All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
As a result of the corrections made, the article can now be accepted.
The authors investigate the function of LRB1 (leucine-rich repeat receptor like kinase 1) and of its homologue LRB2 using double mutant lrb1lrb2 and over-expressors of LRB1. They find that LRB1 involves in the signaling of abscisic acid (ABA) and development.
The English language has been improved a lot and let the main text much more readable. The introduction is well described.
Structure conforms to PeerJ standards. The method part is with full desribed details. The discussion is not fully discussed.
Figures are not good enough for publication. I suggest the authors provide high quality figure to let the editors edit.
This research is fit with the scope of PeerJ.
The experiments are well-designed and performed to a high technical & ethical standard.
Methods described with sufficient detail. Replicate information is provided.
The novelty of this research is not so high. Previous research already showed the function of LRB. This research confirms the function of LRB1 and his homolog of LRB2. The advantage is using double mutant of lrb1/lrb2 to validate the function of each homolog.
All underlying data have been provided; they are robust.
The conclusion is fit the topics after revision.
Dear Authors, I would very much like to ask you to consider experimental design and correction. Please pay special attention to the very thorough comments of the second reviewer.
The authors responded to the comments. Unfortunately, some mistakes occurred in the new text,
l. 112-113 - „There have been recent reports showing that Arabidopsis plant two receptor-like protein kinases, LRR1 and KIN7, whereas lrr1 and kin7 mutants were more sensitive, to drought stress than the wild-type (Chen et al.,2021).“- part of the sentence seems to be missing.
l. 141 – „The germination percentage was calculated according to the seven-day seed exposure index. the germination percentage was calculated with the index of 2 mm of seed radicle exposed.“ – this can be shortened. The germination percentage was calculated according to the seven-day seed exposure index, with the index of 2 mm of seed radicle exposed.“
l. 241 – „as shown in (Fig. 2A).“ - as shown in Fig. 2A.
l. 448 - „LRR1 may involved“ - „LRR1 may be involved“
Experimental desigh is OK.
Conclusions reflect the achieved data.
Overall, the manuscript is much improved. I thank the authors for their changes, which improve the readability of the paper. I would like to comment that the authors did not carefully proofread their revised manuscript because there are formatting errors (some from production of the pdf?), so I am disappointed. Please proof carefully, even the final pdf, its important!
The English still needs improvement. I have tried to help point out needed changes in the Minor section below in the Additional Comments section, but this is not a complete list.
Literature references seem sufficient.
Overall, there are interesting, new data. I have one concern remaining regarding experimentation. Fig 2b is not sufficient as is. It either needs a different assay or removal with experimental additions to Fig 2c (see details on this point in #4 below in Additional comments. ).
There is another concern regarding the text describing Fig 2 experiments. Lines 240-241 and Line 245 in Results and lines 176-177 in M&M. In this study, GUS expression is visualized with histochemical staining with light microscopy based on the blue color in Figure 2a. It is correctly described in the legend to Figure 2(a). However, in the Results section, visualization is described incorrectly: “A stereo fluorescence microscope was used to observe the expression of the GUS gene driven by the LRR1 promoter in various tissues and organs of Arabidopsis, as shown in (Fig. 2A).” It is also incorrectly referred to in the M&M: “Three independent transgenic lines were analyzed and representative images are Shown. Through the Stereo fluorescence microscope observation.”
Fluorescence microscopy cannot be used with the X-GLUC substrate used in this study for visualization of GUS activity, unless I misunderstand the assay. A microscope capable of fluorescence may have been used, but imaging of a fluorescent signal was not- a light image was captured. [note the incorrect sentence structure in the M&M sentence copied directly above, these errors are listed in the next section {below} as needing correction, but here I focus on the needed correction of the experimental protocol.]
More statistical tests are needed in Figures 4c,5c,6c. They are absent in 4c.
The authors could improve the clarity of their conclusions. The specifics are in Additional comments.
1. Comments on the Introduction.
A. The introduction is much improved and provides clear descriptions. However, it still has a very awkward split of information about LRR-RLKs. Lines 86-91 introduce LRR-RLKs- “They are called receptor-like protein kinases (RLKs). The largest subfamily of the RLKs family is the Leucine-rich repeat (LRR-RLKs) subfamily, which includes more than half of the members of receptor-like protein kinases”. Then, again in lines 106-108 LRR-RLKs are introduced as “LRR-RLKs belong to the transmembrane class of receptor protein kinases…”
The information about RLKs and LRR-RLKs should be in one place. I recommend moving the general information about RLKs and LRR-RLKs to follow lines 86-91 so that the general statements come first.
B. I recommend that the section on BR signaling starting at end of line 109 should be the start of a new paragraph.
2. Lines 240-241 and Line 245 in Results and lines 176-177 in M&M. In this study, GUS expression is visualized with histochemical staining with light microscopy based on the blue color in Figure 2a. It is correctly described in the legend to Figure 2(a). However, in the Results section, visualization is described incorrectly: “A stereo fluorescence microscope was used to observe the expression of the GUS gene driven by the LRR1 promoter in various tissues and organs of Arabidopsis, as shown in (Fig. 2A).” It is also incorrectly referred to in the M&M: “Three independent transgenic lines were analyzed and representative images are Shown. Through the Stereo fluorescence microscope observation.”
Fluorescence microscopy cannot be used with the X-GLUC substrate used in this study for visualization of GUS activity, unless I misunderstand the assay. A microscope capable of fluorescence may have been used, but imaging of a fluorescent signal was not- a light image was captured. [note the incorrect sentence structure in the M&M sentence copied directly above, these errors are listed in the next section {below} as needing correction, but here I focus on the needed correction of the experimental protocol.]
3. Lines 240-250. Should be made into one paragraph. The following sentence should be moved to the end of the paragraph because it summarizes the data shown in Figure 2: “The results indicated that LRR1 is widely expressed in multiple organs, and LRR1 is negatively regulated by ABA.” [please put the second LRR in italics, because you are specifically referring to transcriptional control of LRR gene- I am not sure if I put in the italics, the italics will carry over to the reviewers page on the journal website]. And delete the following sentence because its redundant with the sentence above: “These results suggest that ABA may inhibit the LRR1 gene expression.” Overall, the description of the ABA time course is not clear or even correct. “Faded over time” tells me that you put the material in the substrate, and blue color was formed, then the blue color initially there in the same sample reduced in intensity as the material was incubated in the substrate. However, this was not how the experiment was performed. The material was incubated in ABA for different lengths of time, then stained, and I assume all stained for the same length of time. I think the authors’ conclusion is that the staining intensity is lower in seedlings that were incubated in ABA for longer periods of time, suggesting that GUS protein, and GUS expression was lower the longer the seedlings were in high ABA. This sentence must be revised. Change the current description which is: “the color of transgenic plants gradually faded with the increase in ABA treatment time.” to “GUS activity was lower in seedlings treated for with ABA for longer periods of time.” However, I have serious concerns regarding this experiment as detailed in #4.
4. Fig. 2B. The data shown in this figure are not sufficient to support the claim that expression of the LRR1 is negatively regulated by ABA. Only 2 seedlings are shown for each treatment and the staining is splotchy in most of the images – very uneven in intensity. Does that mean something biological? I don’t think so, I worry that the substrate is not evenly infiltrating into the leaves. For this experimental approach to be publishable, a quantitative assay must be used instead of this qualitative assay. There is a fluorescent substrate available for GUS, and so the activity can readily be quantitated from extracts using a fluorescent assay. As presented, the data in figure 2b are not convincing.
However, the data in Figure 2C are more convincing because it is a more direct read-out of gene activity. Barring ABA-dependent changes in RNA stability (which is a possibility), the level of RNA is useful as a read-out of promoter activity. I recommend expanding this experimental approach with more time points.
The text for Fig 2C in the legend needs editing because it is confusing. It currently reads: “The expression levels are presented as relative units with the levels of ABA-treated wild-type seedlings.” I think slight modification will provide a clearer description. Don’t have to say “wild-type” because both samples are WT- [was confused thinking one sample was not wt]. How about saying the expression of LRR1 after ABA treatment is normalized compared to expression without ABA, which is set at a value of 1. But also, there should be an RNA amount control. Was one used here? Typically in qRT-PCR, RNA amount of an RNA not expected to change is determined with the same samples, and the experimental RNA is divided by the control RNA to account for differences in total RNA in the tube (measuring and pipetting errors). Researchers often use actin for the control RNA; the level of actin RNA is determined in each sample (in the machine at the same time with the same master mix) and the actin value is used to normalize to the experimental RNA values (here LRR1) in each sample. The authors have used actin, so this should be included in the qRT-PCR assay at the same time, then the experimental RNA value normalized to the actin from its same RNA to give relative expression to actin. Then the LRR1 RNA from ABA treated seedlings can be divided by the normalized control LRR1 value so the latter value is 1, as presented in the graph. In summary, what is missing is actin normalization.
