Genome-Wide Characterization of Cucumber (Cucumis sativus L.) GRAS Genes and Their Response to Various Abiotic Stresses

Round 1

Reviewer 1 Report

Dear authors!

Critical comments

1) Lines 63-66 and 72-73: Based on the materials of the Introduction, it is not clear why the effect of exogenous treatment by ABA and GA was studied only, but the effect of jasmonic acid on the expression of genes of interest was not studied in this work. It is necessary to explain.

2) Line 102: only 1 method was used to construct the phylogenetic trees (Maximum Likelihood method). It is not clear whether groups change if you build trees using the Maximum-Parsimony or Neighbor Joining method.

3) Lines 143 and 170: possible typographical errors. Different experimental conditions (75 and 200 mM NaCl) are presented. Lines 149, 166-167 and 361-362: similarly, different experimental conditions are presented (13℃, 19℃ and 10℃, 9℃ and 10℃, respectively). It is not clear where the correct data is presented, and if the differences are correct, then for what purpose this is done.

4) Line 149: typographical error. Replace the phrase “treatment. were” to the phrase “treatment were”.

5) Line 155: subsection “2.1. Plant materials” (here “2.8. Plant materials and abiotic stress treatment for qRT-PCR”) should be allocated to a separate subsection and should be the first in the section “2. Materials and methods”. We used three plant lines – genotypes CG25, CG37 and CG104 (Lines 145 and 147). But in the section “2.8. Plant materials and abiotic stress treatment for qRT-PCR” only 2 of the three are presented. You need to describe all three in a separate subsection.

6) Line 159: incorrect phrase. The phrase “salinity (data unpublished)”. Unpublished data cannot be the characteristic of cucumber inbred line CG37 in the section “2. Materials and methods”.

7) Line 274-275: the lines are not specified (their names are absent). The article contains the information about three lines: genotypes CG25, CG37 and CG104 (Lines 145 and 147).

8) Lines 294-295: repeat. This information contained in the text earlier (Lines 145-146; 147-148; 159).

9) Line 299: there is no interpretation of the abbreviation “LT-sensitive”, then it is not used.

10) Line 359: reference number to “(Lu et al., 2020)” absents.

11) Lines 417-455; 202; 215; 238; 266; 267; 297-298; 300; 305; 306; 308; 313; 320: The manuscript should include 8 figures (according to figures legend in lines 417-455), but ones are absent both in the article and in supplementary materials. You need to add 8 figures.

Conclusion: The article is a complete study and can be accepted after minor revision (corrections to minor errors, text editing, figures attaching).

Author Response

Dear reviewer,

We are very grateful for the instructive and critical comments on our manuscript entitled “Genome-wide characterization of cucumber (Cucumis sativus L.) GRAS genes and their response to various abiotic stresses” (ID: Horticulturae-1041328). We also appreciate greatly the instructive and critical comments of the three reviewers, which are very valuable for us to improve our manuscript.

Based on your comments, we have revised our manuscript accordingly. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to comments are as flowing:

1) Lines 63-66 and 72-73: Based on the materials of the Introduction, it is not clear why the effect of exogenous treatment by ABA and GA was studied only, but the effect of jasmonic acid on the expression of genes of interest was not studied in this work. It is necessary to explain.

Response: Thank you, this is a good point. Yes, there are multiple hormones that function in plant abiotic stress response, such as ABA, jasmonic acid, ethylene, etc. In this study, we used ABA-the “stress hormone” and GA-the “growth hormone” as the representatives to study the response of GRAS genes.

2) Line 102: only 1 method was used to construct the phylogenetic trees (Maximum Likelihood method). It is not clear whether groups change if you build trees using the Maximum-Parsimony or Neighbor Joining method.

Response: Thanks for your suggestion. We used the Maximum Likelihood method according to reference 54 in line 110-111. Only the Maximum Likelihood Method was used to construct phylogenetic tree.

3) Lines 143 and 170: possible typographical errors. Different experimental conditions (75 and 200 mM NaCl) are presented. Lines 149, 166-167 and 361-362: similarly, different experimental conditions are presented (13℃, 19℃ and 10℃, 9℃ and 10℃, respectively). It is not clear where the correct data is presented, and if the differences are correct, then for what purpose this is done.

Response: Thanks for your suggestion.

