From the journal RSC Chemical Biology Peer review history

Berlin, 19 October 2021

Dear Dr Mylne:

Manuscript ID: CB-ART-10-2021-000192
TITLE: Inhibition of chloroplast translation as a new target for herbicides

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

************

Reviewer 1

The authors provide another well-written example of discovery of potential herbicides with a new mode of action based on a pharmaceutical compound template. The research described in the paper supports the conclusion of the authors that an analog of linezolid could be used as a herbicide.
I have only a few comments:
Introduction
It may be premature to count tetflupyrolimet as a new herbicide mode of action, as it has not yet reached the marketplace. This is the only herbicide of which I am aware to have been listed as a new mode of action by the Herbicide Resistance Action Committee before commercialization. The company says it expects to launch the product in 2023.
It may be worth mentioning actinonin, a compound that inhibits bacterial, mitochondrial, and plastid (including chloroplast) translation by inhibition of peptide deformylase, which is required to remove the formyl from the N-terminal methionine residue of ribosome-synthesized polypeptides. I think it was patented as a herbicide, but it may have had too much mammalian toxicity for development.
The widely used antimalarial compound, artemisinin, is highly phytotoxic and is likely to have a novel mode of action, although the mode of action in plants has not been clearly determined.
Results and Discussion
I would not say that the symptoms are unique to PSI and PSII inhibitors. Many herbicides cause chlorosis, followed by necrosis. It is hard to clearly connect secondary and tertiary effects of herbicides to primary molecular targets.
A picture is worth a thousand words, but some numerical values for the data of Fig. 4 would be welcome.
I was surprised with the preemergence effects of glyphosate, as it is virtually inactivated by most soils. But, I see that peat, not soil, was used.
An important consideration for a herbicide is the use rate. Considering that 1 g/L might be effective in the field, especially when formulated for maximal activity, one could expect that the use rate could be 100 g/ha, an application volume of 100 L/ha is common. This use rate is similar to many commercial herbicides.
I expect that any patents would be filed by now, if the authors were seeking patent protection. If a patent is filed, this should be mentioned. If not, the authors might mention why not.
It would be nice to determine if linezolid binds to plastid ribosomes, as it does to the bacterial peptidyl transferase center of the 50S ribosomal subunit.
It would also be worth mentioning that bacterial resistance has evolved to linezolid by an amino acid deletion. There is only one case of a codon deletion conferring resistance to a herbicide (the protoporphyrinogen oxidase gene).
The finding that herbicidal analogues of linezolid with no bactericidal activity could be generated was a nice touch.
Experimental procedures
Was glyphosate dissolved in DMSO? It is very water soluble.
What was the source of the technical grade commercial herbicides?
What is the chemical composition of Brushwet?

Reviewer 2

This manuscript is very well written and data is explained clearly. I suggest that the reference 5 be replaced with Dayan, F.E., 2019. Current status and future prospects in herbicide discovery. Plants, 8(9),341.
The authors should also include a reference to actinonin with their reference 32. I suggest this one. Hou C-X and Williams M. Actinonin-Induced Inhibition of Plant Peptide Deformylase: A Paradigm for the Design of Novel Broad-Spectrum Herbicides. In Natural Products for Pest Management. American Chemical Society, pp. 243-254 (2006).
Finally, I have a couple of questions regarding specific experiments.
1-How do the authors explain that these compounds inhibit chloroplast translation but did not inhibit photosytnetic electron transport (Fig 3b), when several components of the photosystem are chloroplast encoded?
2-Demonstration of inhibition of chloroplast protein translation is limited to a gel of 35S-methionine. I would think that more work could be done in this area. An analysis of the chloroplast proteome would clearly establish that translation is inhibited specifically in the chloroplast. Additionally, the authors could use western blot analysis of key chloroplast encoded proteins.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Dear Prof. Dr. Roderich Süssmuth,

Our thanks to you and the two anonymous referees for considering our manuscript. We were grateful for your invitation to revise our manuscript, taking into account the useful comments of the referees.

In the revised manuscript, we have made all new or modified text the colour blue.

We also include a detailed point-by-point with each comment from the referees itemised, the response below and, in in it, using bold format we highlight the key action to the comment.

