Characteristics of Oil Body Development and the Cloning and Expression Analysis of PDAT Genes in Eucommia ulmoides
, Jun Qing 1,2,†, Chenlu Liu 1,3, Qi Wang 1,3, Hongyan Du 1,2, Panfeng Liu 1,2, Lanying Du 1,2, Lu Wang 1,2,* and Qingxin Du 1,2,*Round 1
Reviewer 1 Report
This submission provides a lot of information about a somewhat unusual tree species that yields oil products, rich in linolenic acid.
Overall, I agree with the interpretation of the data but the authors should use the term 'putative' when describing the 'PDAT genes'. This is important because no function has been demonstrated for any of their designated genes and, moreover, the original discovery of PDAT relied on the similarity of its gene sequences with other enzymes.
Specific points:
A recent publication on PDAT provides useful information about the enzyme from previous publications as well. I recommend that it is utilised and, moreover, it shows that manipulation of its activity has notable effects on lipid metabolism---see ref. 39, Marmon et al. also. (Fenyk,S. et al. Biochemical J. (2022) 479, 805-823).
Lines 43-64. It is generally recognised that the Kennedy pathway is the main source of TAG in plants. PDAT provides a subsidiary route useful in certain species---see references in Fenyk et al.
70-74. Alpha-linolenic acid is the precursor of EPA and DHA (not linoleic)
86-88 and elsewhere. It would be very useful to convert harvesting dates to stages in seed development beginning from DAF (days after fertilisation).
99. Note that petroleum ether is a poor solvent for polar lipids and so will preferentially dissolve non-polar lipids and will not be quantitative for total lipids.
114 and elsewhere. Please use the term 'putative' PDAT genes throughout the text and in figure or table legends.
136. ...PDAT deduced protein sequences...
Figure 2. This shows a great variety of oil body sizes (and shapes). Perhaps a comment could be made? There are no error bars in Figure 3A) to confirm this.
213. I suggest that 'genes' is replaced by 'proteins'
227-230. Please explain how the suggested subcellular localisations have been arrived at?
Table 3. Protein 1 has a much lower molecular mass than the others suggesting that it is not a PDAT protein.
254-267. This is a very detailed discussion considering that no functional activity has been demonstrated for any of the genes.
Figure 5. It would be useful to readers to have details of the motifs.
Figure 7. It might be better to list the putative genes in chronological order. It was also rather difficult to see the individual colours.
310. Instead of being a 'unique' protein, PDAT2 may have been wrongly identified.
315-331. One would normally expect an enzyme involved in TAG formation to have good expression before TAG accumulates (see comment about Fig.9)
332 onwards. The responses of gene expression to different stresses is very complex and not really discussed properly. I feel this part of the manuscript could be shortened or even omitted.
Figure 9. Expression of all putative genes is compared to levels at 6/16. But it would be important to give details of the relative expression levels of the seven putative genes with each other.
365. Rephrase 'phospholipid monomolecular'
367. Is lipid degradation involved during seed development? Rephrase?
390. Actually TAG is mainly synthesised in the ER.
416-421. The relative expression of the putative genes should be considered in this discussion.
434-6. How exactly could the findings be used?
438-449. I suggest that the conclusions are made more cautious, especially, since the relative expression of putative genes is not stated. Perhaps PDAT2 and 4 are more significant?
Author Response
Cover Letter
Dear Reviewer,
Thank you for your comments concerning our manuscript entitled “Characteristics of Oil Body Development and the Cloning and Expression Analysis of PDAT Genes in Eucommia ulmoides”(ID: agronomy-1885341). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked with different colors in the paper.
Generally, we are sorry for failing to address some details thoroughly and correctly. Please accept our apologies and thanks for the chance that we can make things right and improve the paper further. This article has been polished by a native English speaker from LetPub. All the revisions are in following pages with answers and details attached, point by point. We hope copy and pasting what we modified in this doc will save your precious time. Again, we appreciate your sharing and time for reviewing, and sincerely wish you all good in your research.
Best regards,
Jian Zhong
Comment 1:A recent publication on PDAT provides useful information about the enzyme from previous publications as well. I recommend that it is utilised and, moreover, it shows that manipulation of its activity has notable effects on lipid metabolism---see ref. 39, Marmon et al. also. (Fenyk,S. et al. Biochemical J. (2022) 479, 805-823).
Reply:Thank you very much for recommending this excellent paper. We have read this paper carefully and gained a lot of useful information and referenced in Introduction and discussion section.
