Identification of the AKCDPK gene family and AkCDPK15 functional analysis under drought and salt stress

Penghua Gao; Ying Zou; Min Yang; Lifang Li; Ying Qi; Jianwei Guo; Yongteng Zhao; Jiani Liu; Jianrong Zhao; Feiyan Huang; Lei Yu

doi:10.1371/journal.pone.0325453

Peer Review History

Original SubmissionDecember 23, 2024
23 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0325453.r001
27 Feb 2025 Decision Letter - Sumita Acharjee, Editor Dear Dr. gao, Please submit your revised manuscript by Apr 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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YJL23010, YJL23005, YJL23007), Yunnan Key Laboratory of Konjac Biology, Yunnan Province Yu Lei Expert Grassroots Research Workstation, Yunnan Provincial Science and Technology Department (grant no. 202201AU070043, 202101BA070001-174), Yunnan Provincial Science and Technology Department (grant no. 202449CE340009), College Student Innovation Training Program Project (grant no.S202411393051, S202411393042)” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. 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This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: Review of Manuscript: Identification of the AKCDPK Gene Family and AkCDPK15 Functional Analysis under Drought and Salt Stress The manuscript presents an interesting study on the AkCDPK gene family in Amorphophallus konjac, highlighting its evolutionary relationships and functional significance in stress tolerance. The scientific content is relevant and well-structured, but there are language and clarity issues that should be improved. Figures title and tables appear within the text, but there are formatting inconsistencies. The figure resolution is poor, making it difficult to interpret the data. Some sections have redundancies and unclear phrasing, which could be refined for clarity. I give a few examples: Lines 91-92: move it to the discussion Lines: 98-99: it “Nine A. konjac seedlings” is it as 9 replicates? If yes, please replicates Line: 100: why “two-month-old seedlings were used? Any reason? Why not one month? lines 105-106: “The samples were collected at 0, 24, 48, and 72 h.” 0 should be converted to “samples were collected before teatment and 24, 48 and 72 h after treatment”. Line 211: “First, transgenic (OE1, OE2, OE3)”, OE should be mentioned as full name when first use. Lines 215- 216: “Next, one-month-old AkCDPK15-overexpressing and WT tobacco strains were subjected to water deficit treatment”. Make it more clear. Line 232: “MW”, five full name when first use. Line 242: Table 1: the table should be in landscape format in order to be able to read the heads. Especially column “GRAVY” Line 247: NJ should be given as full name when first use. Lines 252-255: should be moved from resutls to material and methods. Lines 277-278: should be moved from resutls to material and methods. Lines 282, 284: I think motif means AkCDPKa, if right, then covert motif 13 to AkCDPKa 13. Line 408: “WT strain were significantly shorter than those”. Please give average value or percentage when say shorter than….. Line 467: I recommend to use “abiotic stresses” than “abiotic stressors” Reviewer #2: The manuscript by Gao et al. entitled as Identification of the AKCDPK gene family and AkCDPK15 functional analysis under drought and salt stress screened important CDPK family genes in the Konjac genome, studied their gene structure, evolutionary relationship, synteny-duplication and expression pattern. I have enjoyed reading the MS. This paper would be useful for Konjac improvement however current version is poorly written and rationale for the experiment is missing. I may recommend to accept after the following revision. 1. Mention the importance of Konjac, what are health, medicinal and economic benefits for growers and mankind. Why it is necessary to grown Konjac? 2. Manuscript title doesn’t mention Konjac plant name, the title need to revise. For example, “Identification of the AKCDPK gene family in Konjac and functional analysis of AkCDPK15 under drought” 3. What is the rationale to select 200 mM NaCl and 100 mM mannitol to induce salt and drought stress? How authors selected these concentrations? 4. Line 104; “An inoculation site was selected for each plant”- What does it mean? 5. Did authors include Konjac specific HMM model, which makes the analysis complete? 6. What is the basis of comparing phylogenetic relationship with rice? What are the other closely related tubers genome is available to for comparison and phylogeny? 7. Line 312-313, implying that these AkCDPK genes had experienced strong purifying selective pressure – What is the significance of this strong purifying selective pressure of CDPK genes? 8. Figure resolution is poor, please improve and re-submit. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Sushil S. Chhapekar ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org https://doi.org/10.1371/journal.pone.0325453.r002
