Molecular Plant

Research Article

Targeting Plant Ethylene Responses by

Controlling Essential Protein–Protein
Interactions in the Ethylene Pathway
Melanie M.A. Bisson and Georg Groth*
Institute of Biochemical Plant Physiology, Heinrich-Heine University Du¨sseldorf, D-40204 Du¨sseldorf,
Germany *Correspondence: Georg Groth (georg.groth@hhu.de)
http://dx.doi.org/10.1016/j.molp.2015.03.014

ABSTRACT
The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life
span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane
protein ETHYLENE INSENSITIVE2 (EIN2). Recent studies indicate that in response to ethylene, the extra-
membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus.
Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-
terminus provides an important domain for complex formation with ethylene receptor ETHYLENE RESPONSE1
(ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and
ETR1 is also blocked by a synthetic peptide of the NLS motif. The corre-sponding peptide substantially reduces
ethylene responses in planta. Our results uncover a novel mecha-nism and type of inhibitor interfering with
ethylene signal transduction and ethylene responses in plants. Disruption of essential protein–protein
interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the
potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.

Key words: fruit ripening, ethylene responses, peptide, protein-protein interactions, inhibitor
Bisson M.M.A. and Groth G. (2015). Targeting Plant Ethylene Responses by Controlling Essential Protein–
Protein Interactions in the Ethylene Pathway. Mol. Plant. 8, 1165–1174.

INTRODUCTION insensitivity to the plant. Sequence analysis suggests that EIN2

The plant hormone ethylene is a key regulator for plant growth,
development, and stress adaptation (Johnson and Ecker,
1998). Most of what is known about signal perception and
transduction of the simple unsaturated hydrocarbon ethylene
has been established by genetic studies in the model plant
Arabidopsis thaliana. Decades of research have resulted in the
identification of many elements of the ethylene signal
transduction pathway, including both negative and positive
regulators. At the beginning of the signaling cascade, a family of
endoplasmic reticulum (ER)-located receptors senses the
ethylene signal and functions as negative regulators of the
ethylene response (Bleecker et al., 1988; Chang et al., 1993;
Hua et al., 1995; Hua and Meyerowitz, 1998; Hua et al., 1998;
Sakai et al., 1998). Downstream of the receptors, the
membrane protein ETHYLENE INSENSITIVE2 (EIN2)
implements a positive regulatory role on ethylene signaling
(Alonso et al., 1999). The integral membrane protein holding
similarity to metal-ion transporters was identified as the most
crucial step in ethylene signaling since EIN2 is the only gene
whose loss-of-function mutation confers complete ethylene
shows a pronounced bipartite structure consisting of a hydro-
phobic amino-terminal domain of 12 transmembrane helices
(amino acids [aa] 1–461) and an extensive hydrophilic carboxyl-
terminal part (aa 462–1294) (Alonso et al., 1999). Previous
studies from our laboratory revealed that EIN2 is located at the
ER membrane where it interacts with the kinase domain of the
ethylene receptors via its carboxyl-terminal domain (Bisson et
al., 2009; Bisson and Groth, 2010). As auto-phosphorylation of
the kinase domain is directly related to the binding of the plant
hormone in the membrane domain of the receptors (Voet-van-
Vormizeele and Groth, 2008), interaction of receptors and EIN2
is susceptible to the plant hormone. A nuclear localization signal
(NLS) was identified in the carboxyl-terminal part of EIN2 at aa
1262–1269 (Bisson and Groth, 2011), supporting the idea that
the carboxyl-terminal part of EIN2 is cleaved of the mem-brane
domain and is translocated to the nucleus. Such NLS-
dependent translocation of proteolytically cleaved EIN2 from
Published by the Molecular Plant Shanghai Editorial Office in association with Cell
Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015. 1165

Molecular Plant NLS-Derived Peptide Affects Ethylene Responses

Figure 1. Nuclear Localization Signal (NLS) in EIN2 Is Required for EIN2-ETR1 Complex Formation.
(A–D) Subcellular localization of C-terminal EIN2 479 1294 and EIN2479 1294(DNLS) transiently expressed in Nicotiana benthamina epidermal leaf cells,
analyzed by confocal laser scanning microscopy. (A) DAPI staining of cells expressing NLS-deleted EIN2 479 1294(DNLS)-GFP. Nuclei are indicated by
white arrows. (B) DAPI staining of cells expressing EIN2 479 1294-GFP. Nuclei are indicated by white arrows. Yellow arrows indicate putative ER locali-
zation. (C) Co-localization of EIN2479 1294-GFP and ER-mCherry marker protein at the ER membrane. (D) Cytosolic localization of EIN2 479 1294(DNLS)-
GFP co-expressed with ER-mCherry marker protein. Scale bars in (A–D) represent 5 mm.
(E and F) The NLS domain in EIN2 is essential for EIN2-ETR1 interaction. (E) FRET efficiencies calculated from transient expression of GFP-tagged
EIN2(DNLS) and mCherry-tagged ETR1 in tobacco leaf cells underline that EIN2 lacking the NLS domain is not able to form a tight complex with the
ETR1 receptor. (F) Tryptophan fluorescence-based in vitro protein–protein interaction assay between ETR1 and EIN2 479 1294(DNLS) indicates
unspecific interaction of EIN2 lacking the NLS domain (EIN2 479 1294(DWDNLS)) with ethylene receptor ETR1.

the ER to the nucleus was recently demonstrated by Qiao et al.