Josh Mylne

Referee: 1
[COMMENT #1] Introduction - It may be premature to count tetflupyrolimet as a new herbicide
mode of action, as it has not yet reached the marketplace. This is the only herbicide of which I am
aware to have been listed as a new mode of action by the Herbicide Resistance Action Committee
before commercialization. The company says it expects to launch the product in 2023.
[RESPONSE #1] Based on the Herbicide Resistance Action Committee approval it is safe to say
tetflupyrolimet is a new mode of action that will be introduced, but our original text is too loose
saying “very recent introduction” while concurrently mentioning tetflupyrolimet and
cyclopyrimorate. Only cyclopyrimorate has reached the marketplace.
We have changed the text:
“until the very recent introduction of tetflupyrolimet that targets dihydroorotate
dehydrogenase and cyclopyrimorate that targets homogentisate solanesyltransferase.5, 6”
To now read as follows:
“until the recent introduction of cyclopyrimorate that targets homogentisate
solanesyltransferase5 and the upcoming release of tetflupyrolimet that targets
dihydroorotate dehydrogenase6
.”
[COMMENT #2] Introduction - It may be worth mentioning actinonin, a compound that inhibits
bacterial, mitochondrial, and plastid (including chloroplast) translation by inhibition of peptide
deformylase, which is required to remove the formyl from the N-terminal methionine residue of
ribosome-synthesized polypeptides. I think it was patented as a herbicide, but it may have had too
much mammalian toxicity for development.
[RESPONSE #2] This is another good example of an antibacterial affecting chloroplast processes and
actinonin has been around since 1962. In the introduction we added the text “Another example is
actinonin that inhibits both bacterial and chloroplast peptide deformylase making it antibacterial and
herbicidal” and cited a reference suggest in [COMMENT #15].
[COMMENT #3] Introduction - The widely used antimalarial compound, artemisinin, is highly
phytotoxic and is likely to have a novel mode of action, although the mode of action in plants has
not been clearly determined.
[RESPONSE #3] Based on the contradictory papers all claiming to have found target of artemisinin, it
is likely there is no specific mode of action for it. It is thought the reactive endoperoxide in
artemisinin is cleaved in vivo and the subsequent free radical intermediate alkylates, thus poisoning,
multiple essential proteins. Artemisinin also has poor foliar uptake so only works well in agar media.
As it’s mode of action is unclear we did not add it to the introduction.
[COMMENT #4] Results and Discussion - I would not say that the symptoms are unique to PSI and
PSII inhibitors. Many herbicides cause chlorosis, followed by necrosis. It is hard to clearly connect
secondary and tertiary effects of herbicides to primary molecular targets.
[RESPONSE #4] Growth on linezolid plates better shows the bleaching, but we do not show those in
the paper and appreciate while we only say “symptoms ... similar to PSI and PSII herbicides” we
should have been more precise.
We have changed the text:
“The bacterial mode of action and symptoms of the plants after exposure to 1 (chlorosis,
necrosis), similar to photosystem I and photosystem II herbicides,40 were consistent with the
mode of action of 1, and indeed oxazolidinones, being disruption of chloroplast function.”
To now read as follows:
“The known bacterial mode of action plus the observed plant chlorosis and necrosis were
consistent with 1, and indeed oxazolidinones, disrupting chloroplast function.”
[COMMENT #5] A picture is worth a thousand words, but some numerical values for the data of Fig.
4 would be welcome.
[RESPONSE #5] While we agree quantitation is important, many of the images (esp. the grasses with
a depth of field) do not lend themselves to the ‘flat’ surface area analyses we have done in the past
based on green pixel area and due to the scale of the experiment there were not replicates; only a
dose range with multiple plants per concentration. In the manuscript we do not make quantitative
claims (e.g. three times stronger), only observe what concentrations we saw sensitivity. Variable
germination rates for these species also compounded quantitative analyses. For these reasons, we
respectfully request not to provide numerical data for Figure 4.
[COMMENT #6] I was surprised with the preemergence effects of glyphosate, as it is virtually
inactivated by most soils. But, I see that peat, not soil, was used.
[RESPONSE #6] We note that at high enough doses, herbicides used post-emergence will affect
germination. We also see the pre-emergence herbicide oryzalin at high doses also working postemergence.
[COMMENT #7] An important consideration for a herbicide is the use rate. Considering that 1 g/L
might be effective in the field, especially when formulated for maximal activity, one could expect
that the use rate could be 100 g/ha, an application volume of 100 L/ha is common. This use rate is
similar to many commercial herbicides.
[RESPONSE #7] We agree linezolid was very active:
We have changed the text:
“For other species the efficient dose was 200-800 mg/L”
To now read as follows:
“For other species the efficient dose was 200-800 mg/L, a use rate similar to many
commercial herbicides.”
[COMMENT #8] I expect that any patents would be filed by now, if the authors were seeking patent
protection. If a patent is filed, this should be mentioned. If not, the authors might mention why not.