Also, please delete “target” in the phrase, “control: target materials” I don’t know what that term means. The control RNA here is from seedlings without ABA treatment isolated at time 0. However, a much better control is isolating RNA from seedlings that are incubated for the same length of time as the treatment, but without the ABA (better yet, with added solvent only- the solvent used to dissolve the ABA). Then the time variable is controlled for. If there are any RNA changes from continued growth of the seedlings, this will be controlled for. At a minimum, there should be a time 0, but also RNA from seedlings incubated minus ABA sample for the longest time interval used in the experiment.
5. Lines 320-321. The results of the ABA effect on cotyledon greening in the LRRox2 and LRRox10 lines should be described more clearly. Compare the cotyledon greening of WT to that of LRRox2 and LRRox10 specifically. Also, “Variants” is not an accurate term describing these lines. Modify this sentence: “Interestingly, mutants lrr1-1 and lrr1-2 exhibited less inhibition compared to the wild-type along with LRR1ox2 and LRR1ox10 variants.” Suggest change to “Interestingly, LOF mutants exhibited less inhibition while the OE lines exhibited greater inhibition when both are compared to wild-type.”
6. Discussion, lines 428-437. Here the authors are discussing the single lrr and kin7 mutants compared to the double. They note that there is a closely related protein to LRR1 called KIN7. They say the identity suggests that they “may be functionally redundant.” But they forget to explicitly say that they have data in support of this hypothesis. What I thought they were going to say is that the double mutant shows greater changes than seen for each of the single mutants, indicating that they are partially functionally redundant. That missing both proteins gives a stronger phenotype than missing one. However, the authors fail to make this conclusion. They should directly say, that because the phenotypes of the double mutant appears additive compared to each of the singles, that is, more severe than either single mutant, this result provides evidence that the proteins are partially functionally redundant in these processes.
7. Dicussion, lines 436-437. The authors mention the OE lines. What is confirmed with these lines? It can't be the point about the single vrs double, so this sentence is really out of place and needs further explanation. A separate point is the nature of LRR1’s role (nothing about KIN7 here). What is the nature of its role in ABA signaling? The mutants are less sensitive, suggesting a positive role in ABA signaling. The OE lines provide support for that hypothesis because they are even more sensitive to ABA than WT. So, the authors need to separate the two experiments and their conclusions (double vrs single mutant phenotype and the LRR1 LOF vrs OE phenotype) and for the latter provide more description.
8. Discussion, lines 458. I don’t agree that there is significance to LRR being named a receptor (actually its named a receptor-like) and the bonafide ABA receptor protein. Please delete.
Minor
1. Line 66, Currently reads, “demonstrate their reduced ABA insensitivity”, I think the authors mean, “demonstrate their reduced ABA sensitivity”?The mutants have reduced ABA sensitivity (or increased ABA insensitivity).
2. Lines 84-85, Currently reads, “One problem to be solved in cell signal transduction is that the organism can sense the external signal and produce the response (Zhu et al., 2023).” Suggest modifying so that the sentence expresses a problem …. “One problem to be solved in cell signal transduction is how the organism senses the external signal and produces the response (Zhu et al., 2023).”
3. Line 93, this sentence needs modifying, “Receptor protein kinase plays a bridging role.” “Bridging” between what? Please describe the two proteins/processes that are bridged by receptor protein kinase. If it is meant that it bridges between hormone binding and phosphorylation, then this is confusing because multiple hormones have intracellular receptors. Perhaps this sentence as a general sentence should be removed?
4. Line 95, change “hyper salinity” to “hyper-salinity”
5. Lines 112-144. First the sentence should not be in a separate paragraph, please combine. Next, its meaning is unclear. It currently reads: “There have been recent reports showing that Arabidopsis plant two receptor-like protein kinases, LRR1 and KIN7, whereas lrr1 and kin7 mutants were more sensitive, to drought stress than the wild-type (Chen et al., 2021).” The phrase, “showing that Arabidopsis plant two receptor-like protein kinases, LRR1 and KIN7” …. Showing what? This sentence needs to be revised. Suggest the following, but make sure this suggested revision does not change intended meaning: “A recent report showed that the loss-of-function mutations in two Arabidopsis receptor-like protein kinases, LRR1 and KIN7, were more sensitive to drought stress than the wild-type (Chen et al., 2021).” Note, please spell out LRR1 and KIN7 gene names when first used.
6. Line 115, please modify, “screened T-DNA insertion-deletion mutants”. I think describing them as "T-DNA insertion mutants” is more correct.
7. Line 119, change “cotyledonary regeneration” to “cotyledon greening”.
8. Line 122, change “designed” to “created” Designed means more to plan or draw a model of (as in design a house), but here you actually made the double mutant, more than figuring out how to make the double mutant (another example, designing an experiment is not the same as doing the experiment).
9. Lines 133-134, Not sure what is meant in the sentence regarding the LRR1-GFP, lrr1-1, lrr1-2, etc lines. Seems that only the lrr1/kin7 double was generated by crossing. How was crossing involved in the creation of the other lines in the following sentence? “The LRR1-GFP, lrr1-1, lrr1-2, kin7, and LRR1ox2, LRR1ox10 plants were generated by crossing, construction of the double mutant lrr1kin7 by single mutants lrr1-1 and kin7 of LRR1 and KIN7.” Please correct.
10. M&M, line 139. Please state whether the ABA plate media contained sugar (sucrose or glucose) or not, and if yes, how much. It is important because sugar affects the ABA response.
11. Line 140, the day counting system written here does not match the description in the Results section, line 288. Please make consistent.
12. Line 145, the plasmid pHBT-LRR1-GFP- is not a prokaryotic expression vector, it is a eukaryotic expression vector because it was used to express LRR1-GFP in plants. Please correct.
13. M&M, line 146, the creation of pHBT-GFP is described. But the source of the LRR1 coding region is not- what was the source of the LRR1 sequence for this plasmid? Please name a plasmid used in the enzyme digestion and/or give a citation.
14. M&M section- please give sources and/or citations for the all the backbone plasmids used, such as pCAMBIA13000-GFP, pHBT-GFP, pCAMBIASP+1300, pCAMBIA1381
15. Line 149 and 159 change “Colony” to “colony”
16. Lines 150-151, I don’t understand the sentence that I copied (right below) because the prior text describes construction of the plasmid already from a fragment. This sentence seems redundant-- “Finally, the CDS fragment obtained through double enzyme digestion was recovered and successfully utilized for constructing a transient GFP vector by sequencing.” What plasmid is being described? If pHBT-LRR1-GFP, wasn’t it described in the previous text?
17. Lines 176-177, several formatting problems, please fix. Should be one sentence.
18. Lines 190-191, make one paragraph.
19. Lines 224-225, Please delete the following sentence, because it is not important at all for the study: “The LRR1 gene is located on the fifth chromosome of Arabidopsis (Schmid et al., 2005).”
20. Line 225, “Phylogenetic” should be lower case “phylogenetic”
21. Line 226, shouldn’t it be “LRR-RLKs” instead of “LRRs” because the tree has only LRR-RLKs, not all LRR containing proteins.
22. Line 243, Delete here “and LRR1 is negatively regulated by ABA.” Because you haven’t presented that data yet. The data showing ABA regulation of LRR is in Figure 2C and you haven’t introduced that data yet. The next paragraph describes ABA regulation of LRR1.
23. Lines 260-261, please delete “according to the specificity of intracellular LRR1 expression, the subcellular localization vector” so that the sentence reads: To analyze the subcellular localization of LRR1, the vector pHBT-LRR1-GFP was constructed.”