Revision 1
9 Apr 2025 Author Response Reviewer #1: Review of Manuscript: Identification of the AKCDPK Gene Family and AkCDPK15 Functional Analysis under Drought and Salt Stress The manuscript presents an interesting study on the AkCDPK gene family in Amorphophallus konjac, highlighting its evolutionary relationships and functional significance in stress tolerance. The scientific content is relevant and well-structured, but there are language and clarity issues that should be improved. Figures title and tables appear within the text, but there are formatting inconsistencies. The figure resolution is poor, making it difficult to interpret the data. Some sections have redundancies and unclear phrasing, which could be refined for clarity. I give a few examples: Lines 91-92: move it to the discussion Response: Thank you for your suggestions, which we feel have improved our manuscript. We agree with your suggestion, so based on your advice, we have moved lines 91-92 to the discussion section. Lines: 98-99: it “Nine A. konjac seedlings” is it as 9 replicates? If yes, please replicates Response: We apologise for any confusion. In our experiment, each treatment was set up with three replicates, and each replicate contained nine A. konjac seedlings. This now reads “Each treatment was performed with three replicates, and each replicate contained nine A. konjac seedlings” (Lines 105–106). Line: 100: why “two-month-old seedlings were used? Any reason? Why not one month? Response: A. konjac sprouts relatively slowly, so using two-month-old seedlings ensures that the A. konjac leaves have fully expanded. Therefore, we selected using two-month-old A. konjac seedlings. We have included a brief mention of this rationale (Lines 110–111). lines 105-106: “The samples were collected at 0, 24, 48, and 72 h.” 0 should be converted to “samples were collected before teatment and 24, 48 and 72 h after treatment”. Response: We have made revisions based on your suggestion. The revised section has been highlighted in red font. The new text reads “Samples were collected before treatment and 24, 48 and 72 h after treatment” (Lines 115–116). Line 211: “First, transgenic (OE1, OE2, OE3)”, OE should be mentioned as full name when first use. Response: We have made revisions based on your suggestion. The revised section has been highlighted in red font This now reads “(AkCDPK15 overexpression strain 1 [OE1], AkCDPK15 overexpression strain 2 [OE2], AkCDPK15 overexpression strain 3 [OE3])” (Lines 228–229). Lines 215- 216: “Next, one-month-old AkCDPK15-overexpressing and WT tobacco strains were subjected to water deficit treatment”. Make it more clear. Response: We have made revisions based on your suggestions. The revised sections have been highlighted in red font. Line 232: “MW”, five full name when first use. Response: We have revised this accordingly (Line 252). Line 242: Table 1: the table should be in landscape format in order to be able to read the heads. Especially column “GRAVY” Response: We have altered the table to make it easier to read. Line 247: NJ should be given as full name when first use. Response: We have revised this based on your suggestion. The revision has been highlighted in red. Lines 252-255: should be moved from resutls to material and methods.Lines 277-278: should be moved from resutls to material and methods. Response: We have moved these portions based on your suggestions. Lines 282, 284: I think motif means AkCDPKa, if right, then covert motif 13 to AkCDPKa 13. Response: In the manuscript, 'motif' refers to the conserved motifs contained within the genes of various CDPK family members; therefore, we would like to maintain this as it is. Line 408: “WT strain were significantly shorter than those”. Please give average value or percentage when say shorter than….. Response: We have made revisions based on your suggestions. The revised section has been highlighted in red font. This now reads “In terms of salt stress (200 mM NaCl), the WT strain was substantially shorter than the transgenic strain, with an average height of 0.92 cm compared to 2.13 cm (Fig 10A, B). In terms of mannitol stress (100 mM mannitol), the WT strain was substantially shorter than the transgenic strain, with an average height of 1.86 cm compared to 5.23 cm (Fig 10C, D)” (Lines 436–441). Line 467: I recommend to use “abiotic stresses” than “abiotic stressors” Response: Thank you. We have changed this accordingly. Reviewer #2: The manuscript by Gao et al. entitled as Identification of the AKCDPK gene family and AkCDPK15 functional analysis under drought and salt stress screened important CDPK family genes in the Konjac genome, studied their gene structure, evolutionary relationship, synteny-duplication and expression pattern. I have enjoyed reading the MS. This paper would be useful for Konjac improvement however current version is poorly written and rationale for the experiment is missing. I may recommend to accept after the following revision. 