Cloning and Functional Analysis of NtHDR in ‘Jinzhanyintai’ of Narcissus tazetta var. chinensis M.Roem

Round 1

Reviewer 1 Report

The manuscript: "Cloning and Functional Analysis of NtHDR in ‘Jinzhanyintai’ of Narcissus tazetta var. chinensis M.Roem." represented a solid study for implementation of metabolic engineering tools that could have practical application for improving the quality of many plant species.

The manuscript is well organized and the results are clearly presented.

The following minor changes are recommended and some technical clarifications should be made:

Abstract

Please define MEP. After that, the authors could use abbreviation forms.

Please use italic text for Narcissus tazetta var. chinensis. Also, "var." should not be italicized. This should be implemented for all other species or genus name.   

The first sentence in the abstract should be revised: "The key enzyme, 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase HDR, responsible for the final step in the MEP pathway, was successfully cloned from the petals of Narcissus tazetta var. chinensis, herein referred to as NtHDR (accession number OQ739816)". The HDR gene was cloned, not enzyme.

Pg. 1, Line 15: Please avoid starting the sentence with abbreviation.

Introduction

The authors should briefly present the practical application of floral fragrance volatiles in Chinese Narcissus.

Materials and methods

Pg. 2, Line 87-88: There is technical error in this phrase.

Pg. 2, Line 115-116: The PCR steps should be defined, such as denaturation, annealing, and extension in the following statement: "The amplification procedure was as follows: 95°C for 3 min; 95°C for 15 s, 56℃ for 15 s, 72°C for 2 min (35 cycles); 72°C for 5 min; and 4°C +∞."

Pg. 5, Line 166-167: Please, define if the Agrobacterium are electro- or chemically-competent cells.

Pg. 5, Line 185: Please check "Section 4.6". Did the authors mean Section 2.6?

Pg. 5, Line 185, 202-203: Please provide the full names for the phytohormones BA and NAA, as well for antibiotics Kan and Tim.

Results

Figure 5 Caption: Please check if the HDR expression is presented only for petals or both petals and corona: "(B) Relative expression level of NtHDR at different flowering stages of petals and corona."

Author Response

Response to Reviewer 1 Comments

Thank you for your valuable and thoughtful comments. The detailed corrections were listed below, point by point:

Point 1:

Abstract

Please define MEP. After that, the authors could use abbreviation forms.

Response 1:

Abstract

Thank you for your reminder. We have correct the relevant content in the section “Abstract”. It was described as follows:

The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase, a key enzyme at the end of the methylerythritol 4-phosphate pathway(MEP), was cloned from the petals of Narcissus tazetta var. chinensis‘Jinzhanyintai’ and named as NtHDR (accession number OQ739816).

Point 2:Please use italic text for Narcissus tazetta var. chinensis. Also, "var." should not be italicized. This should be implemented for all other species or genus name. 

Response 2:

Thank you for the reminder. We have modified the latin name of Chinese Narcissus to Narcissus tazetta var. chinensis M.Roem. in the revised manuscript.

Point 3:

The first sentence in the abstract should be revised: "The key enzyme, 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase HDR, responsible for the final step in the MEP pathway, was successfully cloned from the petals of Narcissus tazetta var. chinensis, herein referred to as NtHDR (accession number OQ739816)". The HDR gene was cloned, not enzyme

Response 3:

Thank you for your correction. We have corrected the relevant content in the section “Abstract”. It was described as follows:

The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase, a key enzyme at the end of the methylerythritol 4-phosphate pathway(MEP), was cloned from the petals of Narcissus tazetta var. chinensis‘Jinzhanyintai’ and named as NtHDR (accession number OQ739816).

Point 4:

Pg. 1, Line 15: Please avoid starting the sentence with abbreviation.

Response 4:

Thank you for your reminder, we has modified in the revised manuscript.

Point 5:

Introduction

The authors should briefly present the practical application of floral fragrance volatiles in Chinese Narcissus.