The 75 mM NaCl treatment was used in a gene expression experiment in the publicly available RNA-seq data, deposited in the Cucumber Genome Database (lines 160-161). In contrast, the 200 mM NaCl treatment was used in our in-house, quantitative real-time PCR analysis described (line 173-174). This salt concentration was chosen as the most appropriate for observing a differential response to salinity for cucumber lines CG104 and CG37 based on our previous experiments.

Apologies; there was a misunderstanding, we incorrectly wrote that the low temperature conditions were (13℃, 19℃ and 10℃, 9℃ and 10℃, respectively). We have corrected the conditions: “Low temperature stress (19℃ with 14 h of light/ 10℃ with 10 h of dark for two weeks) was used for RNA-seq and quantitative real-time PCR analysis”.

4) Line 149: typographical error. Replace the phrase “treatment. were” to the phrase “treatment were”.

Response: Thank you for your suggestion. I have revised the sentence “For the low temperature experiment, leaves were sampled from “CG104” (resistant to high temperatures) and “CG37” (susceptible to high temperatures) at 0, 10, 144, 240, and 336 h after a low temperature treatment i.e. (19℃ with 14 h of light/ 10℃ with 10 h of dark).” in line 152-155.

5) Line 155: subsection “2.1. Plant materials” (here “2.8. Plant materials and abiotic stress treatment for qRT-PCR”) should be allocated to a separate subsection and should be the first in the section “2. Materials and methods”. We used three plant lines – genotypes CG25, CG37 and CG104 (Lines 145 and 147). But in the section “2.8. Plant materials and abiotic stress treatment for qRT-PCR” only 2 of the three are presented. You need to describe all three in a separate subsection.

Response: Thank you for your suggestion. I have added the following to section 2.1 on lines 78-87 as follows:

“Three cucumber genotypes were used in this study: “CG104”,”CG37”, and “CG25”. “CG104”is a Japanese-type cucumber that shows resistance to high salt and, high and low temperature stresses. “CG37” is an European-Asian type, that is sensitive to high salt, and, high and low temperature stresses. “CG25”is a North China-type, which is sensitive to high temperatures. “CG104 ” and “CG37” were used for RNA-seq (low temperature-stress) and quantitative real-time PCR Analysis (low temperature-, high temperature- and salt- stresses). “CG104 ” and “CG25” were used for high temperature-stress of RNA-seq. They were all obtained from the Cucumber Research Group within the Institute of Vegetables and Flowers, at the Chinese Academy of Agricultural Sciences, Beijing, China.”

6) Line 159: incorrect phrase. The phrase “salinity (data unpublished)”. Unpublished data cannot be the characteristic of cucumber inbred line CG37 in the section “2. Materials and methods”.

Response: Thank you for your suggestion. We have deleted the phrase “(data unpublished)” in line 173.

7) Line 274-275: the lines are not specified (their names are absent). The article contains the information about three lines: genotypes CG25, CG37 and CG104 (Lines 145 and 147).

Response: Thank you for your suggestion. We have added the specified names “genotypes CG25, CG37 and CG104”, for example, tolerant line CG104 and sensitive line CG37 (low temperature stress) line in 279-280, tolerant line CG104 and sensitive line CG25 (high temperature stress) line in 282.

8) Lines 294-295: repeat. This information contained in the text earlier (Lines 145-146; 147-148; 159).

Response: Thank you for your suggestion. We have revised the phrase to “cucumber lines “CG104” and “CG37”” instead of “cucumber line “CG104” (tolerant to low temperature, high temperature, and salinity) and line “CG37” (sensitive to low temperature, high temperature, and salinity)” in line 302-303.

9) Line 299: there is no interpretation of the abbreviation “LT-sensitive”, then it is not used.

Response: Thank you for your suggestion. We have replaced “LT” with “low temperature” on lines 306-307.

10) Line 359: reference number to “(Lu et al., 2020)” absents.

Response: Thank you for your suggestion. We added the reference number “87” line on line 368.

11) Lines 417-455; 202; 215; 238; 266; 267; 297-298; 300; 305; 306; 308; 313; 320: The manuscript should include 8 figures (according to figures legend in lines 417-455), but ones are absent both in the article and in supplementary materials. You need to add 8 figures.

Response: Yes, there are 8 figures in this manuscript. They were all cited in the“Results” section.