[RESPONSE #8] A patent was not filed although this work was “funded in part by [the startup
company] Nexgen Plants” as we declare in Acknowledgements. Although linezolid is active and a
new mode of action, consultants providing Nexgen Plants with regulatory advice said anything
significantly antibacterial (and esp. an antibacterial in medicinal use) would not pass EU regulations
and be registered as a herbicide. Although by medicinal chemistry we did divorce the herbicidal and
antibacterial activities somewhat, any new analog would need to have no antibacterial activity as
well as face a long and arduous regulatory journey as a new molecule. We do not wish to include
regulatory advice given to Nexgen Plants in a scientific manuscript, but will do so if the editor
demands it also.
[COMMENT #9] It would be nice to determine if linezolid binds to plastid ribosomes, as it does to the
bacterial peptidyl transferase center of the 50S ribosomal subunit.
[RESPONSE #9] This would indeed be nice, but the ribosome is a huge and complex RNA-protein
complex so not easy to reconstitute. The experiment we include herein indicate linezolid acts on
plant chloroplasts as you’d expect based on how it acts on microbes.
[COMMENT #10] It would also be worth mentioning that bacterial resistance has evolved to linezolid
by an amino acid deletion. There is only one case of a codon deletion conferring resistance to a
herbicide (the protoporphyrinogen oxidase gene).
[RESPONSE #10] We added a sentence and new reference to the end of the introduction:
“Bacterial resistance to 1 has arisen as rRNA point mutations as well as an amino acid
deletion in a ribosomal protein (see references in 34).”
[COMMENT #11] Experimental procedures - Was glyphosate dissolved in DMSO? It is very water
soluble.
[RESPONSE #11] Glyphosate is water soluble, but for consistency we dissolved glyphosate in DMSO
as a stock, then diluted with water and used it in the same way as all the other compounds. The
M&M are correct for glyphosate stating generically that “Compounds were initially dissolved in
dimethyl sufloxide (DMSO) at 20 mg/mL and further diluted in water prior to treatments.”
[COMMENT #12] What was the source of the technical grade commercial herbicides?
[RESPONSE #12] We have added text in the methods explaining the sources for the control
compounds as follows:
“Glyphosate and oryzalin were purchased from Sigma-Aldrich (Australia)”
[COMMENT #13] What is the chemical composition of Brushwet?
[RESPONSE #13] Bushwet is an organosilicone surfactant, the active constituent of which is 1020 g/L
polyether modified polysiloxane. We have added the active ingredient with new text. The text
“Brushwet (SST Australia)” now reads “Brushwet (1020 g/L polyether modified polysiloxane, SST
Australia)”
Referee: 2
[COMMENT #14] I suggest that the reference 5 be replaced with Dayan, F.E., 2019. Current status
and future prospects in herbicide discovery. Plants, 8(9),341.
[RESPONSE #14] We have added this reference as suggested.
[COMMENT #15] The authors should also include a reference to actinonin with their reference 32. I
suggest this one. Hou C-X and Williams M. Actinonin-Induced Inhibition of Plant Peptide
Deformylase: A Paradigm for the Design of Novel Broad-Spectrum Herbicides. In Natural Products for
Pest Management. American Chemical Society, pp. 243-254 (2006).
[RESPONSE #15] We have added text and this citation, see [RESPONSE #2]
[COMMENT #16] How do the authors explain that these compounds inhibit chloroplast translation
but did not inhibit photosynthetic electron transport (Fig 3b), when several components of the
photosystem are chloroplast encoded?
[RESPONSE #16] Linezolid treatment only alters translation of de novo chloroplast encoded subunits
and thus electron transfer can still occur via pre-existing photosynthetic subunits. Atrazine on the
other hand specifically blocks electron transfer by associating with the plastoquinone B (QB) binding
site of the D1 subunit of PSII. Also, see [RESPONSE #17] for why we could not wait longer (isolated
chloroplasts are unstable).
[COMMENT #17] Demonstration of inhibition of chloroplast protein translation is limited to a gel of
35S-methionine. I would think that more work could be done in this area. An analysis of the
chloroplast proteome would clearly establish that translation is inhibited specifically in the
chloroplast. Additionally, the authors could use western blot analysis of key chloroplast encoded
proteins.
[RESPONSE #17] In organello translation rates are preferable as this assay only measures the
synthesis rates of de novo chloroplast encoded proteins immediately following treatment.
Immunodetection of chloroplastic encoded proteins would only indicate the steady state
concentrations which are unlikely to change substantially during the short incubation periods.
Furthermore, a proteomic or immuno analysis of isolated chloroplasts following any prolonged
treatment time course would likely be affected by general proteolysis as isolated chloroplasts have a
short lifespan and tend to rupture after only a few hours. As organello translation rates are the ideal
method, no change was made.

Berlin, 10 November 2021

Dear Dr Mylne:

Manuscript ID: CB-ART-10-2021-000192.R1
TITLE: Inhibition of chloroplast translation as a new target for herbicides

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 1

The responses to my comments and suggestions are adequate.