24. Lines 261-263. The following sentence is awkward and should be revised or even deleted: “Due to the fusion expression of GFP and LRR1, the expression sites of the two should be related to each other, and the expression site of GFP is the expression site of LRR1.” I think all readers of the article understand the use of GFP fusions, so I don’t think this sentence is required at all. “Expression site should not be used because its not clear what is meant- expression could be transcription because gene expression is a common term. How about using localization, as in “the intracellular location of GFP chimeras typically mirrors that of the protein fused to GFP.” What is missing in the text, and should be added, is a comparison of the localization of GFP alone vrs LRR1-GFP. This control is in the figure, so a brief comparison between the two seems important. LRR1-GFP fusion protein localizes differently than GFP alone because of LRR1 sequences that direct the fusion to its subcellular locale (that is the assumption of GFP fusions). How about this sentence, “After transient expression in Arabidopsis protoplasts, GFP alone is visible in the cytosol, but the LRR1-GFP fusion is detected only in the plasma membrane, suggesting that the endogenous LRR1 protein is PM-localized.”
25. Line 282, change “was” to “is” so it reads “Its expression is…”
26. Line 284, change “effects of ABA treatment and wild-type,” to “effects of ABA treatment on wild-type,”
27. Figure 4 legend. The legend includes this text: “Student’s t-test, P<0.01”. But in the graph in Fig 4c there are no indications of statistical tests. The information of which samples were compared should be included.
28. Figure 4c, Figure 5c and Figure 6c. The values of the OE lines should be statistically compared to that of WT (via a Student’s t-test) to support the conclusion that OE of LRR1 confers greater ABA sensitivity. In Fig 5C only the LOF lines were compared statistically to WT. Possibly only in germination, if Fig 6c, cotyledon greening in WT and the LRR OE lines are so low, they might not show statistical differences, but the data are the data!
29. Figure 7(A) legend, please state the age of the seedlings at the start of the ABA incubation. Very simply, write, “X-day old seedlings were treated….”
30. Line 344, delete “the” and change “of” to “in” so that it reads, “in Arabidopsis downstream in ABA signaling.” They are not outside of ABA signaling, they are downstream genes that function in ABA signaling.
31. Line 343-344, ABI3, ABI4 and ABI5 should not be in italics because you are referring to proteins here.
32. Line 350, ABI3, ABI4 and ABI5 here need to be in italics, because you are referring to the genes.
33. Lines 350-351. Fix the carriage return error.
34. Line 364, The subsection title, “The Interacting Proteins of LRR1 were Screened by in Vitro Protein Interaction Assay” is not accurate. Y2H and BIFC are in vivo interaction assays, only the GST pull-down is an in vitro assay. The heading also has “interacting/interaction” there twice. Modify to “Proteins binding to LRR1 were identified by in Vivo and in Vitro Protein Interaction Assays”
35. Lines 367-368, Change “is unknown, as well as the associated proteins.” To “as well as the associated proteins, are unknown.”
36. Line 372-373, Delete the whole first part of the sentence, “By observing the application of cotransformed yeast receptor cells on the screening medium of tetrabasic SD yeast” so that the sentence is “We found that both BD-LRRI and BD-KIN7 could interact with AD-PYL6 in yeast cells.”
37. Line 376. Please help the read identify which are the positive control pair by stating the proteins or refer to a specific section of the figure such as (Fig 8b, top row) so we can quickly identify the positive control.
38. Line387, I don’t understand this sentence, “Firstly, the bimolecular fluorescence complementation assay system has no problem.” Please delete, I don’t think this sentence is useful or required.
39. Line 392, delete “blots” and delete “on a chemofluorescence detector” The blots were not observed, the PYL6-His protein was observed. Change to “PYL6-His was observed in the LRR1-GST pull-down and PYL6-His input control lanes.” The second phrase in the original sentence is for the material and methods.
After revision, this article is much more readable. The authors have answered the review with one item by one item. It really improves the quality of this paper.
However, the quality of the figures is not good enough for publication. High resolutions can help readers to get enough information from this paper. I suggest the authors can use the original figures, edit them with Photoshop or Inkscape, save as EPS (Encapsulated PostScript) file, and finally output with high-resolution figures.
Experimental design is fine
This research verified the function of LRB1/2 involved in the ABA pathway and its roles on regulation with developments.
Reviewers still have serious reservations about the merits of the article. Please, point by point, respond to all objections.
This manuscript is on an interesting and important topic, and contains some new information that will be of interest. Some data are clear and well supported. Additional data has strengthened the ms. In the various sections, I detail suggestions to improve the ms and its impact.
1. If I am not mistaken, these proteins were published earlier and given names. The authors should report on previously published data (in the introduction, then tie it all together in the discussion with their own). I also think the authors should use the previously published names. See (Chen et al. 2020 J. Integrative Plant Biology 63:494-509) and references therein. The authors could also look on the TAIR website that contains information on all Arabidopsis genes, and look at the publications associated with each protein (LRB1 and LRB2). These previously published data do not negate, only strengthen, this study.
2. In some of the figures, the font size is too small to see. I am also not sure if the resolution of the text in the figures is high enough. If I zoom in a bit, they look fuzzy (but could be the pdf version I am looking at). For example, in Figure 3, the graph axes are hard to read (some specific suggestions are made below).
3. Needs to be edited for professional English usage- correct term usage.
Confirm that raw data are shared.
The research is within the aims and scope of the journal and the resarch question is defined. The findings are interesting and the data have appropriate controls. There are a number of gaps - insufficient explanation. These are detailed below in order of appearance in the ms.
1. Which lrb1 LOF allele was used in the cross to lrb2? The specific allele used should be mentioned in the methods section. From supplementary Figure 1, I it looks like it is lrb1-1, but this information should be in the main text (methods).
2. The method of how germination was scored should be mentioned in the methods section as well as in the results (it is mentioned in the results section). Please add the description of what was scored as germination to the “Determination of Germination and Greening” section.
3. In the “Determination of Germination and Greening” section (line 155), the authors state they are calculating the germination rate. This is not correct. They are measuring the germination percentage, not rate.
4. Related to #3, in the graphs in Figure 3A,D, the y axis says “germination rate (%)”. This is not correct. The axis should be “germination (%)”. The rate is not the value plotted, but the % germinated at a specific time point (the rate can be calculated from the slope of the line (germination/day), but the y-axis is NOT the rate). I should note that this is an extremely common mistake- and has been published many times incorrectly!
5. The axis legends in the graphs in Figure 3A and 3D are too small. I printed that page out and still could not read the axes nor the legend. They look a bit pixelated, but I don’t know if that if my printer or an issue with the resolution of text. For the y axis, I suggest not putting in as many values. How about just 20, 40, 60, 80 and 100? Then the numbers could be bigger. The symbol sizes for each line are too small and hard to distinguish. More colors should be used for the different genotypes, hard to tell the blue colors apart. The authors should use a simpler font in the graph axes- use a font type called sans serif-type font (without any flourishes, as opposed to serif fonts that have extra lines). Such as Arial or Helvetica.
6. In the Fig 3A and C graphs, the days should correspond to the tick marks on the x axis, not placed in between. The value at day 2, for example, is plotted above the tick mark, so the number 2 should be below the tick mark, not in the intervening space. Please correct.
7. What is T-vision photography? I am not familiar with this term. Could it be explained in the Methods section? I appreciate that the authors were reducing the methods section, but there has to be some description of the microscopy- can be short.
8. If something in the methods can be shortened, I think construction of the double mutant can be deleted. Crossing in Arabidopsis is very standard, so I don’t think a detailed explanation is needed. Just say in the “Plant Materials…” section, “The lrb1-X (which one was it?) and lrb2-1 lines were crossed, F2 progeny were genotyped for the presence of each insertion using primers shown in X and doubly homozygous individuals were identified.” Please let us know which generation was used for the the phenotypic analyses- F3? Or F4?
9. In the germination assays, the authors write that day 1 is the day when the plates were put at room temperature. I would not call that day 1, that is, day zero is when the experiment starts. Day 1 is after 24 hrs of experimental treatment, which is growth at RT. The time course will read very odd to others. Day 1 or one day is considered to be after 24 hrs experimental treatment. I think that is numbering of the graphs in Figure 3, correct? In Figure 1A, almost all germination occurs by day 1- which in your time course would be as soon as they are taken out of refrigeration?
10. Figure 4D, y axis says “Seed germination (%)” but the Fig 4 legend says “(D) Comparison of cotyledon greening between the wild type, mutants and overexpression lines in….” Which is it? Cotyledon greening or germination?