Response 5:

Based on your suggestion, we have added the relevant content in the section “Introduction”. It was described as follows:

Narcissus tazetta var.chinensis is one of the ten famous traditional flowers in China, with a unique sweet and strong fragrance, which can be used to extract the precious essential oil.

Point 6:

Materials and methods

Pg. 2, Line 87-88: There is technical error in this phrase.

Response 6:

Thank you for your reminder. We have modified the relevant content in the section “Materials and methods”. It was described as follows:

The plants were cultivated within a controlled artificial climate chamber, maintaining a temperature of 20°C for 14 h of light exposure, as well as a temperature of 16°C for 10 h of darkness, alongside a humidity level of 70%

Point 7:

Materials and methods

Q:Pg. 2, Line 115-116: The PCR steps should be defined, such as denaturation, annealing, and extension in the following statement: "The amplification procedure was as follows: 95°C for 3 min; 95°C for 15 s, 56℃ for 15 s, 72°C for 2 min (35 cycles); 72°C for 5 min; and 4°C +∞."

Response 7:

Based on your suggestion, we have modified the relevant content in the section “Materials and methods”. It was described as follows:

The amplification procedure was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 56 °C annealing for 15 s, 72°C extension for 2 min (35 cycles); 72°C extension for 5 min; and 4°C +∞.

Point 8:

Materials and methods

Pg. 5, Line 166-167: Please, define if the Agrobacterium are electro- or chemically-competent cells.

Response8:

Thanks for your reminder,the Agrobacterium are chemically-competent cells. We have added the relevant content in the section “Materials and methods”. It was described as follows:

The plant overexpression vector plasmid pBI121-NtHDR-GFP was transferred into GV3101 Agrobacterium tumefaciens chemically-competent cells (Shanghai Weidi Biotechnology, Shanghai, China) via heat stimulation.

Point 9:

Materials and methods

Pg. 5, Line 185: Please check "Section 4.6". Did the authors mean Section 2.6?

Response 9:

Thanks for your reminder. We have changed "Section 4.6" to "Section 2.6" in the section "Materials and methods".

Point 10:

Materials and methods

Pg. 5, Line 185, 202-203: Please provide the full names for the phytohormones BA and NAA, as well for antibiotics Kan and Tim.

Response 10:

Thanks for your reminder. We have added the full names for the phytohormones BA and NAA, as well for antibiotics Kan and Tim in the section "Materials and methods". 

Point 11:

Results

Q:Figure 5 Caption: Please check if the HDR expression is presented only for petals or both petals and corona: "(B) Relative expression level of NtHDR at different flowering stages of petals and corona."

Response 11:

Thanks for your reminder. The NtHDR expression is presented only for petals in Figure 5B. We have modified the relevant content in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Minor comments

Line 44. Notably, the MEP pathway predominantly governs the biosynthesis of monoterpenes[7], which undergo a seven-step enzymatic reaction, where 1-hydroxy-2-  methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) serves as the final step of the reaction catalase. Confusing sentence.

Line 98. The total RNA extraction from… Review the use of the preposition “the” through the manuscript.

Line 153. Clarify how it was calculated relative expression. Was it made in comparison to actin transcript concentration?

Author Response

Response to Reviewer 2 Comments

Thanks a lot for your comments and suggestion concerning our manuscript. The comments and suggestions are all valuable and very helpful for revising and improving our paper. We have considered these comments carefully and have made correction which we hoped meet with approval.

Point 1:

Line 44. Notably, the MEP pathway predominantly governs the biosynthesis of monoterpenes [7], which undergo a seven-step enzymatic reaction, where 1-hydroxy-2- methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) serves as the final step of the reaction catalase. Confusing sentence.

