Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus)
Round 1
Reviewer 1 Report
The study described the identification of three PEPC isoforms in Pitaya. Overall, the mansucript is written in good English, however there are sometimes grammatic errors, so the manuscript should be corrected by a proffessional language editing company or editor.
In the abstract the aim of the study is missing. This is an important part to show the purpose of the study. Please include the aim in the abstract.
The introduction gives sufficient information necessary for the understanding of the research and its results.
The results. The identification oif 3 PEPC isoforms is described, however I cannot find information whether the sequences have been deposited in any of appropriate databases. If this has not been done it should be. If it has been done, accession numbers should be given. The description on the phylogenetic analysis is not clear, was it done twice? Please rewrite this part. Please see the attached file for details.
The results of protein expression, isolation and identification neddes clarification. It is not clear how the presence/absence of N-terminus has been determined. Also on the figure 3, on gel electrophoresis picture there are many bands. The ones that represent PEPC isoforms should be indicated with arrows, currently it is impossible to know which bands are PEPC isoforms. Also please give some more information on the purification, which fractions were best to obtain the biggest amounts of proteins, which were most active... Please rewrite this part.
The sentence of blocking N-termini is not clear. Please rewrite and explain (lines 132-133).
In the part describing enzymatic activities of HuPPC2 and 3 please give the values in brackets. It facilitates understanding of results, going back and forth from the text to the table is not very convenient.
Discussion. Few introductory senetnces should be given as the introduction to the discussion. You start discussion with "Regarding PEPCS in pitaya" which is a bit awkward. The sentence (lines 203-204) is completely unintelligible. Please rewrite. Otherwise the discussion is ok.
Materials and methods are sufficiently described.
Comments for author File: 
Comments.pdf
Author Response
Thank you for your valuable suggestions.
Almost all the details have been revised as instructed.
I added the aim. However, since the number of characters will greatly exceed this, some parts have been deleted from the original part.
Accession No. is shown in Figure 2.
Phylogenetic analysis was performed in two ways: the entire sequence (only 500 residues in the cactaceae family) and approximately 500 residue portion registered in the cactaceae family in all sequences. However, because there is not much difference between them, the former result is shown.
Added to Figure 3.
Regarding purification, we should have published all chromatograms, but due to the limited space available, we chose only the most important ones. After that, all active fractions are used as described.
As for the notation of enzyme activity, you may be right. However, if we list the numerical values of the five items for the two isoforms, it will be rather complicated..
The sentences 203-204 were deleted because they overlapped with the following sentences.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The aim of the study was to clone three cDNAs of phosphoenolpyruvate carboxylase isoforms (HuPPC1, HuPPC211 and HuPPC3) from pitaya plant. The study contains some interesting elements that can be considered for publication after suitable revisions.
Some suggestions:
P3: pitaya
P4: Delete point after the title
P77: A clear and well defined objective is missing at the end of Introduction
L103: Figure should be self-explanatory. Give in full PTPCs
L113: Figure should be self-explanatory. Give in full PEPCs and CAM
L124: Figure should be self-explanatory. Give in full PEPCs and CAM
L139: Table 1 should be explanatory. Give in full PEPC
L139: Table 2 contains very few data. Delete this table and include information in the text.
L148: Table 3: Give in full PEP in the title.
L166: L124: Figure should be self-explanatory. Give in full PEPCs
Table 4 is results and it appears only in the Discussion. You should talked about Table 4 in the Results section.
Author Response
Thank you for your valuable suggestions.
All the points you pointed out have been corrected. However, ‘CAM’ in the legend remains CAM because it becomes redundant when expressed in full expression.
The object was added.
Table 4 is a necessary for ‘discussion’ and should not be included in the results. I made it Supplemental Table 1.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript entitled “Molecular cloning of novel-type phosphoenolpyruvate carboxylase isoforms in Pitaya (Hylocereus undatus)” by Nomura et al is focused on an important fruit called dragon fruit and isolation and characterization of PEPC isoforms taking part in carbon fixation in Cactaceae. The data presented are important for the scientific community working on CAM-type photosynthesis. The manuscript is well written and deserve to be published after addressing the question below.
- Introduction
The introduction is well written and address the main points related to PEP carboxylase and differ type of carbon fixation.
- Results
The results are somehow well presented but some question raised:
Lines-80-82 The authors stated that with PPCF/PPCR degenerate primers obtained 3 different sequences. I would like to know which region did those primers target and also did the authors obtained a single amplicon with the same size containing the 3 PPC transcripts?
The authors wrote that PPC1 is probably expressed at particular conditions and PPC2 and 3 are constitutively expressed. As they isolated the 3 transcripts (PPC1-3) from RNA extracted from lyophilized stems but they failed to detected protein from PPC1. How could the authors comment that? Any ideas about the genome availability of pitaya?
Line 130-132 The authors wrote “After SDS-PAGE and transferring the proteins onto a PVDF membrane, the corresponding bands were cut off and taken for Protein sequencing”. Detailed protocol describing the conditions, staining should be added to the materials and methods for better understanding.
Expression analysis of PPC1-3 would be very helpful to fully describe the role of those isoforms but if I understand this could be part of further analysis.
- Discussion
Some important references should be added and commented accordingly in that part.
Example: https://doi.org/10.3389/fpls.2018.01587; https://doi.org/10.1038/s41598-020-60249-2; https://doi.org/10.1104/pp.114.240283
- Materials and methods
As mentioned above, description of PAGE conditions and sequencing should be added. Also, the protocol applied for malate and aspartate inhibition essay should be added too.
Overall, after addressing all questions and comments mentioned above, the manuscript merit to be published.
Author Response
Thank you for your valuable suggestions.
All three fragments are the same size (1.5kbp). For reference, the amino acid residues corresponding to the degenerate primers are listed in Table S2.
HuPPC1 is expected to be low in quantity. Although it is not quantitative, when the sequences of 32 fragments amplified with degenerate primers were analyzed, the quantitative ratios of 1, 2, and 3 were 3, 12, and 17, respectively. However, we think that the ratio of HuPPC1 will change depending on the environmental conditions.
I added a sentence based on papers presented.
The methods of SDS-PAGE and inhibition experiment were added.
Author Response File: Author Response.docx
