Dear Dr. Patterson:
I apologize for the delay in contacting you about your
manuscript. We have
now received all reviews of your manuscript entitled
"Identification of
Important Regions for Ethylene Binding and Signaling in the
Transmembrane
Domain of the ETR1 Ethylene Receptor of Arabidopsis," which
you may view by
visiting http://submit.plantcell.org. The work you describe is
very
interesting, but your manuscript requires some revision. We will
consider it
appropriate for publication if you return an acceptably revised
version that
is fully responsive to our concerns and questions.
I had many of the same comment/questions as Reviewer 2
(mislabeled Figure 4,
missing controls and the need for some statistical analyses). In
addition,
would you please comment on the following in your response to
me. What is the
basis for concluding that the P110A mutation does not
significantly alter the
ethylene-binding ability of ETR1 (Figure 2)? What is your
explanation for the
effects of altering residues (His and Cys in particular) that are
not
typically found in transmembrane segments and are likely to be
copper
coordinating residues relative to the model for ethylene binding
involving
conjugation of the double bond of ethylene to a bound copper?
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Karen Schumaker
Coeditor, The Plant Cell
PLANTCELL/2006/044537
Identification of Important Regions for Ethylene Binding and signaling in the
Transmembrane Domain of the ETR1 Ethylene Receptor of Arabidopsis
Wuyi Wang, Jeff J Esch, Shin-han Shiu, Hasi Agula, Brad M
Binder, Caren Chang, Sara E Patterson, and Anthony B Bleecker
Date Received: 6 Jun 2006
Article Types: Research Article
TOC Category: Research Article
Corresponding Author: Sara E Patterson
Subject Areas: hormonal regulation; hormone biology; signal
transduction
Supplemental Files: 0
Reviewer 1 Review Comments
Reviewer 2 Review Comments
Reviewer 3 Review Comments
Reviewer 1 Review Comments...
The ethylene receptors are known to function as negative
regulators of
the relatively well-understood ethylene signaling pathway.
Among the
different structural features present in these receptors, the
hydrophobic amino terminus formed by three to four membranespanning
domains is responsible for the interaction between the receptor
and
ethylene and is referred to as the ethylene-binding domain
(EBD). The
ethylene binding properties of the EBD can be studied in yeast
expressing the corresponding EBDs. Interestingly, proteins with
sequence similarity to the ethylene receptors can be found in
non-plant species, and a protein from Synechocystis with
sequence
similarity to ETR1 has been shown to bind ethylene.
In this manuscript, the authors address two interesting
questions:
1) What is the evolutionary origin of the ethylene receptors?
2) What is the structure-function relationship of the EBD?
To address the first question, the authors examined the ethylene
binding properties of an extensive variety of species from all
kingdoms. Representatives of land plant species including ferns
and
bryophytes showed ethylene binding. Among non-land plant
species, only
the algae Chara and cyanobacteria from the Synechocystis clade
were
found to possess ethylene binding activity. These results point to
a
plastidic origin of the ethylene receptors. It would be helpful if in
figure 1 the authors could display a tree showing the
evolutionary
relationship between the species studied.
An extensive search for putative EBD sequences was carried out.
Sequences related to the EBD were discovered not only in
cyanobacteria, but also in other diverse species of bacteria.
Again,
it would be helpful to have an evolutionary tree of these species
to
make it easier for the readers to follow this part of the work.
To better understand the relationship between structure and
function,
41 highly conserved aa of ETR1 were mutated and the binding
activity
of the corresponding EBD in yeast, as well as the in planta
signaling
activity of the mutant EBD forms, were examined. In this study,
specific residues in helixes I and II were found to be critical for
ethylene biding, whereas residues in helix III generally had only
minor effects. In addition to the mutations that abolish ethylene
binding and confer constitutive ethylene insensitivity, several
other
mutations were found that also result in ethylene insensitivity but
do
not affect the binding of ethylene. These results suggest that the
aa
clustered in helix III and bottom of helix I are critical for
transmitting the signal generated upon ethylene binding.
In summary, this is a comprehensive study that provides new
information on the role of specific residues in the EBD of the
receptors. By combining sequence information with the results of
ethylene-binding studies and in planta functional assays, new
insights
into the relationship between structure and function of the
ethylene
receptors have been obtained. Furthermore, the aforementioned
survey
of the ethylene binding capacities of different plant species
suggests
plastidic origin of the receptors.
Reviewer 2 Review Comments...
