Peer Review Report for 10.1105/tpc.16.00104
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Moss Chloroplasts are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids
Takayuki Hirano, Koji Tanidokoro, Yasuhiro Shimizu, Yutaka Kawarabayasi, Toshihisa Ohshima, Momo Sato, Shinji
Tadano, Hayato Ishikawa, Susumu Takio, Katsuaki Takechi, and Hiroyoshi Takano
Plant Cell. Advance Publication June 20, 2016; doi: 10.1105/tpc.16.00104
Corresponding author: Hiroyoshi Takano [email protected]
Review timeline:
TPC2016-00104-BR
TPC2016-00104-BRR1
TPC2016-00104-BRR2
Submission received:
1st Decision:
1st Revision received:
2nd Decision:
2nd Revision received:
3rd Decision:
Final acceptance:
Advance publication:
Feb. 22, 2016
Mar. 15, 2016 revision requested
Apr. 13, 2016
May 23, 2016 accept with minor revision
May 31, 2016
June 1, 2016 acceptance pending, sent to science editor
June 11, 2016
June 20, 2016
REPORT:
(Note: The report shows the major requests for revision and author responses to major reviewer comments.
Miscellaneous correspondence and minor comments are not included. The original format may not be reflected in
this compilation, but the reviewer comments and author responses are not edited, except to correct minor
typographical or spelling errors that could be a source of ambiguity.)
TPC2016-00104-RA 1st Editorial decision – revision requested
March 15, 2016
The editorial board agrees that the work you describe is substantive, falls within the scope of the journal, and may
become acceptable for publication pending revision, and potential re-review. We ask you to pay attention to the
following points in preparing your revision.
In your revision, in addition to other points raised by the reviewers, it will be crucial to include additional negative
controls using EAD-DA staining in murE mutants (if possible in the murF mutant also). Direct detection of moss PG
by, e.g., HPLC methods would further strengthen your conclusions, but this is likely experimentally very challenging
and hence not essential if you are unable to get it to work.
---------------------------------------------------------------------------- Reviewer comments:
Reviewer #1 (Comments for the Author):
Chloroplast evolved from cyanobacterial endosymbionts. As do bacteria, chloroplasts in glaucophyte algae retain
peptidoglycan (PG) layer between the inner and the outer envelope membrane that can be detected by electron
microscopy. However, it has been widely believed that chloroplasts in other lineages of photosynthetic eukaryotes
have lost the PG layer because electron microcopy and other biochemical approaches failed to detect PG in these
organisms.
In contrast, previous studies by the authors and others have found that antibiotics specific to respective enzymes in
the PG synthesis pathway impair chloroplast division in some (but not all) streptophytes including a certain lineages
of land plants and charophycean algae. In addition, the authors have shown that the moss Physcomitrella patens
genome encodes chloroplast-targeted proteins that are supposed to be sufficient for PG synthesis and inactivation of
the gene also impairs chloroplast division.
The similar situation existed in the parasitic bacteria Chlamydiales, in which PG was not detected by conventional
approaches but antibiotics specific to PG synthesis do work ("Chlamydial anomaly"). Recently, a new technique was
developed to detect PG synthesis by using EDA-DA and demonstrated that Chlamydiales possess PG layer.
In this manuscript, the authors applied this new technique to the moss Physcomitrella patens. The results
demonstrate that PG layer surrounds the entire surface of the moss chloroplast. The results are very important to
understand the evolution of chloroplast and the experiments were well executed with appropriate controls. I do not
have any criticisms on the experimental results and the conclusion.
Reviewer #2 (Comments for the Author):
The data in this manuscript strongly suggest that moss chloroplasts are surrounded by DA-DA-containing material.
However, they do not go far enough to show that this material is peptidoglycan (PG). It is formally possible, for
example, that it contains the D-alanine dipeptide but only some or none of the other PG components.
On the positive side, the authors present evidence here and in previous publications that mutants lacking putative
PG-synthesizing and remodeling enzymes phenocopy mutants lacking the Ddl enzyme, as does wildtype treated with
PG-acting antibiotics. An epistasis experiment-either combining the ddl and murE deletions or treating the ddl mutant
with the relevant antibiotics-would bolster their claim that the EDA-DA-labeling around the chloroplasts represents
PG.
As the authors acknowledge in lines 306-309, definitive support for their hypothesis would require biochemical
analysis of the DA-DA-containing material by HPLC or UPLC separation followed by mass spec. This is the standard
way to deduce PG structure in bacteria. In the absence of such data, it is important that they find an alternative way
to characterize the EDA-DA-labeled structures. One method might be to treat the samples with lysozyme and test
whether there is a reduction in signal.