(2012). Similar results on EIN2 processing and subcellular
translocation of the EIN2 C-terminus to the nucleus in response
to ethylene were shown by Wen et al. (2012) and Ju et al.
(2012). Moreover, these studies emphasized a critical role of the
ER-associated protein kinase CONSTITUTIVE TRIPLE
RESPONSE1 (CTR1), which was shown to directly
phosphorylate EIN2 in the absence of ethylene (Ju et al., 2012).
The presence of ethylene results in a lack of EIN2
phosphorylation, which facilitates proteolytic cleavage and
nuclear translocation of the EIN2 C-terminus where it stabilizes
the transcription factor EIN3 and activates ethylene responses.
In this article, we show that the NLS of EIN2 fulfills a dual function.
Besides its known role for nuclear import, it is also fundamental for
complex formation with the ethylene receptor family members at the
ER membrane. In fact, this complex may provide the scaf-fold for
the phosphorylation-dependent proteolytic processing of EIN2. Our
studies on the role of the NLS motif for complex forma-tion between
EIN2 and the ethylene receptor protein ETHYLENE RESPONSE1
(ETR1) further reveal that basic peptides derived from the NLS motif
in EIN2 are potent inhibitors of ethylene signaling. These peptides
are able to block formation of the
1166 Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015.
EIN2-ETR1 signaling complex in vitro and can reduce typical
ethylene responses in planta such as the triple response in
devel-oping seedlings or fruit ripening in climacteric fruits.
RESULTS AND DISCUSSION
The NLS Motif in EIN2 Is Required for Interaction with
Ethylene Receptor ETR1
Previous studies revealed that the isolated EIN2 C-terminus (aa
462–1294) is sufficient to activate ethylene responses in an ein2
loss-of-function background (Alonso et al., 1999). To highlight the
role of the NLS motif in this process, we have analyzed the
subcellular localization of the isolated carboxyl-terminal part of
479 1294
EIN2 (EIN2 ) fused to green fluorescent protein (GFP)
479–1294
and of an NLS deletion mutant (EIN2 (DNLS)-GFP) by
confocal laser scanning microscopy. As expected, the NLS dele-
479 1294
tion mutant of our EIN2 (DNLS)-GFP was found only in the
cytosolic fraction of the cell and no nuclear localization of the
EIN2 C-terminus was observed in these studies (Figure 1A). In
contrast, the carboxyl-terminal EIN2 containing the NLS
sequence was localized at the nucleus, even in the absence of
ethylene (Figure 1B, white arrows). Studies by Wen et al.
NLS-Derived Peptide Affects Ethylene Responses
(2012) using a similar EIN2-CEND corresponding to residues
459–1294 and the related NLS deletion mutant confirm these
localization studies, which conform to the ethylene-dependent
cleavage and translocation mechanism reported for full-length
EIN2 (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012).
479 1294
However, the EIN2 -GFP construct used in our studies
lacking 20 residues at the N-terminus compared with the
construct used by Wen et al. (2012), was not exclusively
localized to the nucleus but also to network-like structures remi-
niscent of ER membranes (Figure 1B, yellow arrows). To clarify
479
the identity of these structures, we co-expressed both EIN2
1294
-GFP fusion proteins together with a red fluorescent
ER-marker protein (ER-mCherry). These studies confirmed that in
addition to its nuclear localization, the isolated EIN2 C-terminus
carrying the endogenous NLS signature also localizes at the ER
membrane (Figure 1C) as shown for full-length EIN2 in previous
studies (Bisson et al., 2009). On the other hand, deletion of the NLS
domain prevents nuclear localization or association of the EIN2 C-
terminus with the ER membrane. The deletion mutant exclusively
localizes to the cytoplasm (see Figure 1D). In
accordance with our results demonstrating ER localization of
479 1294
EIN2 -GFP, recent results from Ju et al. (2015) showed
that the isolated C-terminal part corresponding to residues 432–
1069 of EIN2 from Spirogyra pratensis (SpEIN2-C-term-GFP) also
localizes to the ER membrane in the absence of ethylene. Based on
the results of our localization studies with the isolated EIN2 C-
terminus, we propose that the NLS motif be-sides its function in
nuclear localization also facilitates localization of the extra-
membranous EIN2 domain to the ER membrane. Taking further into
account ER localization of the ethylene recep-tor family (Grefen et
al., 2008), their tight interaction with the EIN2 C-terminus (Bisson
and Groth, 2010) and a sequence identity of 82% for the
Arabidopsis and tobacco receptor protein ETR1 (Supplemental
Figure 1), the results from our confocal microscopy studies imply
that the NLS motif is required for tight interaction of EIN2 with the
ethylene receptor family at the ER membrane. In concordance, Ju
et al. (2015) attributed the observed ER localization of the C-
terminal part of SpEIN2 to complex formation with ER-localized
ethylene signaling com-ponents. In order to test our hypothesis that
the NLS motif in EIN2 facilitates EIN2-ETR1 complex formation, we
performed in planta and in vitro interaction studies with the full-
length EIN2(DNLS) protein and the ethylene receptor prototype
ETR1. Confocal fluorescence resonance energy transfer (FRET)
studies were carried out with GFP-tagged EIN2(DNLS) as donor
and mCherry-tagged ETR1 as acceptor in order to analyze the
inter-action of both proteins in planta (Figure 1E). Complex
formation of EIN2-GFP and ETR1-mCherry observed in previous
studies correlates to a FRET efficiency (FRET eff) of 16.4%, a
number that compares with the positive control (GFP-mCherry EIN2
fusion) indicating tight interaction of both proteins (Bisson and
Groth, 2010). In contrast, a transfer efficiency of only 3.7% was
obtained for the FRET pair EIN2(DNLS)-GFP and ETR1-mCherry.