Comment 2:Lines 43-64. It is generally recognised that the Kennedy pathway is the main source of TAG in plants. PDAT provides a subsidiary route useful in certain species---see references in Fenyk et al.
Reply:We have read the papers you recommended carefully and gained a lot of useful information. In addition, we also referenced this paper in many places and revised the inaccurate expression in text. The modified contents are as follows:
At present, Kennedy pathway is known as the main pathway of oil accumulation, which relies on acyl-CoA and synthesizes TAG mainly under the catalysis of glycer-ol-3-phos-phate acyltransferase (GPAT)[7,8]. In addition, an subsidiary pathway for oil synthesis has been found, which is independent of acyl-CoA. PDAT directly catalyzes the binding of diacylglycerol (DAG) with phospholipids to produce TAG and lyso-phospholipids[9-13].
Comment 3:70-74. Alpha-linolenic acid is the precursor of EPA and DHA (not linoleic)
Reply:Thank you for pointing out this problem. We have checked and revised this error in version.
Comment 4:86-88 and elsewhere. It would be very useful to convert harvesting dates to stages in seed development beginning from DAF (days after fertilisation).
Reply:We are extremely grateful for your suggestion. We have checked and revised this error in version. The revised contents are as follows:
Fruit samples were collected from 60 days after fertilisation(DAF) (when the seeds had fully formed) to 180 DAF (when the seeds had fully matured).
Comment 5:99. Note that petroleum ether is a poor solvent for polar lipids and so will preferentially dissolve non-polar lipids and will not be quantitative for total lipids.
Reply:Thank you very much for your comments. Determination of fatty acid composition and content in this experiment We adopt GC-MS method. The eucommia seed oil was repeatedly extracted with petroleum ether to minimize the test error and we will also consider your comments in future studies.
Comment 6:114 and elsewhere. Please use the term 'putative' PDAT genes throughout the text and in figure or table legends.
Reply: We are extremely grateful for your suggestion. We have checked and revised this error in version.
Comment 7:136. ...PDAT deduced protein sequences...
Reply: Thank you very much for your comments. We have checked and revised this error in version.
Comment 8:Figure 2. This shows a great variety of oil body sizes (and shapes). Perhaps a comment could be made? There are no error bars in Figure 3A) to confirm this.
Reply: Thank you very much for your suggestion. We have revised the description and the revised content are as follows:
There are some differences in the number and shapes of oil bodies in kernels at different developmental stages. The oil body diameters at different developmental stages ranged from 5–8 μm (Fig. 3A). The number of oil bodies and oil content of Eucommia ulmoides seed had the same change trend, with an “S”-shaped trend(Fig. 3BC).
Comment 9:213. I suggest that 'genes' is replaced by 'proteins'
Reply: We are extremely grateful for your suggestion. We have checked and revised this error in version.
Comment10:227-230. Please explain how the suggested subcellular localisations have been arrived at?
Reply: Thank you very much for your comments. Physicochemical properties and subcellular localization prediction of EuPDAT proteins were obtained by online software analysis.( https://web.expasy.org/protparam/ )
Comment 11:Table 3. Protein 1 has a much lower molecular mass than the others suggesting that it is not a PDAT protein.
Reply: We are extremely grateful for your comments. In this study, the sequences containing the unique domain of PDAT gene family in Eucommia ulmoides genome were screened by multiple sequence alignment, and the gene structure of candidate genes was analyzed. Seven candidate genes were found to contain similar gene structures. Therefore, we hypothesized that these 7 genes are PDAT gene family members. However, the reason why the relative molecular mass of PDAT1 protein is much lower, we will further study.
Comment 12:254-267.This is a very detailed discussion considering that no functional activity has been demonstrated for any of the genes.
Reply: We are extremely grateful for your suggestion. We have revised the description and the revised content are as follows:
A total of 10 distinct conserved motifs were found, motif 2 and motif 10 were found to encode the PDAT domain. As illustrated in Fig. 5, most PDAT members within the same clade, especially the most closely related members, usually shared common motif compositions(e.g. EuPDAT4 and EuPDAT7), indicating potential functional similarities among PDAT proteins. Motif 3, Motif 4, Motif 5, Motif 6, Motif 7 and Motif 9 were unique to the members in Clade II, which may be important to the functions of unique EuPDAT III proteins.
Comment 13:Figure 5. It would be useful to readers to have details of the motifs.