1. Mention the importance of Konjac, what are health, medicinal and economic benefits for growers and mankind. Why it is necessary to grown Konjac? Response: We would like to offer our gratitude for reviewing our manuscript. We have made additions in the introduction section, which have been highlighted in red font. The text now reads “The konjac corm is rich in glucomannan, a soluble dietary fibre that has cholesterol-lowering, blood sugar-regulating, detoxifying, and gut health-supporting properties. In addition, konjac also possesses antibacterial and anti-inflammatory effects [19, 20]. Based on these characteristics, the application of konjac in the biopharmaceutical, industrial, and food processing industries is becoming increasingly widespread [21]. As a result, the cultivation area of konjac continues to expand. It is also worth noting that konjac cultivation requires relatively low investment, providing farmers with opportunities to increase their income [22]” (Lines 76–83). 2. Manuscript title doesn’t mention Konjac plant name, the title need to revise. For example, “Identification of the AKCDPK gene family in Konjac and functional analysis of AkCDPK15 under drought” Response: We have revised the title based on your advice. 3. What is the rationale to select 200 mM NaCl and 100 mM mannitol to induce salt and drought stress? How authors selected these concentrations? Response: We have two reasons for choosing 200 mM NaCl and 100 mM mannitol. First, we chose these values based on the results of preliminary experiments: During the testing of different concentrations, we found that 200 mM NaCl and 100 mM mannitol induced the most substantial stress phenotypes in plants. Specifically, we tested NaCl concentrations of 100, 150, and 200 mM, and mannitol concentrations of 50 and 100 mM. Seconed, when reviewing the relevant literature, we found that similar concentrations were used in comparable studies. For example, Yu et al. in their study on rice WRKY gene salt tolerance (doi: 10.1038/s41467-023-39167-0), and Sattar et al. in their study on potato drought stress (doi: 10.3390/plants10050924) both used similar concentrations. 4. Line 104; “An inoculation site was selected for each plant”- What does it mean? Response: “An inoculation site was selected for each plant” means “During PCC inoculation, each plant has only one inoculation site”. The revised sections have been highlighted in red font. 5. Did authors include Konjac specific HMM model, which makes the analysis complete? Response: We apologise for any confusion. We have made the necessary revisions in our manuscript. The revised sections have been highlighted in red font. 6. What is the basis of comparing phylogenetic relationship with rice? What are the other closely related tubers genome is available to for comparison and phylogeny? Response: Since both konjac is a monocotyledonous plant and rice is another widely studied monocotyledonous model plant, we selected rice to compare the phylogenetic relationship. Moreover, since the whole genomes of closely related tuber plants within the Amorphophallus genus have not been published, we did not select other tuber plant genomes for comparison and phylogenetic analysis. 7. Line 312-313, implying that these AkCDPK genes had experienced strong purifying selective pressure – What is the significance of this strong purifying selective pressure of CDPK genes? Response: We have revised this in our manuscript. The revised sections have been highlighted in red font. 8. Figure resolution is poor, please improve and re-submit. Response: We have re-submitted these figures. And in the last, our manuscript has undergone language editing and refinement by Editage. This is our editing certificate. Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0325453.r003
13 May 2025 Decision Letter - Sumita Acharjee, Editor Identification of the AKCDPK gene family and AkCDPK15 functional analysis under drought and salt stress PONE-D-24-59338R1 Dear Dr. gao, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Sumita Acharjee Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #2: Yes ****** Reviewer #2: Author have addressed my comments and I find lots of improvement in the manuscript. The revised manuscript will be helpful for Konjac research. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #2: Yes: Sushil Chhapekar ******** https://doi.org/10.1371/journal.pone.0325453.r004
Formally Accepted
Acceptance Letter - Sumita Acharjee, Editor PONE-D-24-59338R1 PLOS ONE Dear Dr. Gao, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sumita Acharjee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0325453.r005

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