This number is only slightly above the background fluc-tuation of
1.9%, emphasizing that the interaction of ETR1 and EIN2 critically
depends on the NLS domain in EIN2.
To quantify the effect of the NLS sequence on the EIN2-ETR1
interaction, we made use of the intrinsic protein tryptophan fluo-
rescence. Titration of the purified recombinant tryptophan-free
479 1294
carboxyl-terminal part of EIN2 (EIN2 (DW)) to wild-type
Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015. 1167
Molecular Plant
ETR1 was monitored by the associated fluorescence quench that
was used to calculate the dissociation constant (Kd) of the EIN2-
ETR1 complex. In agreement with previous studies, a Kd of 0.4 mM,
corresponding to a highly specific interaction, was ob-
tained for both interaction partners (Bisson et al., 2009). However,
479 1294
when the tryptophan-free EIN2 lacking the NLS sequence
479 1294
(EIN2 (DWDNLS)) was applied in the tryptophan quench-
ing assay, the dissociation constant was substantially enhanced
2
to 21 ± 7.5 mM (c = 0.00015) corresponding to an
approximately 50-fold decrease in EIN2-ETR1 affinity (Figure
1F). The results from our in planta and in vitro studies clearly
demonstrate that the NLS domain in EIN2 is essential for the
interaction of EIN2 with the receptor proteins.
Synthetic Peptides of the NLS Motif Block Interaction of
EIN2 and ETR1
In order to validate our hypothesis that the NLS domain of EIN2 is
required for EIN2-ETR1 complex formation, we used a synthetic
peptide corresponding to the NLS domain (H2N-LKRYKRRL-COOH)
and studied whether this peptide competes with the carboxyl-
terminal part of EIN2 for the binding site in ETR1, thereby disrupting
the EIN2-ETR1 interaction. A random sequence peptide (H 2N-
EFLYMSVN-COOH) determined by the ExPASy Random Protein
Sequence Generator (http://web.expasy.org/ randseq) was used as
a negative control. The specific peptide was named nuclear
localization signal octapeptide 1 (NOP-1) and the control peptide
was termed random octapeptide 1 (ROP-1). The effect of these two
octapeptides on the EIN2-ETR1 interaction was analyzed by our
tryptophan fluorescence-based quantitative assay with the purified
recombinant proteins. Increasing concentrations of NOP-1 or ROP-1
were used in these experiments. Figure 2A illustrates that even at
high concentrations, the affinity between EIN2 and ETR1 is hardly
affected by the random peptide ROP-1. In contrast, we observed
that the affinity of EIN2 for ETR1 is dramatically reduced in the
presence of even minor quantities of NOP-1. Dissociation con-
stants of the EIN2-ETR1 complex show a progressive increase at
increasing concentrations of NOP-1, which strongly supports
competitive displacement of EIN2 by the specific peptide.
To further explore the effect of the NOP-1 peptide on the EIN2-
ETR1 interaction, we repeated our in planta confocal FRET
studies with GFP-tagged EIN2 and mCherry-tagged ETR1 in the
presence of both octapeptides. Figure 2B indicates that the
transfer efficiency between both signaling partners has hardly
changed in the presence of the random sequence peptide ROP-
1 (100 mM) compared with unaffected EIN2-ETR1 complex
formation (Figure 2B, left column). These data confirm that the
random sequence peptide has no effect on the EIN2-ETR1
interaction also in planta. On the other hand, addition of the
same concentration of NOP-1 reduced transfer efficiency to
background level. Hence, in line with our in vitro studies, the
NLS-derived octapeptide competes with EIN2 for ETR1 binding
also in planta and efficiently suppresses EIN2-ETR1 complex
formation.
To analyze the inhibitory effect of NOP-1 on EIN2-ETR1 complex
formation in intact plants, we tested the triple response of dark-
grown Arabidopsis seedlings, which is known as a typical ethylene
response (Guzma´n and Ecker, 1990). As illustrated in
Molecular Plant
Figure 2C, wild-type etiolated Arabidopsis seedlings (ecotype
Columbia) show the typical triple response consisting of an inhibition
of hypocotyl and root elongation, exaggeration of the apical hook,
and swelling of the hypocotyl when treated with the ethylene
precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In the
absence of ACC, seedlings showed normal growth. When grown in
the presence of 200 mM NOP-1, the triple response caused by the
addition of ACC is less pronounced. The apical hook in these plants
is less exaggerated and especially the hypocotyl is longer and
thinner compared with the normal tri-ple response. Application of
the random sequence peptide ROP-1 had no such effect on the
seedlings and plants showed a rather unaffected hypocotyl growth.
However, seedlings treated with either NOP-1 or ROP-1 showed
elongated primary roots compared with seedlings grown in the
presence of ACC alone (Figure 2D). This elongated root growth may
be caused by the high peptide concentration in the growth medium
as previously described for other external proteins and peptides
(Paungfoo-Lonhienne et al., 2008; Lonhienne et al., 2014).