I do have some concerns regarding data interpretation. I don't think any statistical analyses were conducted. in a few cases, tests would strengthen their conclusions. Details below, again in order of presentation in the ms.
1. Figure 1. The interest between LRB1 and LRB2 is at the protein level. The alignment in Fig 1B should be of the two proteins, not DNAs.
2. Supplemental Figure 1. This figure reports on the characterization of the T-DNA mutants. Multiple issues regarding this figure:
a. The diagram of the genes currently in Figure 1C should be placed on the same page with the Supplemental Figure 1 PCR gels so that the reader can compare the reactions with the gene map directly. The primers used for the RT-PCR experiment (supplemental figure 1C,D) should be added to the gene diagrams.
b. The primers for the 2 genes have the same name (LP and RP). This is confusing, especially in supplemental figure 1 panel B. Clearly the same LP and RP are not used in the top and bottom panels. Each primer needs to have a unique name. LP1 and LP2 is fine. Do the two lrb1 alleles use the same LP and RP as well?
c. Since the two lrb1 alleles have different types of insertions (SALK vrs SAIL), their T-DNA primers are likely not the same as well. Only Lba is shown. When was primer LB used?
d. In supplemental figure 1B, which lanes correspond to the double mutant and which to the singles? I don’t understand this panel. Which LP is used in the top set and which in the bottom set of reactions? Please provide more explanation. Why all the individual lanes that are the same, what do they correspond to? DNA from different individuals? Different DNA preps of the same individuals? Please explain in the figure legend.
e. The gels need a few size markers indicated on the side. Just one or two spanning the bands. In panel B, the sizes of a few of the bands in the M lane (I assume that is a size marker lane??- Please say so in the legend) should be tagged in the gel and the number given- in the legend is fine. For the others, please put next to the gel pictures.
f. In the Results section, the authors state that the Supplementary Figure 1 contains the data (lines 249-250), but it does not state what the data are! For example, the lrb1-2 mutant appears to produce a lower level of the RT-PCR band than WT, while the lrb1-1 does not appear to have any band. In addition, lrb2 allele also appears to produce some RNA. These results would seem to indicate that two of the mutants are not nulls, but reduced expression alleles. The authors need to report the expression results in the result section. IN the discussion the authors talk about the mutants (starting at line 451) and how they are down-regulated mutants, but this was never talked about in the results section, so it seems to come out of nowhere in the discussion. The data on the mutants needs to be reported in the results, then the discussion text is appropriate.
g. In panel D, the actin signal is too low to be visible. Could a longer exposure be shown?
3. Lines 253-254, the author write, “The results indicated that LRB1 is expressed in all tissues of Arabidopsis, and LRB1 is negatively regulated by ABA.” But the authors have not really examined all tissues. The level of view is at the organ level (leaves, stems, flower, etc) and expression is not examined in every cell type. Accurate would be to say, “The results indicated that LRB1 is expressed in all organs of Arabidopsis tested, and LRB1 is negatively regulated by ABA.” Or “The results indicated that LRB1 is widely expressed in multiple organs, and LRB1 is negatively regulated by ABA.”
4. Figure 3. The authors conclude that “LRB1 gene knockout plants are not sensitive to ABA..” I disagree, they are less sensitive, but are not insensitive. See graphs. At higher ABA, germination is slowed at high ABA concentrations compared to no ABA. How about, describing them as less sensitive to ABA?
5. Figure 4 legend reads “Cotyledon re-greening assay”. Its not a re-greening, its just greening, correct? So it should read “Cotyledon greening assay”. Please correct in supp data 3 as well.
6. The section of the results that describes the experiment shown in Figure 4 is poorly written. First, it is redundant, with the first paragraph (lines 327-337) and second paragraph (lines 338-350) repeating much the same information. Please condense and state the results only once.
7. Interpretation of Figure 4. The authors write that “mutants lrb1-1 and lrb1-2 displayed uninhibited greening of cotyledons as opposed to the wild-type material.” [referring to growth on 0.4 µM ABA]. I disagree. In the graph the mutant lines show 70-80% inhibition. Is it statistically different than on MS? The authors should test, but I would argue that lrb1-1 and lrb1-2 are slightly inhibited, however, much less affected than WT and the OE lines. But not “uninhibited”.
Here are my comments that are on typos, small text changes, etc. Listed in order of appearance in the ms. There is first a list of meaning suggestions/correction, then another list of typos.
Abstract,
1. (same sentence is in Results section, lines 25 and 26). This sentence is not accurate: “In this study, phenotypic analysis of germination and green cotyledons revealed that the growth of wild type WT Arabidopsis was significantly inhibited by ABA treatment”. The growth inhibitory effects of ABA have been revealed before, and are well-known and published previously many times. I think what the authors mean is that lrb1 and lrb2 mutants exhibit resistance to the inhibitory effects of ABA in germination and cotyledon greening known to occur in WT. Maybe use this sentence instead: Phenotypic analyses revealed that lrb1 and lrb2 mutants are less sensitive to the inhibitory effects of ABA on germination and cotyledon greening that is seen in WT.
2. (same sentence is in Results section, lines 26 and 27). This sentence needs more information. The sentence is “Mutants lrb1 and lrb2 exhibited resistance to ABA and displayed normal growth patterns.” The phrase, “normal growth patterns”, does it refer to under ABA conditions or under normal conditions? Please add clarification. Such as, “normal growth patterns under control conditions”.
3. Introduction, line 62. In the discussion of the history of isolation of abi mutants, the authors write that they were “confirmed to be insensitive to ABA”. In reality, they are NOT insensitive, but significantly less sensitive to ABA, not truly insensitive. The name is not strictly correct (I guess they were insensitive at the concentration of ABA used in the genetic screens at the time). I ask the authors to look at early data- from example, the abi1-1 is very much less sensitive, but does show inhibition of germination (at 2 days) with 10 micromolar ABA (see Figure 4, Plant Cell 11:2897), and for abi4 and abi5 see figure 2 The Plant J. 5:765-771), LOF in abi4 and abi5 alleles show sensitivity to ABA at ~5 and higher micromolar exogenous ABA in 5-day germination assays. I think this is an important point. Some double mutant combinations do show insensitivity, but none of the singles listed are truly insensitive (see also Fig 7, Plant Physiol. 94: 1172-1179). These mutants are not insensitive, but show a reduced sensitivity. Please modify to be correct. For example, simply saying, “to be less sensitive to exogenous ABA”
4. Results, lines 62-63. The description of the abi alleles needs more information. There is only 1 type of ABI1 and ABI2 allele that gives a dominant ABA hyposensitivity, specifically found in the abi1-1 and abi2-1 alleles. (the same point mutation in both proteins results in reduced binding of the ABA-receptor complex to the ABI protein, preventing ABA’s inhibitory effect). The other alleles in these same genes (ABI1 and ABI2) are loss-of-function (LOF) alleles and have a different phenotype. They are mildly ABA-hypersensitive (I think by itself the LOF of abi2 does not have an altered ABA sensitivity, but I haven’t looked this up). So I think that the authors should list the specific alleles- change “Such as the dominant mutant ABA-insensitive1 (abi1) and (abi2) and the recessive mutant of… ” to “such as the dominant alleles of ABA-INSENSITIVE 1 (abi1-1) and ABI2 (abi2-1) and the recessive alleles of…”
5. The next sentence should be modified to be accurate (same ideas as in #4 above), Modify “While abi1 and abi2” to “while abi1-1 and abi1-2” [in italics] and modify [in lines 65-66] from “demonstrate their ABA insensitivity” to “demonstrate their reduced ABA sensitivity”.
Minor Changes to suggest (these are listed in order they appear in the manuscript). All are proposed wording changes to correct errors, typos, English usage, or unclear writing.
1. Abstract, Methods section, line 20-21, The first sentence does not use standard expression. Please modify the following: “The homozygous identification method was employed to generate T-DNA insertion mutants of Arabidopsis.” Modify, for example, to “Homozygous T-DNA insertion alleles for LRB1 and LRB2 were isolated.”
2. Abstract (Results section, line 27), the sentence starting, “the double lrb1lrb2 showed reduced responsiveness to ABA.” The first word needs to be capitalized. “The”
3. Abstract, lines 32-33. Delete “sites”, modify to “tissues.” Or better “across all developmental stages and tissues tested.” Because you likely did not test all!