Response 1:

Thanks for your reminder,we want to express the importance of HDR as a catalytic enzyme in the last step of MEP pathway reaction. Based on your reminder, our modifications are as follows:

The monoterpenoids are mainly generated through the MEP pathway[7]. Notably, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) serves as is the catalytic enzyme in the last step of the MEP pathway, which precisely regulates the generation of the mixture of IPP and DMAPP[8,9,10].

Point 2:

Line 98. The total RNA extraction from… Review the use of the preposition “the” through the manuscript.

Response 2:

Based on your reminder, we have removed  “the”.

Point 3:

Q:Line 153. Clarify how it was calculated relative expression. Was it made in comparison to actin transcript concentration?

Response 3:

The relative expression of the NtHDR was calculated using the 2^-△△Ct method.It is compared to the actin transcript cycle threshold, which is the number of cycles experienced by the fluorescence signal within each reaction tube to reach a set threshold.

Author Response File: Author Response.pdf

Reviewer 3 Report

Major Comments -

1) The tertiary protein structure predictions were constructed via SWISS-MODEL, but also recommend comparing structure similarity with recent start-of-the-art AI tools (such as AlphaFold). This has an added benefit of identify low and high confidence structural features in the protein.

2) L240-245 mention using retention times reported in literature to identify volatile compounds, but also recommend using standards to verify compound identification, especially for the most abundant/significant ones

3) Fig 5 - add description in the caption for the normalization basis for 'relative expression' (y-axis), in all sub-figures. Do a,b,c represent different p-value thresholds? Not clear, please elaborate in the caption.

4) L314 - add website reference

5) How many significantly altered volatiles were found between wild-type and transgenic plants? Is it possible to employ statistical tests (such as GO enrichment,principal component analysis, etc) to understand more about the respective metabolite profiles?




Minor  Comments -

1) L87, L110, 155, 339 - Fix in-text referencing

2) L370 - fix spaces

Author Response

Response to Reviewer 3 Comments

Thank you for your valuable and thoughtful comments. We have studied comments carefully and have made correction which we hope to meet with approval.

Point 1:

The tertiary protein structure predictions were constructed via SWISS-MODEL, but also recommend comparing structure similarity with recent start-of-the-art AI tools (such as AlphaFold). This has an added benefit of identify low and high confidence structural features in the protein.

Response 1:

Thank you very much for your suggestion, which we have adopted. In our use of AlphaFold, we found that it is really useful and powerful. Thanks again.

Point 2:

L240-245 mention using retention times reported in literature to identify volatile compounds, but also recommend using standards to verify compound identification, especially for the most abundant/significant ones.

Response 2:

Thanks for your advice, your suggestions are very meaningful and enrich our article. Regretfully, due to the limited time of this revision, the supplementary experiments have not been fully completed, so the content related to the use of standards to increase the verification of compound identification has not been added.

Point 3:

Fig 5 - add description in the caption for the normalization basis for 'relative expression' (y-axis), in all sub-figures. Do a,b,c represent different p-value thresholds? Not clear, please elaborate in the caption.?

Response 3:

Thanks for your reminder. Different small letters indicated significant difference at p < 0.05 level by Duncan test. We have modified the relevant content in the revised manuscript.

Point 4:

 L314 - add website reference.

Response 4:

Thanks for your reminder. We have modified the relevant content in the revised manuscript.

Point 5:

How many significantly altered volatiles were found between wild-type and transgenic plants? Is it possible to employ statistical tests (such as GO enrichment,principal component analysis, etc) to understand more about the respective metabolite profiles?

Response 5:

Thank you very much for your suggestion. Such suggestions are very meaningful. Unfortunately, the program we used measured 7 kinds of volatile substances related to flower fragrance, and only 3 kinds of substances with significant differences. Such a small base for statistical tests is not necessary. Secondly, we think it is more necessary to analyze the volatile matter of transgenic Narcissus. However, due to the long genetic transformation cycle and immature system of Narcissus tazetta var.chinensis, all the experiments we tried failed.

Author Response File: Author Response.pdf