Statistical analysis of the hypocotyl length of the seedlings given in
Figure 2D confirms that the ethylene response of the Arabidopsis
seedlings is indeed affected in a concentration-dependent manner
by the specific peptide NOP-1. To validate this observation, the
hypocotyl length of dark-grown seedlings in the presence of ACC
and various concentrations of NOP-1 and ROP-1 was analyzed. As
illustrated in Supplemental Figure 2, the presence of NOP-1 leads
to significant elongated hypocotyl growth at different concentrations,
whereas ROP-1 was uninfluential. Based on these results, we
conclude that the Arabidopsis seedlings, known from previous
studies to assimilate external peptides and proteins (Paungfoo-
Lonhienne et al., 2008), also assimilate the peptides NOP-1 and
ROP-1 from the growth medium. In consequence, assimilated NOP-
1 was able
1168 Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015.
NLS-Derived Peptide Affects Ethylene Responses
Figure 2. NLS-based Peptide NOP-1 Inhibits
Ethylene Responses In Vitro and In Vivo.
(A) Kd values of the EIN2-ETR1 complex obtained
by titration of ETR1 with EIN2479 1294(DW) as
a function of synthetic octapeptides NOP-1 and
ROP-1, respectively. NOP-1 strongly de-creases
complex formation of EIN2-ETR1 in a
concentration-dependent manner; ROP-1 had no
effect.
(B) FRET-based in planta interaction assay
performed in the presence of the synthetic octa-
peptides. NOP-1 prevents EIN2-ETR1 complex
formation in tobacco cells, ROP-1 was unin-
fluential. EIN2-ETR1 complex formation in the
absence of any peptides was used as positive
control.
(C) Effect of 200 mM octapeptides on the triple
response of dark-grown wild-type Arabidopsis
(ecotype Columbia) seedlings. NOP-1 leads to a
less pronounced triple response. ROP-1 had no
such effect.
(D) Synthetic peptide NOP-1 inhibits ethylene
response (hypocotyl length) in Arabidopsis in a
concentration-dependent manner. Asterisks
represent statistical significance (*P < 0.05
compared with the positive control [no NOP-1]).
to inhibit EIN2-ETR1 complex formation resulting in a reduced
tri-ple response of the seedlings.
Structure Activity Analysis of the Inhibitory Effect of the
NLS Motif on EIN2-ETR1 Complex Formation
To identify the molecular structures in the NLS motif that destabi-
lize EIN2-ETR1 complex formation, we set up a fluorescence-based
screening assay to study the EIN2-ETR1 interaction in a format
providing larger screening potential than the tryptophan quenching
assay. The assay is readily adaptable to medium- or even high-
throughput screening, includes a negative control, and permits
quantitative correlation of the peptide sequence and composition
with the potency to disrupt EIN2-ETR1 complex formation. To this
end, purified recombinant receptor protein
ETR1 was labeled with fluorescent dye Alexa 488, while the puri-
479 1294
fied recombinant EIN2 protein was labeled with fluores-
cent dye Alexa 568 (Panchuk-Voloshina et al., 1999). The
emission spectrum of the resulting ETR1-Alexa 488 partially over-
479 1294
laps with the excitation spectrum of the labeled EIN2 -
Alexa 568 (Figure 3A) allowing energy transfer (FRET) from the
479 1294
ETR1-Alexa 488 donor to the EIN2 -Alexa 568 acceptor.
Defective EIN2-ETR1 complex formation results in an increased
distance of Alexa fluorophores in both binding partners and a
related reduced FRET from the donor to the acceptor, which is
manifested by a decreased emission signal of the EIN2
479 1294
-
Alexa 568 acceptor. Figure 3 emphasizes that this reduced
acceptor emission is indeed observed with the fluorescence
labeled EIN2 in the presence of 100 mM NOP-1 peptide. In
contrast, 100 mM control peptide ROP-1 had no effect on
acceptor fluorescence (Figure 3B). None of the peptides affects
the fluorescence of the isolated Alexa dyes (Figure 3C)
underlining that the observed reduction with the labeled
NLS-Derived Peptide Affects Ethylene Responses Molecular Plant
Figure 3. FRET-Based In Vitro Assay to
Monitor the Inhibition of EIN2-ETR1 Inter-
action.
(A) Overlapping emission and excitation spectra
of Alexa dye-labeled recombinant proteins ETR1
and EIN2479 1294.
(B) Inhibitory peptide NOP-1 (100 mM) leads to
decreased energy transfer from ETR1-Alexa 488
to EIN2479 1294-Alexa 568, as shown by reduced
acceptor fluorescence. Control peptide ROP-1
(100 mM) was ineffective.
(C) Fluorophore control measurements empha-
size that NOP-1 had no effect on isolated dyes.
(D) Calculation of the IC50 value of NOP-1 for in-
hibition of EIN2-ETR1 complex formation, deter-mined via the FRET-based
in vitro assay. All data represent the mean of three independent mea-
surements ± SD.

As illustrated in Supplemental Figure 3,

proteins reflects a defect in EIN2-ETR1 complex formation.
Next, our FRET-based assay was used to quantify the inhibitory
effect of the NOP-1 peptide on EIN2-ETR1 complex formation.
Figure 3D illustrates how the IC50 (the concentration of NOP-1
inhibiting complex formation to 50%) is derived from the FRET
signals measured at different NOP-1 concentrations. From
these data, the IC50 for NOP-1 was calculated to be 93.6 ± 28.6
mM. Similar IC50 values have been described for other small
molecule compounds inhibiting the interaction between
Interleukin-2 and its receptor protein IL-2R (Braisted et al.,
2003; Arkin and Wells, 2004).