4. Introduction, line 66-67, “As diversion factors” should be not in italics and should be “As transcription factors”.
5. Lines 74-75. Not sure what the “respectively” refers to. But the PYR/PYL name came from mutant screening (Park et al) and the RCAR name from Y2H (May et al). So the sentence should read “using mutant screening and yeast two-hybrid methods, respectively (Park et al, 2009; Ma et al 200)
6. Line 75, modify “confirmed that it plays” to “confirmed that they play” There are multiple ABA receptors in most species. And who is “they” in “They confirmed”? How about changing the sentence to “The central role of PYL/PYR/RCAR proteins in ABA perception was subsequently confirmed by the crystal structure of the PYR1-ABA complex (Santiago et al 2009).
7. Line 76, change “done” to “written”
8. Line 78, change “reactive” to “responsive” and add the abbreviation (ABF) and modify “ABA reactive element binding proteins” to “ABA-Responsive Element Binding Proteins” and add abbreviation (AREB), so should read, “ to activate ABA responsive binding factors (ABFs)/ABA-Responsive Element Binding Proteins (AREBs) (Zhao et al., 2018).
9. Line 87, modify “We call them” to “They are called”
10. Line 92, Modify “RPK1 is LRR-RLKs isolated from Arabidopsis” to “RPK1 is one LRR-RLK in Arabidopsis.”
11. Line 92, Change “expression was” to “expression is”
12. Line 93, change “hypersaline” to “hypersalinity”.
13. Lines 93-94 seem redundant to sentence that starts in line 92.. The regulation of expression of RPK seems to be mentioned twice. Combine information into one sentence and put two references together.
14. Line 102, should be LRRs (plural).
15. Line 248, delete “positively”.
16. Line 248, use “approximately 1000 bp upstream” because they are all not at exactly 1.000 kb upstream.
17. Line 300, delete “disinfected”
18. Line 301-302, modify to “on MS medium plates with two different concentrations of ABA, either 0.4 µM or 0.8 µM.”
19. Line 302. The term “control check” is not a standard term in English. I suggest just use control.
20. Line 327, delete “seed”. Doesn’t make sense with it in.
21. Line 327, should read “mutant lrb1-1 and lrb1-2 and overexpression lines LRB1ox2 and LRBox10.
22. Line 401, Figure 6B looks at in vivo interaction, not in vivo. I also think the experiment shows they do interact in vivo, so please correct to “LRB1 and PYL6 have the possibility of interacting in vitro” to “LRB1 and PYL6 interact in vivo”
23. Line 430, This sentence doesn’t make sense to me: “While research on early growth and development in Arabidopsis lags behind that of above-ground plant parts numerous studies have shown the combined regulation of this process by internal and external factors.” The combined regulation of what process? Please revise because the meaning is not clear.
24. Line 451, mutants should be Mutants
25. Line 476-477, should be yeast two-hybrid assay. (hybridization is not the correct term- they are hybrid proteins, not hybridization). Complementary should be complementation.
26. Title to figure 6 (in the pdf). The Figure 6 title is the Figure 5 title repeated.
The English has improved a lot after revision. The logic is much better than the previous versions. The authors should answer the review with one item by one item.
However, the qualities of figures are not good enough.
In the title, the word of ‘Participates’ is a bit too strong. The authors don’t have evidences how and where LRB1 participates ABA pathway. In the main text, it is shown ‘The cascades of LRR- RLK receptor protein kinases are involved in ABA-regulated plant growth and development’ at line 244, as well as in conclusion part.
All of text labels and figures are still not good enough with quality for of publication. These are not font or size issues, but resolutions. The readers should be able to see them clearly.
The figure legends are shown twice, in the result part and with figures.
Suppl. Fig. 1 should be labelled as ‘Suppl. Fig. 1’ , not ‘Figure 1’. The ‘LP’ ‘RP’ … are not explained in legend. B should be mis-labelled and the wt control with no signal. C LRB2 is expressed. D. Actin control is not good. I can not get the conclusions that the T-DNA mutation can completely down LRB1 and LRB2. Please clarify it.
Species Latin names (Arabidopsis) should be in italics.
Line 27, ‘the’ should be ‘The’
Line 129 ‘disinfected with 0.1% mercury’. It is horrible and mercury can not be dissolved in water. Is it ‘0.1% mercuric chloride’?
Line 134, should ‘by’ be ‘from’?
Line 132-138, a so long sentence mixes different types of phrase, please rewrite it.
Line 142, ‘LRB11’ should be ‘LRB1’.
Line 148, 202 and 213, ‘used primers’ is not a good word.
Line 214, ‘Supplementary Table 2.’ should be ‘Supplementary Table 2’.
Line 228, please give the full name of ‘N. benthamiana’
Line 246, ‘high relative degree’ should be ‘relatively high degree’
Fig 1. A-B order is wrong. B. ugly alignment. C. what do colors mean and where is exact T-DNA position? E. please directly use ‘control’ instead of ‘CK’, and where are the data from different time of ABA treatment?
Line 275, ‘and to find the protein associated with LRB1’ this purpose is not clear and it is hard to reach by GFP vector. And this sentence is a bit overlap with line 281. Since LRB1 is expressed everywhere, does GFP show signal in the leaf cell membranes?
Line 297 ‘and to find the protein associated with LRB1’ weird phrase here.
Line 305, ‘Statistics were collected for 6 days’ what is it?
Line 308, ‘no significant difference’, line 310 and 312 ‘higher’, how to test and what is the value.
Line 370-371, what is the statistics of ‘significantly lower’?
Fig. 2. A. the number labels are not well located. The bars are different scales, does it mean different cell size?
Fig. 3. A and D have poor and undistinguished colors. Poor resolution of seed germination is hard to get a conclusion.
Fig. 4,5,6, the legends have Garbled characters.
Line 327, please re-write ‘wild-type, mutant lrb1-1 and lrb1-2 overexpression lines seeds LRB1ox2and LRB1ox10.’
Line 328, how to test ‘significant differences’? what is the result?
Line 349, there is no evidence for ‘signal transduction process of ABA’?
Line 369, remove the bracket of ‘As shown in (Fig. 5A & 5B)’.
Good experimental design
Some kind of novelty.
Reviewers have rightly pointed out many weaknesses in the article. I ask that the authors address all objections. It is required that The response letter show all responses with details.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services if you wish - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title). Your revision deadline is always extended while you undergo language editing. #]
The authors met most of the comments and changed the text substantially. However, still some mistakes remained. Moreover, the English style still does not meet the standards of scientific texts.
l. 18, 115 - Leucine-rich receptor-like kinase – it should be “leucine-rich repeat receptor-like kinase”
l. 22 - instead of “the predominant expression” it should be “expression pattern”
l. 27 – skip “However,”
l. 63 - ABA-responsive – Abbreviation of abscisic acid is always in capital letters
l. 64 – insensitive to ABA, ABA-insensitive (abi1)
l. 65 – it should be “Arabidopsis thaliana (L.) Heynh)“ - full name should be mentioned at least once
l. 101 – genes should be in italics
l. 110 – „of genes ERATA, CLV1, and BRI1“ – „of receptor genes ERECTA, CLV1, and BRI1“
l. 111 – „are more intensively in-depth“ – „are recently intensive“
l. 120 – „double mutant material lrb1lrb2 for both LRB1 and LRB2“ – „„double mutant lrb1lrb2“
l. 137 – „μmolm-2s-1“ – „μmolm-2s-1“ (-2 and -1 should be in upper index)
l. 154 – „Construction and Plant System“ - Construction of what?
l. 182 - 183 - „cultured overnight and oscillated incubation final to OD600=1.2“ - cultured overnight with shaking to final OD600=1.2
l. 185 – „add L77“ – L77 was added
l. 186 – „Arabidopsis was transfected with newly bolstered Arabidopsis“ ??
l. 209 – „Construction of Overexpressed Genes and Screening of Transgenic Plants“ – Construction of vectors with LRB1 gene…
l. 250 – 259 – This part should be re-written in the same style as the other parts in MM, not as a laboratory protocol (Prepared before use, add 9.8 mL,…)
l. 265, 276 – „Primers used (Supplementary Table 1).“ – The used primers are shown in Supplementary Table…
l. 321 - „The heatmaps presented herein“- I do not see any heatmap
l. 324 – sentence: „It was further determined that LRB1 is involved in ABA signaling pathway“ can be skipped
l. 332, 334, – „CK: target materials 50 μM ABA at 0 h.“ CK: control (0 μM ABA at 0 h).