Reviewer 2 Report

Dear Authors,

your extensive work is very appreciate and interesting, however, please pay attention to some minor technical corrections as follows:

1. Line 77 - Adjust font size (bold?/no bold?)
2. Line 81, 82, 83,94,118,125,132,133,134 - Font size check/adjustment
3. Line 128,180 - Check / correct left indent
4. Line 149 - Sentence should be revised
5. Line 170 - Distance between number and comma - reduce
6. Line 192,193, 196-235 - Font size check/adjustment
7. Line 209 - Correct left indent
8. line 235 - Check font size
9. Line 280 - Check left indent
10. Line 331 - Check font size/type
11. Line 348 - Left indent - Check
12. Line 366 - Left indent check
13. Reference 61 - Second row - indent adjustment

I have no other suggestions or corrections.

Excellent work.

Best regards.

Author Response

Dear reviewer,

We are very grateful for the instructive and critical comments on our manuscript entitled “Genome-wide characterization of cucumber (Cucumis sativus L.) GRAS genes and their response to various abiotic stresses” (ID: Horticulturae-1041328). We also appreciate greatly the instructive and critical comments of the three reviewers, which are very valuable for us to improve our manuscript.

Based on your comments, we have revised our manuscript accordingly. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to comments are as flowing:

Line 77 - Adjust font size (bold?/no bold?)

Response: Thank you for your suggestion. The size and bold has been adjusted in line 77.

Line 81, 82, 83,94,118,125,132,133,134 - Font size check/adjustment

Response: Thank you for your suggestion. We have revised the font size in line 78,88,106,114,123,129,135,144,163,184.

Line 128,180 - Check / correct left indent

Response: Thank you for your suggestion. We have corrected left indent in line 130, 185.

Line 149 - Sentence should be revised

Response: Thank you for your suggestion. We have revised the phrase “cucumber line “CG104” and “CG37”” instead of “cucumber line “CG104” (tolerant to low temperature, high temperature, and salinity) and line “CG37” (sensitive to low temperature, high temperature, and salinity)” in line302-303.

Line 170 - Distance between number and comma – reduce

Response: Thank you for your suggestion. It has been revised in line 177.

Line 192,193, 196-235 - Font size check/adjustment

Response: Thank you for your suggestion. We have adjusted font and size in line 197-338.

Line 209 - Correct left indent

Response: Thank you for your suggestion. It has been corrected in line 205.

line 235 - Check font size

Response: Thank you for your suggestion. It has been corrected in line 238.

Line 280 - Check left indent

Response: Thank you for your suggestion. It has been corrected in line 288.

Line 331 - Check font size/type

Response: Thank you for your suggestion. It has been corrected in line 320.

Line 348 - Left indent – Check

Response: Thank you for your suggestion. It has been corrected in line 356.

Line 366 - Left indent check

Response: Thank you for your suggestion. It has been corrected in line 373.

Reference 61 - Second row - indent adjustment

Response: Thank you for your suggestion. We have revised reference 61.

Reviewer 3 Report

In this paper the authors aimed at identifying and characterizing the transcription factor genes belonging to the GRAS family in cucumber, starting from the information available for other species and surveying the Cucumis genome for possible orthologues. A detailed analysis of the expression and activation patterns by abiotic stress is undertaken and reported.

The manuscript provides novel information about GRAS genes, whose role and functioning was not characterized in Cucumis, and the data are a useful starting point for improving stress tolerance of this species by selection and genome editing.

The topic is adequately introduced and the experimental pipeline is correct. The clearliness of the results is good, although some minor improvements could be done for a faster understanding. The conclusions are adequately supported by the results.

The paper is certainly worth of publication; there are only some minor errors to be corrected and minor changes that should be done for a better comprehension. Also a general revision of the english would help.

Here below I detailed the corrections I suggest:

L15: “Cucumis sativa” should be “sativus” (as correctly typed elsewhere in the manuscript)

L31-32: This sentence doesn’t sound clear to me; please reformulate

L39: the scientific name of poplar is Populus sp., not Prunus mume

L45: genera names should be in italics

L70: “identified” should be replaced by “analyzed” or “characterized”

L91-97: not clear, please clarify

L118: Strictly speaking, synteny and colinearity are non synonyms, although they’re ofter used as if they were. Synteny is location on the same chromosome, colinearity is location on the same chromosome in the same order. Therefore I would use “and/or” instead of “i.e.”

L131: is “curcumber” correct or is it a typing error for “cucurbit”?

L138-139: What does “male/female” stand for? Male/female whole plant?

L149: a sentence was left incomplete after revising

L162-172: in general the description of stress treatments is not well detailed, it should be clarified. L166-167: how were the stress conditions? A single 14+10h cycle? L170: how was the salinity treatment done?