To identify specific residues or positions within the NOP-1 pep-
tide affecting EIN2-ETR1 complex formation, we employed
alanine scanning mutagenesis (Cunningham and Wells, 1989),
a step-by-step substitution of individual residues in the NOP-1
peptide by alanine. To this end, we constructed a set of eight
alanine substitution peptides (AP) (Figure 4A) and tested for
their inhibitory effect on EIN2-ETR1 complex formation in our
FRET-based in vitro assay. Concentrations of 100 mM were
used for all alanine peptide variants as this concentration corre-
lates with the IC50 obtained for the NOP-1 peptide.
The potency of a particular peptide variant to inhibit EIN2-ETR1
complex formation was estimated in relation to the effect caused by
the NOP-1 peptide. Figure 4 shows that all APs inhibit complex
formation to a similar extent, although substitution of Arg-3, Lys-5,
or Arg-7 seems to affect inhibition somewhat more strongly. In
general, all substitution peptides compare with the NOP-1 response
and clearly outperform the ROP-1 control (Figure 4B). As a
representative of all alanine substitution peptides, AP-1 was tested
for the effect on the hypocotyl length of dark-grown Arabidopsis
seedlings in response to ACC in vivo.
Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015. 1169
AP-1 leads to significant elongated
hypocotyl growth, similar to the effect
observed by NOP-1 (Figure 2C and 2D and
Supplemental Figures 2 and 3). Taken
together, alanine scanning did not clearly
identify a particular residue or position in
NOP-1-mediating inhibition of EIN2-ETR1
complex formation. However, the high content of NOP-1 in basic
amino acids (62.5%) corresponding to a highly positively
charged peptide under physiological conditions and the
somewhat reduced inhibitory effect observed when Arg-3, Lys-
5, or Arg-7 were substituted suggest that charge and related
chaotropic characteristics of NOP-1 may provoke the inhibitory
effect on EIN2-ETR1 complex formation. These characteristics
are hardly affected by substitution of a single residue at a time,
as done in our alanine scanning approach.
To further explore the specificity of the NLS motif in inhibiting the
EIN2-ETR1 interaction, we constructed a second set of peptides
(Figure 4C). In these peptides, we extended the NOP-1 sequence
by residues located upstream in the NLS motif of EIN2. We selected
a stepwise extension by four residues resulting in NLS
dodecapeptide 1 (NDP-1, aa 1258–1269 in EIN2), NLS hexadeca-
peptide 1 (NHDP-1, aa 1254–1269 in EIN2), and NLS icosapep-tide
1 (NIP-1, aa 1250–1269 in EIN2) and studied the effect of these
peptides on EIN2-ETR1 complex formation. To further un-derline
the requirement of the NLS core motif used in our previous
experiments, we also synthesized the peptide peri-NLS octapep-tide
1 (PNOP-1) corresponding to the eight residues directly up-stream
of the NLS motif (aa 1254–1261 in EIN2) that were used for the
stepwise extension of NOP-1. This set of peptides was tested in our
FRET-based assay for their effects on EIN2-ETR1 complex
formation. Our studies show positive correlation of sequence length
and inhibition, as the EIN2-ETR1 interaction is more efficiently
blocked with increasing peptide length (Figure 4D). Similar to the
random octapeptide ROP-1, the octapeptide PNOP-1 consisting of
residues adjacent to the NLS core motif in EIN2 has no effect on the
EIN2-ETR1 interaction (Figure 4D). In vivo studies analyzing the
response of hypocotyl growth to ACC in the presence of NDP-1 and
PNOP-1 support
Molecular Plant
tion than the NOP-1 peptide, corresponding to the endogenous NLS motif of EIN2. Peptides SHOP-1, SHOP-2, and YSOP-4 are less efficient. Control
peptide AOP-1 consisting essentially of acidic residues was ineffective. All data represent the mean of three independent measurements ± SD.
these results (Supplemental Figure 4). NDP-1 (200 mM) leads to
significant elongated hypocotyl growth, whereas PNOP-1 was
uninfluential. These results underline that inhibition of the EIN2-
ETR1 interaction is specifically related to the NLS motif, while
adjacent residues in the EIN2 sequence have no impact on the
stability of the EIN2-ETR1 complex. The stronger inhibitory effect
observed with the extended peptides in the FRET assay might be
related to the fact that these peptides are more resistant to degra-
dation or form more stable secondary structure elements.
The studies presented so far provide compelling evidence that
the NLS sequence of EIN2 (aa 1262–1269) is required for EIN2-
ETR1 complex formation and show that synthetic peptides of
this sequence inhibit EIN2-ETR1 interaction. To further substan-
tiate the specificity of this sequence for this reaction, we de-
signed another set of peptides (Figure 4E) based on the six
classes on NLS motifs known as the recognition sequence for
importin a proteins (Kosugi et al., 2009). This set of peptides is
characterized by a high content of positively charged, basic
residues, a common characteristic of the various NLS motifs
(Tinland et al., 1992; Hicks and Raikhel, 1993; Kosugi et al.,
2009). When analyzed in our FRET-based interaction assay
1170 Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015.
NLS-Derived Peptide Affects Ethylene Responses
Figure 4. Structure Activity Analysis of the
Inhibitory Effect of the NLS Motif on EIN2-
ETR1 Complex Formation.
(A and B) Impact of single alanine substitutions
in inhibitory NLS peptide NOP-1 on EIN2-ETR1
interaction. (A) List of sequences and physico-
chemical characteristics of alanine substitution
peptides of NOP-1. Alanine substitutions are
highlighted in red. (B) Inhibitory effect of 100 mM
substitution peptides on EIN2-ETR1 complex
formation measured by FRET-based in vitro
assay. Complex formation is affected to a similar
extent as the control peptide NOP-1.