Reviewer #3 (Comments for the Author):
The data shown in this paper provide the first direct evidence for the presence of peptidoglycan in the chloroplasts of
Physcomitrella patens. The authors demonstrated that the ∆PpDdl knockout line lacking the Ddl enzyme conserved
from the bacterial peptidoglycan biosynthetic pathway showed macrochloroplasts indicative of a severe defect in
chloroplast division. They then went on to show that D-Alanine:D-Alanine dipeptide (DA-DA), the putative PpDdl
enzymatic product, specifically rescued the ∆PpDdl phenotype when supplemented into the growth medium and that
a labeled form of the dipeptide ethynyl-DA-DA (EDA-DA) developed recently to label peptidoglycan in bacteria
complemented the chloroplast phenotype and localized to the chloroplast perimeter. These experiments provide
strong evidence for the presence of a peptidoglycan wall surrounding chloroplasts in P. patens. Some revisions are
needed, however.
Major revisions
-A key omission of the paper is the lack of a negative control for EDA-DA labeling experiment shown in Fig 4B.
Couldn't the authors use one of their other mur mutants, such as murE, which would presumably be defective in
peptidoglycan synthesis, as a negative control? Presumably EDA-DA labeling at the chloroplast perimeter would not
be detected in these mutants. This would provide further evidence that EDA-DA probe is specific for peptidoglycan
detection in P. patens and strengthen the conclusions of the paper.
-In their previous paper (Machida et al 2006), the authors indicate the presence of two Ddl genes in P. patens, but
imply in the methods that genome sequencing data suggest the presence of only a single gene. For clarity, this
information should be included in the first section of the results.
-The authors state in line 170 that WT protonemal cells had an average of 45 chloroplasts (43.4 +/- 6.49), but state in
lines 197-199 that "addition of 1 mg/mL of DA-DA dipeptide to ∆PpDdl plants recovered normal phenotype with a
chloroplast number of 22.9 +/- 9.8." There is a large discrepancy between these numbers, suggesting that rescue of
the phenotype was not actually complete. Additionally, the chloroplasts in the DA-DA-treated ∆PpDdl plants in Fig.
3A appear slightly larger than those in WT, at least in the lower full cell in the image. Similarly, lines 222-223 state
that "EDA-DA was able to complement the giant chloroplast phenotype, similar to DA-DA." In Arabidopsis and other
angiosperms, chloroplast numbers are proportional to cell size. Does a similar relationship exist in protenemata of P.
patens? Were cell sizes in the in the DA-DA- and EDA-DA-treated ∆PpDdl plants the same size as WT cells and was
complementation of the ∆PpDdl chloroplast phenotype actually incomplete? Partial complementation would not
invalidate the conclusions of the paper, but better quantification of chloroplast size and/or number should be provided
so that the extent of complementation can be accurately assessed.
-In lines 204-206, the authors state that addition of 0.1 mg/mL DA-DA partially complemented ∆PpDdl plants but did
not show any micrographs or quantitative comparison to plants treated with 1 mg/mL DA-DA. Please show these
data.
TPC2016-00104-RAR1 1st Revision received
April 13. 2016
Reviewer comments and author responses:
Reviewer #1 (Comments for author):
[No major comments for revision].
Reviewer #2 (Comments for author):
Point 1. An epistasis experiment - either combining the ddl and murE deletions or treating the ddl mutant with the
relevant antibiotics - would bolster their claim that the EDA-DA-labeling around the chloroplasts represents PG.
RESPONSE: According to Reviewer’s suggestion, we added the photos and data on chloroplast number of the
∆PpDdl mutant treated with D-cycloserine (Fig. 3D). As expected, chloroplast number was the same to that in the
∆PpDdl mutant. We added the sentences in the Results section as follows.
"Treatment with D-cycloserine resulted in the macrochloroplast phenotype in the wild-type plants of P. patens (Fig.
3D) (Katayama et al., 2003). We examined the effect of this antibiotic on chloroplasts of ∆PpDdl (Fig. 3D). An
average chloroplast number of ∆PpDdl treated with D-cycloserine for 1 week (2.20 ± 1.7) was the same to that of
this mutant in the medium without antibiotic, suggesting that inhibitor and disruption of the PpDdl gene affected the
identical peptidoglycan biosynthetic pathway."
The sentence was added in Figure legend of Fig. 3: "(D) Wild-type and ∆PpDdl plants were grown on BCDAT
medium with 100 μM D-cycloserine (DCS). Scale bar = 20 μm."