Reply: Thank you very much for your suggestion. We have supplemented the relevant content of the motif, as follows :
A total of 10 distinct conserved motifs were found, motif 2 and motif 10 were found to encode the PDAT domain. As illustrated in Fig. 5, most PDAT members within the same clade, especially the most closely related members, usually shared common motif compositions(e.g. EuPDAT4 and EuPDAT7), indicating potential functional similarities among PDAT proteins. Motif 3, Motif 4, Motif 5, Motif 6, Motif 7 and Motif 9 were unique to the members in Clade II, which may be important to the functions of unique EuPDAT III proteins.
Comment 14:Figure 7. It might be better to list the putative genes in chronological order. It was also rather difficult to see the individual colours.
Reply: Thank you very much for your suggestion. We have modified Figure 7 in version to make it easier to view the content corresponding to different colors.
Comment 15:310. Instead of being a 'unique' protein, PDAT2 may have been wrongly identified.
Reply: We are extremely grateful for your comments. In the analysis of protein interaction network, we found that EuPDAT2 had no interaction with any other protein, speculating that this protein may be a unique protein in Eucommia ulmoides. Of course, we also agree with you, probably because of misidentification during interaction analysis.
Comment 16:315-331. One would normally expect an enzyme involved in TAG formation to have good expression before TAG accumulates (see comment about Fig.9)
Reply: Thank you very much for your comments. We agree with you. We have described in detail the gene expression levels that may be involved in the oil accumulation process.The content are as follows:
The expression of EuPDAT2, EuPDAT4, EuPDAT5, and EuPDAT7 increased first and then decreased with the passage of time, and the expression levels were the highest at 140 DAF, which was consistent with the highest oil content period of E. ulmoides seeds.
Comment 17:332 onwards. The responses of gene expression to different stresses is very complex and not really discussed properly. I feel this part of the manuscript could be shortened or even omitted.
Reply: Thank you very much for your suggestion. We agree with you that the response mechanism of different genes under different stresses is very complex. Therefore, in this section, we analyzed some expression-specific genes and shortened other content.
Comment 18:365. Rephrase 'phospholipid monomolecular'
Reply:We are extremely grateful for your suggestion. We have revised the description in reference to Fenyk et al. The revised contents are as follows:
The oil body is the smallest organelle in plants, and is mainly composed of triacylglyc-erol (TAG), phosphatidylcholine molecular , and oil body protein.
Comment 19:367. Is lipid degradation involved during seed development? Rephrase?
Reply:Thank you for pointing out this problem. According to the results, the number of oil bodies increased first and then decreased gradually. Therefore, the description here is inappropriate and has been revised. The revised contents are as follows:
With the development of seeds, the number of oil bodies gradually increases and then slowly decreases andWith the increase in the number of oil bodies during seed devel-opment, the oil body distribution gradually gathered from the cell wall to the center of the cell.
Comment 20:390. Actually TAG is mainly synthesised in the ER.
Reply:Thank you very much for your comments. We have read the relevant papers carefully and revised them.
Comment 21:416-421. The relative expression of the putative genes should be considered in this discussion.
Reply:We are extremely grateful for your suggestion. We have revised the description and the revised content are as follows:
This study found that EuPDAT genes had different degrees of response to low temper-ature, ethylene and methyl jasmonate treatment. Among these EuPDAT genes, the expression of EuPDAT1, EuPDAT4 and EuPDAT5 were obviously induced by almost all stress, and especially the expression of EuPDAT1 was upregulated more than 13-fold after the Ethylene treatment, significantly higher expression than other genes.
Comment 22:434-6. How exactly could the findings be used?
Reply:Thank you very much for your comments. We have revised the description and the revised content are as follows:
Taken together, these findings can not only provide a reference for further exploring the law of oil accumulation in Eucommia ulmoides, but also provide a theoretical basis for directional cultivation of new varieties of Eucommia ulmoides with high oil content.
Comment 23:438-449. I suggest that the conclusions are made more cautious, especially, since the relative expression of putative genes is not stated. Perhaps PDAT2 and 4 are more significant?
Reply:We are extremely grateful for your suggestion. We have revised the description and the revised content are as follows:
According to the correlation analysis, it was found that the expression of EuPDAT2, EuPDAT4 and EuPDAT7 were significantly positively correlated with the change of kernel oil content in different periods. Therefore, it was speculated that EuPDAT2, EuPDAT4 and EuPDAT7 may play important role in oil accumulation.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript by Zhong et al is an interesting study. It is well written. I only have some minor suggestion to improve it.