(C and D) Impact of NLS peptide length on EIN2-
ETR1 interaction. (C) List of sequences and
physicochemical characteristics of synthetic
peptides corresponding to the NLS motif in EIN2
extended by residues adjacent to this motif. The
NLS motif is highlighted in bold; the adjacent N-
terminal residues are illustrated in italics.
(D) Inhibitory effect of 100 mM peptides on EIN2-
ETR1 complex formation measured by FRET-
based in vitro assay. Inhibition correlates with
sequence length. EIN2-ETR1 interaction is more
efficiently blocked with increasing peptide length.
Control peptide PNOP-1 corresponding to resi-
dues adjacent to the NLS motif in EIN2 has no
effect on the EIN2-ETR1 complex.
(E and F) Impact of peptide variants derived from
plant NLS motifs on EIN2-ETR1 interaction.
(E) List of sequences and physicochemical char-
acteristics of synthetic peptide variants derived
from plant importin recognition sequences.
(F) Inhibitory effect of peptide variants on EIN2-
ETR1 complex formation measured by FRET-
based in vitro assay. In general, all variants
inhibit complex formation. However, synthetic
peptides YSOP-1 and YSOP-2 show a more
pronounced inhibition of the EIN2-ETR1 interac-
(Figure 4F), all NLS-derived peptides inhibit EIN2-ETR1 complex
formation, although peptides like YSOP-1 and YSOP-2 were
somewhat more efficient than NOP-1, while peptides SHOP-1,
SHOP-2, and YSOP-4 showed a less pronounced inhibitory effect
on EIN2-ETR1 interaction. The fact that all NLS-derived peptides
tested in our study are inhibitory on the EIN2-ETR1 com-plex
formation, although to a different extent, reflects the sequence
redundancy of the peptides able to interfere with EIN2-ETR1
complex formation. Along these lines, the results sup-port the idea
that inhibition of EIN2-ETR1 complex formation is mediated by the
cationic, chaotropic characteristics of the peptides rather than by an
explicit sequence. Bearing in mind that EIN2-ETR1 complex
formation takes place at the kinase domain of the receptors (Bisson
and Groth, 2010), which is negatively charged when
phosphorylated, we propose that the basic, cationic peptides
directly compete with EIN2 for the ETR1 binding site. To
substantiate this hypothesis, we generated an additional control
peptide consisting of 62.5% acidic residues called acidic
octapeptide 1 (AOP-1, Figure 4E). The related interaction studies
confirm that this negatively charged peptide was ineffective in
inhibiting the EIN2-ETR1 inter-action (Figure 4F), underlining the
crucial impact of basic residues
NLS-Derived Peptide Affects Ethylene Responses
Figure 5. Impact of NLS Peptides on Tomato Fruit Ripening.
(A) NLS-based peptide NOP-1 leads to a delayed ethylene response in
living plants. The impact of synthetic peptides NOP-1 and ROP-1 on fruit
ripening of climacteric tomato fruits is given in the left panel. Graphical
summary of the fruit ripening process of climacteric tomato fruits treated
with synthetic peptides is illustrated in the right panel. Green represents
an immature fruit, red a mature fruit.
(B) Graphical summary of tomato ripening of surface-treated fruits with
alanine substitution variants (APs) of NLS peptide NOP-1. All APs
showed an effect on fruit ripening.
Molecular Plant
in the synthetic peptides for inhibition of complex formation.
These results are supported by in vivo data studying the
response of hypocotyl growth to ACC in the presence of YSOP-
3 and AOP-1 (Supplemental Figure 5). YSOP-3 (200 mM) leads
to significant elongated hypocotyl growth, whereas AOP-1 was
uninfluential.
Bioassay for Studying the Structure–Activity
Relationship of NLS-Derived Peptides on Ethylene
Responses
To substantiate the agronomic potential of our findings, we
applied the NOP-1 peptide on developing tomato fruits aiming to
elucidate the effects of the synthetic peptide on the ripening
process, which is strongly controlled by ethylene gas. As shown
in Figure 5A, surface treatment of developing green tomato
fruits with 200 mM NOP-1 significantly delays fruit ripening. A
related delay in ripening was not observed in untreated tomato
fruits or in fruits treated with 200 mM ROP-1 as control peptide.
Therefore, in line with the results that NOP-1 but not ROP-1
affects the triple response of Arabidopsis seedlings (Figure 2C
and 2D), NOP-1 proved to be a potent inhibitor of ethylene
responses in different developmental processes in living plants
and plant tissues. In further studies, we used tomato fruit
ripening as a suitable bioassay for analyzing the effect of our
various synthetic NLS-derived peptides in planta. In line with
results from the FRET-based in vitro assay, we observed a
delay in fruit ripening for all APs that was comparable with the
delay caused by the NOP-1 peptide (Figure 5B).
Next, we tested the effect of the three larger peptides containing
additional residues from EIN2 adjacent to the NLS motif for their
inhibitory effect on tomato ripening. Although all three peptides
(NDP-1, NHDP-1, NIP-1) showed delayed ripening, the potency
of the peptides negatively correlated with their length (Figure
5C). While the icosapeptide NIP-1 showed only minor in-
hibition, ripening in the presence of the dodecapeptide NDP-1
and the hexadecapeptide NHDP-1 was reduced to about the
same extent as with NOP-1. The reason for the negative
correla-tion of sequence length and inhibition, which is in
contrast to the results obtained in our FRET-based in vitro
assay (Figure 4D), might be related to a less efficient uptake of
larger peptides by the fruit or a more efficient degradation of
these peptides. To substantiate the significance of the NLS
sequence in the synthetic peptides, we also tested the control
peptide PNOP-1 consisting of the residues adjacent to the NLS
motif in EIN2. As in the in vitro assays, this peptide showed no
effect on tomato fruit ripening, emphasizing the requirement of
the NLS motif to inhibit ethylene responses in vitro and in vivo.