L181: “mRNA was treated with…” looks incomplete; unless you refer to HiScriptfiIII Supermix

L201-202: see comment for L118; this is generally valid fot the whole manuscript

L230-231: CsGRAS6 has only 2 exons. The gene with 3 exons is CsGRAS5

L247-248: not clear/convincing (probably grammar incorrect); please reformulate

L251: CsGRAS13 is missing in Fig. S3

L260: Genes of the HAM family also show a high expression level in roots and stems

L303: CsGRAS4 is missing in Fig. S6

L311: lower should be higher

L313: CsGRAS4 is missing in the figure; did you mean CsGRAS2?

L322-325: CsGRAS2 and CsGRAS26 are missing in Fig. S9, please check or add “(not shown)”

L348-365: the sense is not clear, please revise the English

L374: “function in” should be deleted

In general, the reference to figures and supplementary materials in the text should be revised, as there are a number of errors. Here I listed some (also including some other points to fix):

L184-185: Primers’ sequences are listed in Table S9, not S8; the amplicon sizes and primer locations are not reported

L224: reference to Table S5 should be to S6

L229: reference is to Fig. S2b, not S2c

L244: check reference to Tab. S7

L259: reference to Table S7 should be to Table S8

Supplementary material:

Fig. S1: what do the different colors in chromosome banding mean?

Fig. S2: In the legend, a), b) and c) should be put a the beginning of each corresponding subsection. Moreover, the color explanation in S2c is wrong; (UTRs are yellow and CDSs are green, as correctly indicated in the figure legend)

Fig. S5: What are the values in the heatmap? Expression fold changes between resistant and susceptible varieties or pure expression indices (please clarify). In the caption, “R=resistant and S=susceptible” doesn’t refer to any part of the figure

In general, the manuscript should be checked carefully for typing and grammar errors. Here I listed some (I cannot report all):

L60: “leads” should be “lead”

L68: “response TO abiotic stress”

L89-90: the verb is missing

L111: “were displayed” should be “was displayed”

L226: “a similar” should be “similar”

L272: “tolerance” should be “tolerant”

L285: “acted” should be “acting”

Author Response

Dear reviewer,

We are very grateful for the instructive and critical comments on our manuscript entitled “Genome-wide characterization of cucumber (Cucumis sativus L.) GRAS genes and their response to various abiotic stresses” (ID: Horticulturae-1041328). We also appreciate greatly the instructive and critical comments of the three reviewers, which are very valuable for us to improve our manuscript.

Based on your comments, we have revised our manuscript accordingly. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to comments are as flowing:

L15: “Cucumis sativa” should be “sativus” (as correctly typed elsewhere in the manuscript)

Response: Thank you for your suggestion. We have corrected “Cucumis sativus” in line 14.

L31-32: This sentence doesn’t sound clear to me; please reformulate.

Response: Thank you for your suggestion. We have revised the sentence “Transcription factors (TFs) play important regulatory roles in diverse plant biological processes for fitness.” in line 30-31.

L39: the scientific name of poplar is Populus sp., not Prunus mume

Response: Thank you for your suggestion. We have corrected “Populus sp.” in line 39.

L45: genera names should be in italics

Response: Thank you for your suggestion. We have corrected genera names as italics in line 44-45.

L70: “identified” should be replaced by “analyzed” or “characterized”

Response: Thank you for your suggestion. We have replaced “identified”, with “characterized” on line 70.

L91-97: not clear, please clarify

Response: Thank you for your suggestion. We have revised this part “Third, two independent sets of candidate GRAS genes were merged, and the repetitive and redundant genes were removed manually. In order to ensure candidate gene accuracy, conserved domains for GRAS proteins were further analyzed using NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART (http://smart.embl-heidelberg.de/). The non-redundant genes were used for subsequent analysis.” in line 101-105.

L118: Strictly speaking, synteny and colinearity are non synonyms, although they’re ofter used as if they were. Synteny is location on the same chromosome, colinearity is location on the same chromosome in the same order. Therefore I would use “and/or” instead of “i.e.”

Response: Thank you for your suggestion. We have replaced “i.e.” with “and” in line 126.

L131: is “curcumber” correct or is it a typing error for “cucurbit”?

Response: Thank you for your suggestion. We have changed “cucumber” to “cucurbit” in line 138.

L138-139: What does “male/female” stand for? Male/female whole plant?

Response: Thank you for your suggestion. We have used the word “male flowers” instead of “male”, “female flowers” instead of “female” in line 146-147.