l. 332 - „GUS staining cotyledon gradually fades“ - cotyledon GUS staining gradually faded
l. 338-341 – description of suppl. figures should be better in the text than in Figure 1 legend
l. 350 - why is „confocal“ repeated?
l. 355 – „LRB1-GFP transgenic fusion expression plants“ – it is enough: LRB1-GFP transgenic plants
l. 363 – „of Arabidopsis plants“ – in Arabidopsis roots
l. 365 – „Subcellular localization Transient transformation experiments of protoplast showed that LRB1 was located on the cell membrane.“ - Subcellular localization of LRB1 on the cell membrane of transiently transformed protoplasts
l. 369 – „Subcellular localization A permanent SP1300-LRB1-GFP vector was constructed to infect Arabidopsis, and the root cell experiment showed that LRB1 was located on the cell membrane.“ - Subcellular localization of LRB1 on the root cell membrane determined by using a permanent SP1300-LRB1-GFP vector
l. 375 – „LRR-RLKs“ - LRR-RLK
l. 378 – „single mutation“ - single mutants
l. 378 – „overexpressed genes on MS“ – overexpression lines cultivated on MS…
l. 380 – „Refrigerate at 4 ℃ for 2-3 days.“ – The seeds were refrigerated at 4 ℃ for 2-3 days.
l. 380 – „it was cultured … it was taken out..“ – they were cultured … when they were taken out …was considered as the first day..
l. 385 – the germination rate of wild-type WT was observed to be lower following treatment with 0.8μM ABA (Fig. 3A)“ - The germination rate of wild-type WT decreased after treatment with 0.8 μM ABA (Fig. 3A) – this sentence concerning WT should be moved to l. 382, before description of mutants (not mutant material)
l. 390, 405, 410 – „overexpression materials“ - overexpression lines
l., 395 – „Participate in regulating seed germination with LRB1 and LRB2 in the ABA Response Pathways“ – Participation of LRB1 and LRB2 in regulation of seed germination during the ABA response
l. 407 – „the cotyledon of the material turned green“ – all cotyledons turned green
l. 410 – „wild-type, resulting“ - wild-type response, resulting
l. 430 - „Cotyledon re-greening assay. (A) Through 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM ABA concentration gradient treatment,” - (A) The effect of ABA (0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM) on cotyledon greening.
l. 433 – „After 0.4 μM ABA treatment, a comparison of cotyledons green between the wild type, mutants, and overexpression lines in 9-d-old seedlings“ - Comparison of cotyledon greening between the wild type, mutants, and overexpression lines in 9-d-old seedlings after 0.4 μM ABA treatment
l. 434 – „CK: target materials“ CK: control
l. 442 – 444 – „However, evidence for gene expression is lacking. To prove the function of LRB1 in the ABA signaling pathway, we performed real-time quantitative qRT-PCR detection.“ - To prove the function of LRB1 in the ABA regulation of gene expression, qRT-PCR analysis was performed.
l. 458 – 459 – „ABA treatment, so the germination rate and green cotyledon of single mutant lrb1-1 and lrb2 were significantly higher than those of wild-type WT. This is consistent with the results of previous assays.“ - …ABA treatment, which is in accordance with the fact that the germination rate and cotyledon greening of single mutants lrb1-1 and lrb2 were significantly higher than those of wild-type WT.
l. 462 - 463 – „Seedling material, 0.8 μM (±) ABA treatment 9 days. CK: target materials 0 μM (±) ABA treatment 9 days. (B) Seed material, 5 μmol/L (±) ABA treatment 3 days. CK: target materials 0 μM (±) ABA treatment 3 days.“ – Seedlings treated with 0.8 μM (±) ABA for 9 days. CK: control (0 μM (±) ABA, 9 days). (B) Seeds treated with 5 μM (±) ABA for 3 days. CK: control (0 μM (±) ABA, 3 days).
l. 469 – „ However, the upstream and downstream relationship of LRB1 and its homologous protein LRB2 in the ABA signaling pathway is not known, and which protein is associated with it.“ - However, the mechanism of the function of LRB1 and its homologous protein LRB2 in the ABA signaling pathway is not known, as well as the associated proteins.
l. 473 – 475 – „…bait proteins, and using protein phosphatase ABI1 and ABI2 in ABA signaling pathway, protein kinase SnRK2.1 to SnRK 2.10, ABA receptor PYL1 to PYL9 is the target protein: AD-ABI1, AD-ABI2, AD-SnRK(2.1～2.10), and AD-PYL(1～9) were used to screen target proteins interacting with BD-LRB1 and BD-LRB2.“ - …bait proteins. Protein phosphatases ABI1 and ABI2 (repressors of ABA signaling pathway), protein kinases SnRK2.1 to SnRK 2.10 and ABA receptors PYL1 to PYL9 were used as the target proteins (AD-ABI1, AD-ABI2, AD-SnRK(2.1～2.10), and AD-PYL(1～9)) to check their potential interaction with BD-LRB1 and BD-LRB2.
l. 484 – skip „Ben´s“
l. 488 – „fluoresce is very strong“ - fluoresce very strongly
l. 539 – „double mutant lrb1lrb2 of LRB1 and LRB2“ - double mutant lrb1lrb2
l. 540 – „lrb1-1, lrb1-2, lrb2, and lrb1lrb2 were not sensitive to ABA compared with WT“ - lrb1-1, lrb1-2, lrb2, and lrb1lrb2 were less sensitive to ABA
l. 543 -544 – „that the insensitivity of LRB1 to ABA was caused by LRB1 gene mutation, and LRB1 may participate in the regulation process of early growth and development in Arabidopsis by positively regulating ABA response.“ - that LRB1 may participate in the regulation of early growth and development in Arabidopsis by positively affecting ABA response.
l. 548 – „LRB1 was inhibited by ABA“ - LRB1 was down-regulated by ABA
l. 549 – „were consistent with the data“ – which data?
l. 552 – „regulation of ABA on the growth” – ABA regulation of the growth
l. 553 – 555 – „signaling on early growth and development in Arabidopsis. However, LRB1, a member of the Arabidopsis leucine-rich receptor protein kinase family, is involved in ABA-regulated early growth and development in Arabidopsis through what signaling cascade? – this should be skipped, discussion needs to be more concise, not repeating the things over and over
l. 557 – „feel“ – sense
l. 563 – „In quantitative qRT-PCR assays“ - should be skipped, function of TFs is not related to qRT- PCR assays
l. 567 - 569 – „we observed significant downregulation of these gene expressions in mutant materials: (lrb1-1, lrb2) compared to wild-type material WT after ABA treatment,“ - significant downregulation was observed after ABA treatment in expression of these genes in mutants (lrb1-1, lrb2) compared to WT
l. 570 – „normal“ – unaffected
l. 575 – „The interaction between LRB1 and PYL6 in the ABA signaling pathway was detected by yeast two-hybridization“ - The interaction between LRB1 and PYL6 in the ABA signaling pathway was detected by yeast two-hybridization, bimolecular fluorescence complementary and GST pull-down assays. The results indicated that PYL6 may be the substrate of LRB1 phosphorylation activation. Thus, LRB1 may be involved in the regulation of ABA signaling pathway affecting the early growth and development of Arabidopsis through interaction with PYL6. (The logical flow of the text should be kept. Repeating should be avoided.)
l. 580-585 skip „These results suggest that LRB1 may be involved in ABA regulation of early growth and development in Arabidopsis. It was found that LRB1 interacts with PYL6 by bimolecular fluorescence complementary and GST pull-down assays. Further phenotypic analysis showed that the lrb1-1 was not sensitive to ABA, which was consistent with the previous phenotype, indicating that LRB1 may be involved in the ABA signaling pathway to regulate the early growth and development of Arabidopsis through interaction with PYL6.
l. 596 – 597 –„ LRB1 positively regulates the early growth and development processes of Arabidopsis in the ABA signaling pathway,“ - LRB1 positively regulates the effect of ABA signaling pathway on early growth and development of Arabidopsis
l. 598-599 – „downregulation of the LRB1 gene caused the early growth and development of Arabidopsis to be insensitive to ABA and affected by ABA concentration“ - downregulation of the LRB1 gene decreased Arabidopsis sensitivity to ABA
Original research, well defined.