Finally we tested several of the basic peptides derived from the
various NLS motifs for their inhibitory potential on fruit ripening
(C) Graphical summary of the ripening process of tomato fruits treated
with NLS peptides of increasing length. Maturation of tomato fruits is less
delayed with increasing length of the NLS peptide applied. Control pep-tide
PNOP-1 corresponding to residues adjacent to the NLS motif in EIN2 had
no effect on maturation.
(D) Graphical summary of tomato fruit maturation of fruits surface treated
with peptide variants derived from plant NLS motifs. Control peptide AOP-1
consisting essentially of acidic residues had no effect on fruit ripening.
Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015. 1171
Molecular Plant
(Figure 5D). The results from these studies are in line with the
related results from the FRET-based in vitro assays with these
peptides (Figure 4F). Peptides SHOP-1 and SHOP-2, which
have a lower inhibitory effect on EIN2-ETR1 complex formation
than NOP-1, show a related reduced delay in tomato fruit
ripening (Figure 5D). Likewise, peptides YSOP-1 and YSOP-3,
which have a stronger inhibitory effect on EIN2-ETR1 complex
formation than NOP-1, cause enhanced delay of fruit ripening.
Tomato fruits treated with the acidic control peptide AOP-1
showed no effect on tomato ripening consistent with their lack of
response in the in vitro assay.
In summary, our study provides compelling evidence that NLS-
based peptides are able to decrease ethylene responses in planta
and thus might act as novel and potent inhibitors of ethylene
signaling. The potential molecular basis of the inhibitory effect
caused by the NLS-based peptides probably correlates with their
high positive charge at physiological conditions due to their high
content of basic residues and competition with EIN2 for ETR1
binding. The EIN2-ETR1 complex may provide the scaffold for EIN2
phosphorylation by CTR1 and is therefore essential for sub-sequent
signaling events. Hence, the disruption of EIN2-ETR1 complex
formation by the NLS-based peptides leads to reduced ethylene
responses, such as the Arabidopsis triple response or tomato fruit
ripening tested in this study.
METHODS
Cloning of Expression Vectors for In Planta and In Vitro
Interaction Assays between Arabidopsis Proteins, Expression,
and Purification of Recombinant Proteins
Expression vectors encoding for EIN2479 1294
-GFP and the NLS deletion
EIN2(DNLS)-GFP were prepared using the Gateway system (Invitrogen). A
DNA fragment encoding for EIN2 479 1294 was transferred into vector
pABindGFP (Bleckmann et al., 2010) following the manufacturer’s
manual. In order to delete the NLS sequence from EIN2 variants,
constructs encoding for EIN2 479 1294(DW), EIN2-GFP, and EIN2 479 1294-
GFP (Bisson et al., 2009; Bisson and Groth, 2010) were used as
templates following the Stratagene Quick change protocol. Mutagenesis
0 Labeling was performed according to the manufacturer’s manual for amine
forward primer was 5 -AAG AGA ATT TGG CTT CGG TTT CGA ATA
0 0
AAC CAG TAG GTA T-3 ; reverse primer was 5 -ATA CCT ACT GGT
0
TTA TTC GAA ACC GAA GCC AAA TTC TCT T-3 . The preparation of
ETR1 expression vectors is described in Bisson et al. (2009) and Bisson
and Groth (2010). Expression and purification of recombinant ETR1 and
EIN2479 1294 variants for in vitro measurements are given in Bisson et al.
(2009) and Scharein et al. (2008).
Peptide Synthesis
All peptides used in this study were purchased commercially. All
peptides were ordered with the following characteristics: >90% purity
level, N-ter-minal free amine, C-terminal amidation. Octapeptides NOP-1
and ROP-1 were obtained from the Molecular Proteomics Laboratory,
BMFZ Du¨ssel-dorf. Peptide set 1 (APs of NLS peptide) was obtained
from JPT Peptide Technologies, peptide set 2 (extension of NLS motif
according to the EIN2 sequence) was obtained from GenScript, and
peptide set 3 (additional NLS peptides derived from various NLS motifs)
was obtained from ThermoScientific. For measurements, lyophilized
peptides were dissolved in the respective measurement buffer at
appropriate concentrations.
Confocal FRET-Based Analysis and In Vitro Tryptophan
Fluorescence Interaction Assay between ETR1 and EIN2
Transient expression of fluorescence tagged Arabidopsis EIN2 and ETR1
variants in Nicotiana benthamiana epidermal leaf cells was performed as
1172 Molecular Plant 8, 1165–1174, August 2015 ª The Author 2015.
NLS-Derived Peptide Affects Ethylene Responses
described in Bisson et al. (2009). The confocal FRET-based in planta
inter-action assay for ETR1-EIN2 complex formation was performed in
tobacco leaf cells as described in Bisson et al. (2009). For FRET
measurements, data were collected with as many as 15 cells. GFP and
mCherry signals were scanned simultaneously at an excitation
wavelength of 488 nm for GFP and 561 nm for mCherry. GFP and DAPI
signals were scanned separately at 405 nm excitation wavelength for
DAPI. Emission wavelengths were 420–480 nm for DAPI, 479–550 nm
for GFP, and 572–637 nm for mCherry. For measurements in the
presence of synthetic octapeptides, NOP-1 or ROP-1 was dissolved in
water to a con-centration of 100 mM and infiltrated into the tobacco leaf
1 h prior to the start of the confocal microscopy measurements.