L149: a sentence was left incomplete after revising

Response: Thank you for your suggestion. We have corrected the sentence “For the low temperature experiment, leaves were sampled from “CG104” (resistant to high temperatures) and “CG37” (susceptible to high temperatures) at 0, 10, 144, 240, and 336 h after a low temperature treatment i.e. (19℃ with 14 h of light/ 10℃ with 10 h of dark).” in line 152-155.

L162-172: in general the description of stress treatments is not well detailed, it should be clarified.

Response: Thank you for your suggestion. We have added the detail information, “Fifty seeds were soaked at 55 ◦C for 2 min, and were then transferred to 28 ◦C for 1 d. Germinated seeds were sown in a 32-well seedling plate with substrate. After sowing, the cucumber seedlings were cultivated in a light incubator (29℃ with 14 h of light/ 20℃ with 10 h of dark; light intensity 190~600 μmol/m²·s) until the two-leaf stage. Then, the seedlings were subjected to different stress treatments, including low temperature, high temperature, and salinity stress. Conditions for the low temperature treatment were: 19℃ during the 14 h of light, and 10℃ during the 10 h of dark. Leaves were sampled at 0, 10, 144, and 336 h after the temperature treatment. For the high temperature treatment, the incubator was maintained at 50±1℃, and leaves were collected 0, 10 and 60 min after the application of the stress. For the salinity treatment, leaves were harvested 12, 48, and 96 h after watering seedlings with a 200 mM NaCl solution. Growth temperatures were 29℃during the 14 h of light, and 20℃during the 10 h of dark. For all experiments, light intensity was maintained at 190~600 μmol/m²·s”

 in line 165-176.

L166-167: how were the stress conditions? A single 14+10h cycle?

Response: Thank you for your comment. Growth temperature was 19℃ during the 14 h of light, and night time temperature was 10℃ during the 10 h of dark and other condition were normal. It is 14+10h cycle (line 170).

L170: how was the salinity treatment done?

Response: Thank you for your question. We revised the sentence “Cucumber seedlings at the two-leaf stage were treated by watering with 200 mM NaCl solution” on line 174.

L181: “mRNA was treated with…” looks incomplete; unless you refer to HiScriptfiIII Supermix.

Response: Thank you for your suggestion. We have revised the sentence “The first-strand cDNA was synthesized using HiScriptfiIII RT SuperMix (Vazyme Biotech).” (line 186-187).

L201-202: see comment for L118; this is generally valid fot the whole manuscript

Response: Thank you for your suggestion. We have revised the phrase “and” instead of “i.e.” in line 206.

L230-231: CsGRAS6 has only 2 exons. The gene with 3 exons is CsGRAS5

Response: Thank you for pointing this out. We have revised the sentence “CsGRAS5 has three exons, the largest number of all the GRAS genes.” in line 233.

L247-248: not clear/convincing (probably grammar incorrect); please reformulate

Response: Thank you for your suggestion. We have revised the sentence “Therefore, they are likely to be key genes that respond to a variety of plant processes, although this need to be confirmed.” in line 253.

L251: CsGRAS13 is missing in Fig. S3

Response: Thank you for your suggestion. We have deleted CsGRAS13 on line 255.

L260: Genes of the HAM family also show a high expression level in roots and stems

Response: Thank you for your suggestion. We have revised the sentence “members of PAT1, HAM and SCL3 subfamily were highly expressed in vegetative organs.” in line 267.

L303: CsGRAS4 is missing in Fig. S6

Response: Thank you for pointing this out. CsGRAS4 is in Figure 5m (line 312).

L311: lower should be higher

Response: Thanks you for your suggestion. We have revised the sentence, “However, the expression of 4 genes (CsGRAS2, CsGRAS11, CsGRAS26, CsGRAS33) in “104” were lower than that of “CG37” at 10 or 60 min (Figure 6).” in line 317-319.

L313: CsGRAS4 is missing in the figure; did you mean CsGRAS2?

Response: Thank you for pointing this out. We have revised the phrase It should be “CsGRAS2” instead of “CsGRAS6”, This was added to line 321.

L322-325: CsGRAS2 and CsGRAS26 are missing in Fig. S9, please check or add “(not shown)”

Response: Thank you for noticing this. We have deleted the sentence, “Interestingly, CsGRAS2 showed down-regulated expression after GA treatment while up-regulated expression after ABA application”.

