Genome-Wide Identification and Expression Analysis of the TCP Genes in Jatropha curcas L. Reveals Its Roles in Involvement of Leaf Shape

Round 1

Reviewer 1 Report

Jatropha curcas L. is an important non-wood forest tree that has been widely spread throughout the world in tropical and subtropical regions. In order to understand the leaf development regulation, the authors systemically studied, for the first time, the Jatropha TCP genes, which were demonstrated to participate in leaf development by genetic and molecular studies in many other plant species.

This is a carefully studied work, and the manuscript is of considerable significance in its clear findings, rich in data and diverse。I enjoyed reading and found the manuscript interesting. 

I only have a few concerns that could be addressed to improve this manuscript.

First, the introduction part can make a more in-depth analysis of the description of the TCP gene family and the current research status, so that readers who are new to this content can more quickly understand the preface results and current problems of the research direction.

Second, in the phylogenetic tree analysis, it would be more perfect to provide the gene registry numbers of the various species used in the figure at least in the supplementary materials.

In the expression profiling of genes during the development of different organs, it is recommended that the authors provide the data that make up the heatmap to the supplementary data in the form of tables.

 

In biological nomenclature, the genes, genus and species names need to be italicized.

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Reviewer 2 Report

The importance of the industrial use of the Jatropa curcas plant in the production of biofuel is outstanding. For this reason, it is worth dealing with the plant.

However, the authors did not conduct research on the seeds, but compared the morphological characteristics of the leaves in two subspecies.

I have some suggestions to improve the article:

1.       The entire article is characterized by the fact that there are few cited literature references. The authors also make statements in the introduction that they do not support with references: „Given its untapped potential for sustainable production of food and bioenergy, and studies related to Jatropha are now attracting extensive attention”.

2.       „The morphological diversity of leaves helps plants adapt to environmental changes, and differences in the shape of leaves lead to different photosynthetic efficiency of plants.”

This claim cannot be verified. The difference between the photosynthetic efficiency of plants mostly stems from whether they are C3 or C4.

3.       3. The materials and methods chapter as a whole is characterized by thoughtless editing. The sequence of the performed experiments is not logical.

4.       „An unrooted phylogenetic tree based on the full length protein sequences alignments was constructed using MEGA 7.0 and a phylogenetic tree of the JTT+G model was constructed using the maximum likelihood method (ML).”

References are missing from all used softwares.

Likewise here: „Multiplex alignment analysis of the TCP protein domain using Clustal X and visual analysis using Jalview 2.11.1.0 software.”

Likewise here: „Based on the transcripts of the whole genome of Jc, the expression levels of transcripts in roots (SRR5974855), stems (SRR5974856), young leaves (SRR5974846), female flowers (SRR5974844), male flowers (SRR5974843), young seeds (SRR5974841), semi-mature seeds (SRR5974842), mature seeds (yellow) (SRR5974857), mature seeds Ⅱ (blackbrown) (SRR5974858) were calculated by Kallisto Package of R. Integrated these results into a cluster heatmap by TBtools.”

5.       In chapter 2.4, no reference is made to the place of origin of the transcripts. If they come from a database, the deposit number given by the public database must be provided.

6.       In chapter 2.5, the description of the plant material is missing. Planting conditions of the plant species used, sample collection data, dates, number of repetitions, sample processing data, etc.

7.       The following description can be found in chapter 2.5: „Baby leaves, apical new leaves, initial leaf shape, leaf shape tiny (leaf width less than 2cm), reddish or red. Young leaves, second only to young leaves, with complete leaf morphology, concave veins, large leaf shape, light green or wrinkled leaf reddish. Intermediate leaves, slightly higher than functional leaves, leaf shape slightly larger than young leaves, green or bright green. The position of the functional leaf is close to the lower end of the morphology of the sample plant, showing green or dark green. Bar=5 cm.”

This could be another subsection called morphology. The structure here is also illogical.

8.       The Results chapter contains inaccurate statements in many places: „18 non-redundant JcTCP genes were identified in 7 out of 11 Jatropha chromosomes after removing redundant sequences.” respectively „In addition, 18 JcTCP genes were annotated as JcTCP1 to JcTCP18 on the basis of their distributions in the genome and relative linear orders in each chromosome.”

Identified genes and annotation results are not found in the article.

9.       „…that most JcTCP proteins contained amino acids” -  All proteins are made up of amino acids.

10.   "Multiple sequence alignment was performed with DNAMAN."  - The reference is missing.

11.   Table2. note contains the same sentence twice.

12.   Figures 7. A and 7. B are crowded and blurry when enlarged. However, they contain essential information.

13.   „The publicly available transcriptome data was used…” - There is no reference to the public database with a deposit number in chapter 3.7.

14.   We know nothing about the starting material of the transcriptomes used (where they come from, under what conditions, etc.).

15.   „The heatmaps were generated using the Jctropha Curcas L. transcriptome data.”  -The description of the experiment is not included in the materials and methods chapter. We generate a heat map in silico. What program did you use for this, where are the output data?

16.   „The color gradient indicated the change in transcript levels as expressed in ‘reads per kilobase per million of mapped reads’ (RPKM) (a) and log2 values (b).” - Authors write about RPKM values, but there is no indication of what exactly these values mean (they indicate differences between gene expression levels) and how they were calculated (formula). The basic data required to calculate the RPKM values ​​are generated as the output of computer analyzes (e.g. with the Geneious program). Where did you get the RPKM data?

17.   Figure 9 is unnecessary because it is not informative at all.

18.   On the basis of what was Figure 10 made? In how many repetitions? The axes are not uniform. Images are too small. If the method was RTq-PCR, where are the machine output data? If the figures were made differently, how?

19.   The caption of Figure 10 does not reflect what is depicted.

20.   When analyzing Figure 10, it is repeatedly mentioned that the differences are significant or not significant. From what do they think this? No statistical analysis is available.

21.   In many places, the statements made by the authors in the Discussion chapter are not supported by relevant references. Without it, you should not make assumptions.

22.   „Furthermore, using the public transcriptome…” - The public database deposit number is missing.

23.   „By using RT-qPCR, the relative expression characteristics of TCP gene at different stages of two shape leaves development were verified.” - This is a serious methodological error. RT-qPCR is used to validate the results of in silico gene expression analyses. However, that is not what they are used for here.  

24.   In several places, the authors mention the performance of in silico gene expression analyses, but in the Materials and Methods chapter, they do not detail how the experiment was performed. In addition, neither the article nor the Supplementary files contain any output data that is generated as a result of the experiment.

25.   Gene expression results are derived from transcriptomes from a public database. For RT-qPCR, however, their own plant material is used. Confusion of conclusions drawn from results from two different plant materials is also a methodological error.

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Reviewer 3 Report

The authors identified and analyzed the TCP genes in J. curcas. The abundance analysis contents are listed in this paper. The results of this paper contributed to the further understanding of TCP genes in the leaf development of J. curcas and wood plants. I have some questions as follows,

1. the title name is "genome-wide", but the protome data is used to search the TCP genes. why "genome-wide" and how the TCP genes located in chromosome?

2. In the section of 2.4, where is the expression data come from?

others miniors comments are in the pdf text. 

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Author Response

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Round 2

Reviewer 2 Report

Dear Editorial Board,

 

the authors made the necessary corrections. This is how I recommend publishing the manuscript.

Reviewer 3 Report

The authors answered my questions.

Some figures are not clear, for example, Figure 7, et al.

In my opinion the article is adequately described in all sections and Results are clearly presented and discussed. The article can be accepted for publication in the present form.