And, following sentences were inserted into the Methods section. We counted the number of chloroplasts only in
subapical cells because chloroplast number varied according to the growth of apical cells. We added this
information. "For the antibiotic treatment, wild-type and ∆PpDdl plants were grown on BCDAT solid medium with
100 μM D-cycloserine for 1 week. To measure chloroplast number, we counted the number of chloroplasts in
subapical cells after 1 week cultivation because protonemata undergo apical growth."
Point 2. As the authors acknowledge in lines 306-309, definitive support for their hypothesis would require
biochemical analysis of the DA-DA-containing material by HPLC or UPLC separation followed by mass spec. This is
the standard way to deduce PG structure in bacteria. In the absence of such data, it is important that they find an
alternative way to characterize the EDA-DA-labeled structures. One method might be to treat the samples with
lysozyme and test whether there is a reduction in signal.
RESPONSE: As the Editors pointed out, it is not easy to determine detailed structure of moss chloroplast
peptidoglycan. This project is now in progress. At present, we have determined the method for isolation of intact
chloroplasts in our lab. Bacterial peptidoglycan can be purified by the simple boiling method with SDS. On the other
hands, because no one can purify peptidoglycan from land plants, we have to pay close attention whether isolated
material is plastid peptidoglycan or not. In addition, amount of isolated chloroplasts is not too high in comparison
to bacterial culture. To determine fraction including peptidoglycan during purification, click reaction to EDA-DA
probably in the plastid peptidoglycan will be useful. We usually use EDA-DA at the concentration of 1 mg/mL for
complementation. Therefore, if we want to use 1 L culture of protonemata, 1 g of EDA-DA is needed. Although the
chemists in our Faculty can make EDA-DA, we cannot use large amount of EDA-DA (and it’s expensive). Therefore,
we are now trying several methods to purify peptidoglycan from small scale of isolated chloroplasts as pilot
experiments. When purification method is decided, we will determine the structure of moss peptidoglycan from the
large volume of protonemal culture. We think that more experiments are needed to purify moss chloroplast
peptidoglycan. We want to show the results in our next report. Lysozyme treatment of isolated chloroplast was also
under way. We observed the reduction of signal during lyzozyme treatments. In addition, we want to investigate
resistance of isolated chloroplasts to osmotic stress in hypotonic solution in details. We hope to report these
results with purification of peptidoglycan in the near future.
Reviewer #3 (Comments for author):
Point 1. A key omission of the paper is the lack of a negative control for EDA-DA labeling experiment shown in Fig
4B. Couldn't the authors use one of their other mur mutants, such as murE, which would presumably be defective in
peptidoglycan synthesis, as a negative control?
RESPONSE: We added the photos of the results of the click reaction for the murE knockout mutant inoculated with
EDA-DA containing medium (Fig. 4B). No green fluorescence was observed in the murE mutant cells. We have
revised the text in the Results section as follows.
"No fluorescence was detected in the protonemata of the ∆PpDdl line complemented with DA-DA. The addition of 1
mg/mL EDA-DA to ∆PpMurE plants did not recover the macro chloroplast phenotype and no fluorescence was
detected in the protonemata used for the click reaction."
Figure legend of Fig. 4 has been revised.
"∆PpMurE plants were also cultured in the medium containing 1 mg/mL EDA-DA. No fluorescence was detected in
the protonemata of the ∆PpDdl complemented with DA-DA and of the ∆PpMurE cultured with EDA-DA.
And, following sentence was inserted into the Methods section.
"∆PpMurE plants grown in the medium supplemented with 1 mg/mL EDA-DA for 9 days were also used."
Point 2. In their previous paper (Machida et al 2006), the authors indicate the presence of two Ddl genes in P.
patens, but imply in the methods that genome sequencing data suggest the presence of only a single gene. For
clarity, this information should be included in the first section of the results.
RESPONSE: According to the Reviewer’s comment, we entered the explanation in the Results section as follows.
"Previously, we identified two Ddl cDNAs with minor differences from the full-length cDNA library of P. patens
(Machida et al., 2006). The updated genome sequence of P. patens (Rensing et al., 2008) showed that only one gene
exists in the genome (cDNA accession number: AB194083). Another cDNA (AB194084) may have been generated by
a PCR error. Therefore, we analyzed one Ddl gene (PpDdl) in the P. patens genome."
Point 3. The authors state in line 170 that WT protonemal cells had an average of 45 chloroplasts (43.4 +/- 6.49), but
state in lines 197-199 that "addition of 1 mg/mL of DA-DA dipeptide to ∆PpDdl plants recovered normal phenotype
with a chloroplast number of 22.9 +/- 9.8." There is a large discrepancy between these numbers, suggesting that
rescue of the phenotype was not actually complete.