In the Introduction part it would be nice to read how that oil content of E. ulmoides is utilised. Is it pressed similar to olives or just the seeds are used as medication or in cooking?
line 86: “once” has to be in lower case letter.
line 211: “bobies” has to be bodies.
Figure 4 legend: what do the yellow and burgundy circles represent?
lines 254-267 and Figure 5: what type of motifs were identified in the genes? Would be advisable to show them in a table.
line 292: I suppose “Fig. 6” should Fig. 7.
line 304: full stop is missing after the word “interacted”.
line 305: delete dot in front of AT3G44830.
part 2.6.: how gene expression was normalised using the refence gene actin? Regarding this, at Figs 9, 11, 12 and 13 is the “Relative expression value” the actin normalised value?
Figs. 9, 11, 12 and 13: I would not say “extremely”. It is a very relative thing, that what someone calls “extreme”. P<0.01 is quite normal, not “extreme”. Just say: “* and ** indicate significant differences at the P < 0.05 and P < 0.01 levels, respectively” or something like that.
Author Response
Cover Letter
Dear Reviewer,
Thank you for your comments concerning our manuscript entitled “Characteristics of Oil Body Development and the Cloning and Expression Analysis of PDAT Genes in Eucommia ulmoides”(ID: agronomy-1885341). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked with different colors in the paper.
Generally, we are sorry for failing to address some details thoroughly and correctly. Please accept our apologies and thanks for the chance that we can make things right and improve the paper further. This article has been polished by a native English speaker from LetPub. All the revisions are in following pages with answers and details attached, point by point. We hope copy and pasting what we modified in this doc will save your precious time. Again, we appreciate your sharing and time for reviewing, and sincerely wish you all good in your research.
Best regards,
Jian Zhong
Comment 1: In the Introduction part it would be nice to read how that oil content of E. ulmoides is utilised. Is it pressed similar to olives or just the seeds are used as medication or in cooking?
Reply: Eucommia seed oil is a kind of high-quality vegetable oils rich in various nutrients and pharmacological components, which is widely used in medical, health care and diet fields. Therefore, eucommia seed oil can be used as both a medicinal component and an edible oils.
Comment 2:line 86: “once” has to be in lower case letter.
Reply: Thank you for pointing out this problem. We have checked and revised this error in version.
Comment 3:line 211: “bobies” has to be bodies.
Reply:We are extremely grateful for your suggestion. We have checked and revised this error in version.
Comment 4:Figure 4 legend: what do the yellow and burgundy circles represent?
Reply: We are extremely grateful for your suggestion. We have added the legend below the Figure 4 in the revision. The content are as follows:
The different colored arcs indicate different groups (or subgroups) of PDAT domains. The green, yellow, burgundy and blue circles represent PDAT domains from E. ulmoides, Ricinus communis, Carya cathayensis and Arabidopsis, respectively.
Comment 5:line 292: I suppose “Fig. 6” should Fig. 7.
Reply: Thank you for pointing out this problem. We have checked and revised this error in version.
Comment 6:line 304: full stop is missing after the word “interacted”.
Reply:We are extremely grateful for your suggestion. We have checked and revised this error in version.
Comment 7:line 305: delete dot in front of AT3G44830.
Reply:Thank you for pointing out this problem. We have checked and revised this error in version.
Comment 8:part 2.6.: how gene expression was normalised using the refence gene actin? Regarding this, at Figs 9, 11, 12 and 13 is the “Relative expression value” the actin normalised value?
Reply:The expression of reference genes usually does not change at different developmental stages and under different stress treatments. The measurement of the reference gene is to homogenize the initial amount of different samples during the analysis process, and then use the expression of the internal reference gene as a reference to represent the expression of the gene to be tested. The expression of each gene was calculated by 2^-ΔΔCt method.
Comment 9:Figs. 9, 11, 12 and 13: I would not say “extremely”. It is a very relative thing, that what someone calls “extreme”. P<0.01 is quite normal, not “extreme”. Just say: “* and ** indicate significant differences at the P < 0.05 and P < 0.01 levels, respectively” or something like that.
Reply:We are extremely grateful for your suggestion. We have checked and revised this error in version. “* and ** indicate significant differences at the P < 0.05 and P < 0.01 levels, respectively”.