Underlying data are provided. The conclusions are well stated now.
Discussion should be more concise.
BASIC REPORTING
The response letter seems to be not professional to the reviewers. The authors just said that they made some changes, but not with details. In particular, the reviewers gave several items of comments or opinions, but the authors only used one sentence to say that they changed. This lets the reviewers be difficult to follow the revisions. It would be better that the authors could answer the comments one by one and mark down where the changes are. I would start the second review if the authors can give the right response letter. Otherwise, it would waste the time of reviewers.
The English language can be improved more. Some words or sentences should be more accurate. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service.
The introduction improved a lot. However, the logic of the text still need be improved..
The method part can be shrunk, especially known method can cite literatures. The discussion is not fully discussed.
The quality of Figures are not enough. Please edit the figures with professional editor. Figure legends are missing.
This research is fit with the scope of PeerJ.
The experiments are well-designed and performed to a high technical & ethical standard.
Methods described with sufficient detail.
The novelty of this research is not so high. Previous research already showed the function of LRB. Here confirms the previous research. The advantage is using double mutant of lrb1/lrb2 to validate the function of each homolog.
All underlying data have been provided; they are robust.
The conclusion is not too much. It is just correlated with the results.
Dear Authors,
The reviewers did a tremendous job and suggested a lot of corrections that must be done to make the article suitable for further proceedings. I would like to ask you to improve the style of the article and thoroughly correct the language.
[# PeerJ Staff Note: The review process has identified that the English language should be improved. PeerJ can provide language editing services - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title) #]
The authors prepared mutant of LRB1 (leucine-rich repeat receptor like kinase 1) and of its homologue LRB2 as well as double mutant lrb1lrb2 and over-expressors OE2-LRB1 and OE10-LRB1. Using prepared lines they studied the involvement of LRB1 in the signalling of abscisic acid (ABA). Both LRB1 and LRB2 were found to interact with ABA receptor PYL6. LRB1 and LRB2 were on plasma membrane and expressed in roots, stems and petioles, also hypocotyls, flowers , cotyledons and seeds.
The English very often does not meet the professional standard (e.g. l. 25 – double mutant, not „twin“)
Latin names (Arabidopsis thaliana) need to be in italics (l. 24, 124, 300, 302, 580, 586), thaliana should not start with capital letter (l. 29, 293, 353, 579)
l. 89 – „It is well known that plants have something important for their growth and development, hormones.“ The sentence should be re-written, e.g. Plant growth and development are regulated by phytohormones.
l. 167 – „overexpressed plants“, „overexpressed seeds“ – genes can be overexpressed
l. 508 – „cross-linking effect of LRR-RLKs in the regulation of ABA signal transduction“ - cross-linking is usually a connection between ligand and carrier by chemical bond
The Introduction includes misleading sentences
l.67 – „ABI1 and ABI2…negatively regulate ABA signaling..by protein phosphatase“ – ABI1 and ABI2 are phosphatases (PP2C)
l. 71 – „ABA receptors are signal regulators upstream of the ABA signaling pathway“ – This is not correct, ABA receptors are integral part of ABA signalling pathway.
l. 73 – „In 2009, Ma and Park (Ma et al., 2009; Park et al., 2009) identified a new ABA receptor, PYR/PYL/RCAR protein, in Arabidopsis by using yeast two-hybrid and mutant screening methods, respectively“ – more correctly: In 2009, two independent groups identified ABA receptors, PYR/PYL/RCAR proteins (14), in Arabidopsis by using yeast two-hybrid and mutant screening methods, respectively (Ma et al., 2009; Park et al., 2009).
l. 77 – “proteokinase”? protein kinase
l. 78 – factors, …proteins
l. 80 - ..“signal transduction“ in nucleus, in cytoplasm of guard cells, ABA activates outward ion channels which results in stomata closure.“ The ABA effect in cytoplasm should be also mentioned.
l. 92 - LRR-RLK
l. 98 – 105 – „Studies have shown that the expression levels of most ABA-induced genes…. significantly reduced in rpk1 mutants. Therefore, the function of RPKl is regulated by the expression of ABA-induced genes, and the position of RPK1 is labeled upstream of the ABA signaling“ If ABA- induced genes are suppressed in rpk1 mutant, they probably do not affect RPK1, but the situation is vice versa. Why the authors suppose that RPK1 is upstream of the ABA signalling and not a part of it?
l. 106 – more common style is: ligands are binding to the receptors (not LRR-RLK is bound to ligands, receptors are quite often localized in plasma membrane)
l. 108 – CLV – Do you mean CLV1 and CLV2 (receptors) or CLV3 (peptide hormone)? What is downstream factor of brassinosteroid receptor BRI?
l. 109 – Factors are “concentrated”?
l. 117 – „clarifying the correlation between LRB1 and ABA“? Do you mean relationship?
The study contains original research within the scope of the journal.
Experimental design is O.K.
The mutants and overexpressor lines were obtained and used reasonably.
The authors obtained novel data, unfortunately, their presentation is sometimes confusing.
Results
l. 316 – „The GUS gene driven by the LRB1 promoter was expressed in germinating roots, hypocotyls, flowers, cotyledons, and seeds of Arabidopsis thaliana were all expressed“ „expressed“ is shown twice.
l. 317 – It is not clear whether Fig. 1D concerns ABA treatment of LRB1-GUS plants.
l. 318 – 319 – „(E) Quantitative PCR results of expression of LRB1 gene in wild type WT1 mutants lrb1-1, Overexpressed materials OE2-LRB1 and OE10- 319 LRB1 treated with 50 µM ABA for 12 h. – Fig. 1E shows just WT and mutant.
Method description suffers from many mistakes (see below). It fails to provide relevant information.
l. 126 – „double-steaming“ ? Do you mean double-distilled?
l. 127 – „0.1% liters of mercury“ I do not understand what was used
l. 128 – „0.6% MS solid medium“ Do you mean that all components of MS medium were used in the ca 167x dilution in comparison with full MS medium?
l. 181 – „Agrobacterium strains….were...oscillated“?
l. 191 – „planted on MS medium plate with 0.6% concentration of ABA. The control group was 0.6% MS medium plate without ABA“ – Fig. 1D, E shows ABA at 50 µM concentration
l. 195 – „monoclonal variant lrb1-1…“ monoclonal are antibodies
l. 204 – „using a primer with specific primers“?
l. 227 – „add 100 L N, n-dimethylformamide“ ?
l. 229 – „Mix 100 µL 5 mmol/L potassium ferricyanide and 100 µL 5 mmol/L potassium ferricyanide“ ?
l. 244 – „then 2 µL, SYBR premix EX Taq 10 µL, ROX dye (control dye) 0.4 µL“ - 2 µL of what?
l. 248 – „. 55±°C“ ?
The conclusions are not justified by the data.
The interpretation does not seem to reflect the reality.
l.579 – „LRB1 was activated by ABA“ - but Fig. 1D shows that ABA has a negative effect on LRB1 expression.
l. 581 – „Aba-activated LRB1 is involved in regulating downstream ABI3/4/5 expression by the ABA signaling pathway“ – This conclusion is not justified by the results. The data seems to show that interaction of LRB1 and PYL6 is necessary for the signal transduction. It can be assumed that specific PYL6 phosphorylation is necessary for the action (as e.g. in the case of PIN proteins)
The strong point is preparation of the mutants and overexpressor lines.
Link of LRB1 to ABA signalling pathway is very interesting.
The interpretation of the data is a weak point.
The formal style of the manuscript does not seem to be acceptable.
The entire manuscript needs to be edited for clarity and for the use of appropriate scientific terms. The figure legends are especially unclear and need additional description of the experiment and editing for clarity. In addition, there is some repetition (in M&M and in Discussion in particular).
Raw data are included.
Figure 1A is not of publishable quality. Data need statistical tests.
Primary research is within the aims and scope. The research does cover a gap in our knowledge.
The topic of the manuscript is on an interesting and important subject matter, and contains some new information. Some data are clear and well supported. The results that LRB1 is localized to the PM (Fig 2A) and that PYL6 interacts with LRB1 and LRB2 (Fig 6) are convincing.
The methods are not clearly written and are redundant in part. This section needs extensive editing. Some approaches are completely absent (GUS vector, how tree was made, as examples.