In lines 204-206, the authors state that addition of 0.1 mg/mL DA-DA partially complemented ∆PpDdl plants but did
not show any micrographs or quantitative comparison to plants treated with 1 mg/mL DA-DA. Please show these
data.
RESPONSE: According to the Reviewer’s suggestion, we added a photo of protonemal cells cultured with 0.1
mg/mL DA-DA (Fig. 3A). The average chloroplast number in the subapical cells of ∆PpDdl grown with 0.1 mg/mL
DA-DA was 9.2 ± 4.6, half of that in the mutant plants grown with 1 mg/mL DA-DA. We have revised the text in the
Result section as follows:
"While the addition of 0.1 mg/mL D-Ala did not recover the phenotype of the ∆PpDdl plants, 0.1 mg/mL DA-DA did
partially recover the phenotype with the chloroplast number of 9.2 ± 4.6 (Fig. 3A).
[The following] sentence was added to the Figure legend of Fig. 3. "∆PpDdl plants were also grown with 100 μg/mL
DA-DA (x0.1 DA-DA)."
And, the text in the Methods section was revised: "The ∆PpDdl plants were also grown on BCDAT solid medium
supplemented with 100 μg/mL DA-DA, 1 mg/mL DA-LA, or 1 mg/mL LA-DA for 1 week."
When ∆PpDdl was cultured with 5 or 10 mg/mL DA-DA, plants did not grow healthy and chloroplast number was not
recovered. We supposed that D-Ala generated from DA-DA affected cell growth. So, higher concentration of DA-DA
could not be used for the recovery of chloroplast number. In the plaque assay experiments on Chlamydia treated
with D-cycloserine, DA-DA could not recover the plaque formation completely (Liechti et al. 2014). Dipeptide DA-DA
has to be in stroma, because PpDdl located in stroma generates UDP-MurNAc-pentapeptide. Therefore, DA-DA has
to transport from outside of the cells to stroma through cell membrane and chloroplast envelopes. Import system of
dipeptides is not known in plants, as far as we know. It is interesting that DA-DA dipeptide reaches to stroma to
recover the macrochloroplast phenotype. We speculated that import of DA-DA to stroma is limited. This
restrictionmay be a reason why DA-DA complementation is incomplete. We added the sentences in the Discussion
section, as follows:
"The average chloroplast number in ∆PpDdl grown on the medium supplemented with 1 mg/mL DA-DA was about
half of that in the wild-type plants. Because PpDdl located in stroma (Fig. 1E) has to use DA-DA for peptidoglycan
biosynthesis, import of DA-DA from outside of the cell to stroma may be limited for recovery of chloroplast
number."
TPC2016-00104-RAR1 2nd Editorial decision – accept with minor revision
May 23, 2016
On the basis of the advice received, the board of reviewing editors would like to accept your manuscript for
publication in The Plant Cell. This acceptance is contingent on revision based on the comments of our reviewers. In
particular, please consider the following:
In your revision, please address the point raised by reviewer #1 as to "p. 8, line 204-206. The authors should
describe that the addition of 1.0 mg/mL DA-DA "partially" recovered phenotype of the delta-Ddl plants (22.9+-9.8
Cps; wild type, 45 Cps ) ". Further it is recommended that the text is seen by a professional editor or a native speaker
to correct some issues with use of the English language.
---------------------------------------------------------------------------- Reviewer comments:
Reviewer #1 (Comments for the Author):
I think that the authors have successfully addressed most of the points raised by reviewers.
p. 8, line 204-206. The authors should describe that the addition of 1.0 mg/mL DA-DA "partially" recovered
phenotype of the delta-Ddl plants (22.9+-9.8 Cps; wild type, 45 Cps ).
Reviewer #3 (Comments for the Author):
The authors have satisfactorily addressed this reviewer's concerns and it appears nearly all those of the other
original reviewers. Other than some minor editing for language, I consider the manuscript ready for publication in
TPC. It will make a nice contribution as a Breakthrough Report.
TPC2016-00104-RAR2 2nd Revision received
May 31, 2016
To deal with the comment from the Reviewer #1, we added the word “partially” in the text for explanation of DA-DA
recovery (line 218, p. 11).
The English in this revised manuscript has been checked by the Scientific editing service Textcheck.
TPC2016-00000-RAR1 3rd Editorial decision – acceptance pending
June 1, 2016
We are pleased to inform you that your paper entitled "Moss chloroplasts are surrounded by a peptidoglycan wall
containing D-amino acids." has been accepted for publication in The Plant Cell, pending a final minor editorial review
by journal staff.
Final acceptance from Science Editor
June 11, 2016