In the first part of this manuscript, Wang et al. describe the
identification of ethylene receptor-like sequences in species of
different kingdoms and the use of a bioinformatics approach to
get
insight into the potential origin and distribution of ethylene
receptors. The bioinformatics data are well done and show that
ethylene-binding domain (EBD) containing sequences are found
largely
in land plants, Chara and a group of cyanobacteria. The
bioinformatics approach is accompanied by ethylene binding
assays
using crude material from selected organisms.
In the second part of the manuscript, the authors describe their
use
of analysis to identify additional amino acid residues in the EBD
of
ETR1 which might be crucial for ethylene binding and/or signal
transmission to the cytoplasmic transmitter domain of the
ethylene
receptor.
The overall impression of the manuscript is very positive.
However,
I have some criticisms and points the authors should address:
Major comments:
•
The authors mention, without showing the available data,
that “all of the ETR1 mutants formed homodimers in yeast and
could
be converted into monomers by treatment with DTT, indicating
that
the mutations did not disturb the native structure of the receptor
and that the ETR1 mutants were still membrane-bound proteins”
(page
8/9). As these data are so crucial for the interpretation of
functional results later on, I would recommend including them as
a
supplemental figure, which should also contain a short
description
of the method.
•
For the same reason the authors should present the
immunoblot results of ethylene receptor expression, which they
used
for protein quantification (!), in Figure 1.
•
Regarding the hypocotyl length determination, there are no
statistics given (e.g. how often was the experiment repeated,
how
many seedlings were measured, SD values).
•
A major drawback of the manuscript is the missing protein
expression data for the transgenic lines expressing the different
ETR1 mutants. I would like to see immunoblot data for at least
some
representative lines of each response group.
•
The authors should explain why they switched to the ers12
etr1-7 double mutant for the investigation of the S98A and
P110A EBD
mutants.
•
Figure 4B does not show rosette size data as mentioned in
the text but results of hypocotyl length measurements. Please
solve
this problem.
Minor comments:
•
The paper “Chang and Mount, 2002” is not mentioned in
the
REFERENCES
•
The authors write that the etr1-6 etr2-3 ein4-4 mutant has
a
constitutive ethylene-response phenotype. However, in Figure 3,
they
show that this mutant is still responsive to ethylene (air: 9 mm;
ethylene: 3 mm)! Please comment on this.
•
For non-specialist readers, please give a substantiation of
the AVG experiment and a short interpretation of the results.
•
Please explain in more detail why a “large number of
mutations that cause a drastic….ethylene binding” allows the
suggestion “that the transduction of……requires only subtle
changes
in steric structure”.
•
The last paragraph is very difficult for non-specialist
readers to understand. As the introduction of a possible
intermediate state (state II) of ethylene receptors is crucial for
the understanding of receptor function and the mutant
phenotypes, an
in-depth discussion should be provided.
Reviewer 3 Review Comments...
The pathway of hormone signal transduction for ethylene has
been
advanced through genetic studies identifying multiple
components of
ethylene signaling including a family of receptor molecules.
While
numerous steps in the pathway have been identified and ordered
based
on epistatic interactions, the biochemistry of ethylene signaling
remains largely unknown. Here the authors provide a
comprehensive
mutational analysis on the ethylene binding domain (EBD) of the
ETR1
EBD and assay ethylene binding capacity of a large collection of
EBDs
derived from genes representing diverse taxa. This latter activity
was based on an informatics assessment of EBD prevalence and
yielded
results consistent with the prior hypothesis that the origin of
plant
ethylene receptors traces back to symbiotic acquisition of a
cyanobacterial genome. The mutational analysis clarifies the
borders
of the EBD, confirms helicies I and II as the basis of the ethylene
binding pocket and has facilitated development of a more in
depth
model of receptor function and functional states. The manuscript
is
well written and while the amount of work is considerable, the
experimental analysis is relatively straight-forward and based on
systems that are well established in the lab. As such it is not
surprising that the data is clear and of high quality. The
manuscript
itself is very well written and a tribute to the former mentor of
the
group. The only point I would ask the authors to address is state
II
of their model. I am not quite sure I follow the logic that uses the
fact of mutations uncoupling receptor/ligand binding and receptor
pathway suppression to infer the existence of such a state in
normal
biochemical activity of receptors. I may be missing something
here but
it seems that states I and III are clear and II appears a bit
forced.
The authors should clarify their logic in the Discussion.