acknowledgments
We thank members of Chory Lab for stimulating discussion
on this project. We thank Drs. David O’Keefe and Xuelin Wu
for comments and text editing; Drs Zuyu Zheng and
Wenrong He for providing seeds of ethylene-related mutants;
Dr Rongfeng Huang for providing ESE1ox; Dr Yogev Burko
for providing cop1-4, Dr Björn C. Willige for providing pifq,
and Dr Zhi-yong Wang for providing bzr1-1D seeds. We
thank James Moresco and Jolene Diedrich for technical support. We thank Dr Mark Zander and Dr Ming Zhou for the
help in ChIP-seq.
Funding
This research was supported by Howard Hughes Medical
Institute, National Institutes of Health (NIH) funding
(R35GM122604), and the U.S. Department of Energy funding
(DE-FG02-04ER15540) to J.C. X.Z. is supported by the
Hundred-Talent Program of Zhejiang University and the
Fundamental Research Funds for the Central Universities
(2020QNA6020), Zhejiang University, China. RNAsequencing and mass spectrometric analyses were performed
at the Next Generation Sequencing Core and Mass
Spectrometry Core of the Salk Institute for Biological Studies
supported by NIH-National Cancer Institute Grant CCSG:
P30 014195, the Chapman Foundation, the Helmsley
Charitable Trust, and the Helmsley Center for Genomic
Medicine.
Conflict of interest statement. The authors declare no conflict
of interests.
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