For the tryptophan fluorescence quenching interaction assay between
ETR1 and tryptophan-free variants of EIN2, see a detailed description in
Bisson et al. (2009). For measurements in the presence of synthetic
octapeptides, respective concentrations of NOP-1 or ROP-1 were pre-mixed
479 1294
with receptor protein ETR1 prior to titration with EIN2 (DW).
Data were analyzed with the GraFIT program (Erithacus Software) by
using a model assuming 1:1 stoichiometry of both binding partners. For
the in vitro fluorescence interaction assay, all data represent the mean ±
SD of three independent measurements.
Triple Response Assay and Statistical Analysis
The triple response assay with wild-type Arabidopsis seedlings (ecotype
Columbia) was performed as described in Bisson and Groth (2010). For
treatment with synthetic peptides, NOP-1 or ROP-1 was added to the
agar at appropriate concentrations before the seeds were plated. After 8
days of growing in the dark, the plates were opened and scanned at 150
dpi. The hypocotyl length of the seedlings was subsequently deter-
mined using Adobe Photoshop CS3. To prove statistical significance, we
used GraphPad Prism 6.05. Fisher’s least significant difference test by
ANOVA was performed with an a value = 0.05 and as many as 25
seedlings.
FRET-Based In Vitro Interaction Assay
479 1294
For the FRET-based in vitro assay, recombinant ETR1 and EIN2
proteins were labeled with Alexa 488 or Alexa 568 (Life Technologies). For
labeling, proteins were dissolved in a buffer containing 50 mM potas-sium
phosphate (pH 8.0), 300 mM NaCl, and 0.015% (w/v) Fos-choline 16.
reactive Alexa dyes. After labeling, free dyes were removed using a PD
MiniTrap G-25 column (GE Healthcare Life Sciences) following the
manufacturer’s instructions and proteins were transferred into a buffer
containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5% (w/v) glycerol,
0.015% (w/v) Fos-choline 16, and 13 cOmplete ULTRA mini protease in-
hibitor (Roche). Next, ETR1-Alexa 488 and EIN2 479 1294-Alexa 568 were
mixed to a final concentration of >1.5 mM each and transferred to a white
384-well microplate. For measurements in the presence of synthetic pep-
tides, the protein complex was pre-mixed with the respective peptide before
measurement started. FRET measurements were carried out in a microplate
reader (Infinite M200 Pro, Tecan) at 455 nm excitation wave-length, while an
emission spectrum from 565 nm to 645 nm was recorded. FRET efficiencies
were calculated as the percentage of decreasing emission at 610 nm
compared with measurements in the absence of the inhibitor according to the
following equation:
Inhibition FRETeff [%] = (100/F0) 3 (F0 FI)
where F0 is the acceptor fluorescence at 610 nm without inhibitor and F I
is the acceptor fluorescence at 610 nm in the presence of inhibitor. The
data represent the mean of three independent measurements.
To calculate the IC50 of NOP-1 on EIN2-ETR1 complex formation, the
FRET-based in vitro assay was performed in the presence of increasing
concentrations of NOP-1 until saturation was reached
NLS-Derived Peptide Affects Ethylene Responses
(up to 20 mM NOP-1). Inhibited fluorescence at saturation conditions
was set to 100% and the IC 50 value was determined by the GraFIT
program (Erithacus Software). All data represent the mean of three
independent measurements ± SD.
Surface Treatment of Climacteric Tomato Fruits with Synthetic
Peptides
Immature, green cherry tomato fruits (Solanum lycopersicum variety
Juanita or Solanum pimpinellifolium) were harvested from the panicle
and cleaned with water. Then 1 ml of a 200 mM stock of the respective
peptide dissolved in a buffer containing 50 mM Tris-HCl (pH 8.0), 300
mM NaCl, 5% (w/v) glycerol, and 0.015% (w/v) Fos-choline 16 was
applied to the tomato fruit surface with a brush. The tomato fruit surface
was air-dried for 30 min at room temperature. At least three tomato fruits
per experiment were tested. The control tomato fruits remained
untreated or were treated with buffer alone. After drying, the tomato
fruits were transferred into a vial and closed in a gas-permeable
manner. Tomato fruits were subsequently stored at room temperature in
the dark. The fruit ripening process was documented every 24–48 h until
the maturity of all tested tomato fruits was observed.
SUPPLEMENTAL INFORMATION
Supplemental Information is available at Molecular Plant Online.
FUNDING
This work was partially supported by a grant from the Deutsche For-
schungsgemeinschaft to G.G. within the SFB 590 ‘‘Inherent and adaptive
differential processes’’ at Heinrich-Heine University Du¨sseldorf.
ACKNOWLEDGMENTS
We thank Dr. Sabine Metzger and her team at the Molecular Proteomics
Laboratory, BMFZ Du¨sseldorf, for performing peptide synthesis and Dr.
Yvonne Stahl for discussion concerning the confocal microscopy. No
conflict of interest declared.
Received: September 4, 2014
Revised: March 9, 2015
Accepted: March 30, 2015
Published: April 2, 2015
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