The manuscript is deficient in several areas:
1. The authors use several T-DNA mutants. Verification data should be included (in supplemental is fine). That is, the PCR reactions testing for presence of WT and the insertion junction fragment should be shown. The lines used in the experiments should have their genotypes published.
2. More importantly, the authors need to show the data that the insertions result in a reduction of LRB RNA and the over-expression lines have increased LRB mRNA relative to WT. I am very surprised that T-DNA insertions about 1 kb away from the ATG would affect expression. It doesn’t seem likely. For this reason, the authors must show data that the insertions reduce expression of the RNA from the gene. Several quantitative PCRs reactions spanning parts of the coding region (maybe include the predicted 5’ UTR) should be performed with cDNA from WT, lrb1-1, lrb1-2 and lrb2 and the over-expression lines. And how much more RNA is made in the OE lines? Is the a relationship between ABA sensitivity and the amount of over-expression? Some evidence of over-expression should be provided.
3. Construction of the GUS expression vector used in Figure 1C,D is not described in the material and methods- or at least I could not find it. A description needs to be included- or separated into a section so that it can be found.
1. Fig 1. Several concerns.
A. Panel A. This could be a problem of my version only, but the resolution of panel A is too low. I cannot read the gene numbers. The numbers at the branch points are too small. When I blow them up, the resolution is too poor and I cannot read them at all.
B. Panel A, again. This panel compares LRB with other proteins in one species. I can see that its purpose is to identify additional family members with a high % identity to LRB1, but I don’t understand this statement in the Results section: “homology rate of the genes LRB2 and LRB1 was quite high, reaching about 80%”. I don’t understand the term "homology rate". Also, the method for construction of this tree should be given in the material and methods section. Linking to the home page of TAIR does not help the reader find where this tree is, nor how constructed.
C. Panel B. Both insertion mutants for LRB1 are listed, but their positions are not indicated on the gene line. Their positions relative to the ATG should be indicated on the lines. The orange boxes could be much small and for LP and RP and their positions relative to ATG indicated. The line should be to a scale (at least upstream of ATG).
D. Panel C, D. What gene fused to GUS? The legend doesn’t say. Add LRB1 in the legend.
E. Panel D. Showing one seedling is not sufficient. Here the authors could extract protein and measure GUS activity to get a quantitative measure. Or stain a pool of 5 seedlings for each treatment. The text in the legend is confusing: “Applying 50 µM ABA at 2h, 6h, and 12h time gradients” doesn’t sound the same as described in the Results section: “were treated with 50 µM ABA for 2h, 6h and 12h, respectively” Please make the same description in both places.
F. I think panel E did not get copied correctly. Only LRB1 RNA is included under control and 50µM ABA treatment. But the legend says that expression in lrb1 and the two OE lines are included, and they are not. The legend doesn’t have a correct sentence.
G. Panel E. The y axis is “Relative Expression”. Relative to what? Please include in legend and in the material and methods. I don’t see a reference gene in the “Real-Time..” section.
2. Figure 2C. What is cytoderm?
3. Fig. 3. In this experiment the germination of WT, mutant and over-expression lines are compared in germination ability over time under control and in the presence of different concentrations of the germination inhibitor, ABA. There are several concerns regarding this figure.
A. This explanation is not clear. The authors write, “the mutant was significantly different from the wild type, and the cotyledon of the material in 0.6%MS medium with ABA....” Which is “the mutant”? Which “cotyledon”? Several mutants were in the experiment. Please clarify which mutant and which cotyledon.
B. I recommend that the authors take more time points between day 1 and 2 under control conditions. There could be a slight difference in germination between the lines that would show up if the measurements were 6 hrs apart rather than 24. A delay of several hours could be observed that won’t be seen with 24 hr time points.
C. I also suggest that a higher ABA concentration be used to see if can distinguish the single mutants from the double.
D. The authors write (line 365), “The germination rate of double mutant material lrb1lrb2 was significantly higher than that of single mutant materials lrb1-1 and lrb1-2”. I do not see this difference in the figure. The double looks the same as the lrb1 single. [I think there is a typo- is lrb2 meant instead of lrb1-2?]. And there are no statistical tests to support the conclusion made by the authors.
E. I recommend changing the X axes. Day 0 is time zero when there is no germination (right after taking them out of the cold). Day 1 should be after 24 hrs of RT treatment. Calling as “day 1” the time point immediately after taking the plates out of the cold is not established convention- and is confusing!.
4. Figure 5 legend. The description- the value of control is taken as “1”, not 100%. And bottom value on the y axis should be “1” not “0”. A value cannot be relative to “0”.
5. Figure 6. The figure legend really needs more information. How are the proteins visualized? Which are input and which are from the GST-pulldown sample? What is the amount of input relative to the amount that is recovered from the interaction assay. Typically, a percent of the input is loaded on the same gel as the pulldown to compare the efficiency. It is not clear whether this control was done.
Minor.
1. The convention for the plant name is Arabidopsis thaliana (small “t” and italics). If you just refer to the plant as Arabidopsis, use capital “A” and no italics. Only need to give full name one time. Please correct usage throughout the manuscript.
2. Fig 1C. The results section (line 299 and 305) refers to this as “Fluorescence microscopy”. Visualizing GUS staining is not “Fluorescence microscopy”.
3. Fig 3B,D. The y axis is currently, “Germination rate (%)”. However, what is plotted is the percent germination, not a rate. The y axes in these graphs should be changed to “% Germination”. (then the line produced in the graph becomes the rate).
4. Line 214, I think its pCAMBIA, not pCMBIA.
5. The plasmid description of the fusion of LRB1 with GFP is not correct. It should be pHBT-LRB1-GFP to make it clear that there is a coding region fusion between LRB1 and GFP.
6. Line 296, what is “south mustard”?
7. The discussion mentions “insensitivity of LRB1 to ABA seed growth and development was caused by LRB1 gene mutation…” However, the experiments do not look at seed development, only seed germination and seedling growth.
8. The discussion writes “The results indicated that LRB1 regulates the down regulation of ABI3, ABI4, and ABI5 genes after downregulation” I am not sure what this means.
The English language should be improved to ensure that an international audience can clearly understand your text. For example, which is “regulated by ABA“ is not clear in the title“, and the species name should be ‘’Arabidopsis thaliana’’. Some words are not scientific words, for example, ‘twin mutant’ at line 25 should be ‘double mutant’, and overexpression lines of ‘OE2-LRP1’ should be ‘LRP1ox2’. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service.
The introduction is well described. However, there is a gap between LRR-RLK subfamily and the topics of LRB1 and LRB2. The biological questions are not asked by the authors.
Structure conforms to PeerJ standards. The method part is a bit longer with too much details. The discussion is not fully discussed.
Figures are poorly organized and some are in low quality. Figure legend 2,4 and 5 are saved as pictures and mixed with figures. The legend is not well described and some labels are not explained.
Raw data is supplied.
This research is fit with the scope of PeerJ.
Research questions were not introduced after the literature summary. The research studied two homologous genes, but many times only talked LRB1.
The experiments are well-designed and performed to a high technical & ethical standard.
Methods described with sufficient detail, a bit too much. Replicate information is not provided.
The novelty of this research is not so high. Previous research already showed the function of LRB. Here confirms the previous research. The advantage is using double mutant of lrb1/lrb2 to validate the function of each homolog.
All underlying data have been provided; they are robust.
The conclusion is not too much. It is just correlated with the results.
This research confirms the function of LRB1 and his homolog of LRB2. They use backward genetics method to verify the gene function with different techniques. All the different directions of experiments support the gene function. The experiments are well designed and well performed. However, the writing is not so good. The logical structure is not so clear for this paper. Sometimes, the language is not clear and not professional.
As always there are a number of unclarities in the manuscript which I would like the authors to
elucidate.
At line 113, does ‘seed growth’ mean ‘plant germination’?
At line 89, the sentence is not clear.
At line 132, ‘by MS solid medium’ is not clear.
At line 139, it is not accurate word of ‘bioinformatics’.
At line 191, ‘The control group was 0.6% MS medium plate without ABA’ should remove 0.6%.
At line 297, what is ‘homology rate’?
At figure 1,3,4,5, what does ‘CK’ mean?
At figure 1a, I can not see clearly the figure.
At figure 1c, d, the scale bar is missing or not clear.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.