L348-365: the sense is not clear, please revise the English

Response: Thank you for your suggestion. I have revised this part,

“In this study, many cucumber GRAS genes were differentially regulated by salinity, and by low and high temperature stress. These responses to different stresses may occur via common or different regulatory mechanisms. For example, CsGRAS26 was down-regulated by high temperature stress, but was up-regulated by low temperature and salt stresses, while in contrast, CsGRAS33 was down-regulated by low temperatures, but was up-regulated by high temperature stress. These data indicate that these genes i.e. CsGRAS26 and CsGRAS33, respond to identical stresses via different mechanisms. However, in another example, CsGRAS2 and CsGRAS18 were down-regulated by all four stress treatments, which suggests they might share common regulatory mechanisms or pathways which is distinct from CsGRAS26 and CsGRAS33. Interestingly, no expression changes were detected in CsGRAS6 and CsGRAS8 under any of the stress treatments applied. This study also highlights the widespread involvement of GRAS genes in multiple environmental adaptation. Recently, the GRAS gene family in cucumber was found to have a potential role in cold tolerance [87]. In that study, a short-term (6 d), extreme cold stress (4℃) was used, and they found that Csa_1G408720 (DELLA) was involved in regulating the CBF-regulatory cold pathway. In our study, we exposed cucumber seedlings to a long-term, mild cold stress (19℃ with 14 h of light/ 10℃ with 10 h of dark for 14 d), similarly, CsGRAS2 (Csa1G408720) responded to low temperature stress as well as other abiotic stress (e.g., high temperature-, and salt- stresses).”. (line 356-372)

L374: “function in” should be deleted

Response: Thank you for your suggestion. We have deleted “function in” in line 381.

In general, the reference to figures and supplementary materials in the text should be revised, as there are a number of errors. Here I listed some (also including some other points to fix):

L184-185: Primers’ sequences are listed in Table S9, not S8; the amplicon sizes and primer locations are not reported

Response: Thank you for your suggestion. We have changed “Table S8” to “Table S9”. And I have added amplicon sizes “such that each amplicon size varied from 100~150 bps” in line 188 and primer locations in Table S9.

L224: reference to Table S5 should be to S6

Response: Thank you for your suggestion. It has been changed to “Table S6”.

L229: reference is to Fig. S2b, not S2c

Response: Thank you for your suggestion. It has been changed to “Table S2b”.

L244: check reference to Tab. S7

Response: Thank you for your suggestion. Table S7 is correct.

L259: reference to Table S7 should be to Table S8

Response: Thank you for your suggestion. We have revised the “Table S7” to “Table S8”.

Supplementary material:

Fig. S1: what do the different colors in chromosome banding mean?

Response: Thank you for your suggestion. We have added “Red colors in chromosome banding represent higher gene density, blue colors in chromosome banding represent lower gene density.” in the supplementary legends.

Fig. S2: In the legend, a), b) and c) should be put at the beginning of each corresponding subsection. Moreover, the color explanation in S2c is wrong; (UTRs are yellow and CDSs are green, as correctly indicated in the figure legend)

Response: Thank you for your suggestion. We have revised the position of a), b) and c) and color explanation.

Fig. S5: What are the values in the heatmap? Expression fold changes between resistant and susceptible varieties or pure expression indices (please clarify). In the caption, “R=resistant and S=susceptible” doesn’t refer to any part of the figure

Response: Thank you for your suggestion. The values in the heatmap indicate the expression indices, they were listed in Table S8. We have added “CS: Chilling stress, R: Resistant line, S: Susceptible line at the bottom of the picture. Log2 transformed values were used to generate the color-coded heatmap.” in legends of Fig. S5.

In general, the manuscript should be checked carefully for typing and grammar errors. Here We listed some (I cannot report all):

L60: “leads” should be “lead”

Response: Thank you for your suggestion. We have revised the word “lead” in line 61.

L68: “response TO abiotic stress”

Response: Thank you for your suggestion. We have added “to” in line 68

L89-90: the verb is missing

Response: Thank you for your suggestion. We have revised the sentence “32 Arabidopsis and 48 rice GRAS proteins were used as queries to search the cucumber genome using the BLASTP program.” in line 99-100.

L111: “were displayed” should be “was displayed”

Response: Thank you for your suggestion. It has been changed to “was displayed” in line 119.

L226: “a similar” should be “similar”

Response: Thank you for your suggestion. It has been changed to “similar” in line 229.

L272: “tolerance” should be “tolerance”

Response: Thank you for your suggestion. It has been changed to “tolerance” in line 280.

L285: “acted” should be “acting”

Response: Thank you for your suggestion. It has been changed to “acting” in line 293.