Identification of the Oleosin Genes and Functional Analysis of CeOle4 Gene in Cyperus esculentus L.

Comments 1: Lines 132- 134: The two sentences are unnecessary (remove them).

Response 1: Thank you for your suggestion. We have revised this sentence. The purpose of the article is to investigate the function of the Oleosin gene in tiger nut.

Comments 2: Lines 132- 134: The two sentences are unnecessary (remove them).

Response 2: Agree. The two sentences have been deleted in the revised manuscript.

Comments 3:Figure 3: What are SH, T, U & SL? The legend has to include these acronyms too.

CeOl 6,7, 8 & are 9 distantly related to CeOl 1, 2,3, 4 & 5. Also, the phylogenetic distance between CeOl 1 and the relatively closely related CeOl 2,3,4 & 5 genes is more than the distance between CeOl6 and CeOl 7, 8 &9. How could you explain such phylogenetic distances that seems gene families from the same species are closer to the families in different species than their own.

Lines 171-173: ‘CeOle6-9 likely 171 play a role in the synthesis of oleic acid, total fatty acids

(FAs), and phosphatidic acid (PA). 172 CeOle1-5 are likely associated with neutral lipid synthesis’. What was your evidence as far as your experiments were concerned? OR you aimed to conclude from other reports without citing reference? If the later is the case, please cite appropriate reference.

Response 3: Thank you for your suggestions. We assessed the distance results based on the phylogenetic tree of the oil body protein family genes. The phylogenetic distance reflects the evolutionary relationships between different genes or gene families. In different species, gene families may experience varying evolutionary rates and genetic drift, which can result in some families appearing closer in evolutionary terms to genes in other species. For instance, gene families within the same species might undergo different evolutionary pressures or functional changes, leading to distinct positions on the phylogenetic tree compared to gene families in other species. The functional differences between CeOle6-9 and CeOle1-5 might reflect different selective pressures and functional needs during their evolutionary process.

Reference: Zhang W, Xiong T, Ye F, Chen JH, Chen YR, Cao JJ, Feng ZG, Zhang ZB. The lineage-specific evolution of the oleosin family in Theaceae. Gene. 2023 Jun 5;868:147385. doi: 10.1016/j.gene.2023.147385. Epub 2023 Mar 22. PMID: 36958508.

We obtained the corresponding conclusions from the references and inserted them into the revised manuscript.

Reference: (1) Parthibane V, Iyappan R, Vijayakumar A, Venkateshwari V, Rajasekharan R. Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions. Plant Physiol. 2012 May;159(1):95-104. doi: 10.1104/pp.112.197194. Epub 2012 Mar 20. PMID: 22434039; PMCID: PMC3375988. (2) Niu R, Chen F, Liu C, Duan X. Composition and Rheological Properties of Peanut Oil Bodies from Aqueous Enzymatic Extraction. J Oleo Sci. 2021 Mar 4;70(3):375-383. doi: 10.5650/jos.ess20247. Epub 2021 Feb 12. PMID: 33583919.

Reference: Zhang W, Xiong T, Ye F, Chen JH, Chen YR, Cao JJ, Feng ZG, Zhang ZB. The lineage-specific evolution of the oleosin family in Theaceae. Gene. 2023 Jun 5;868:147385. doi: 10.1016/j.gene.2023.147385. Epub 2023 Mar 22. PMID: 36958508.

We obtained the corresponding conclusions from the references and inserted them into the revised manuscript.

Reference: (1) Parthibane V, Iyappan R, Vijayakumar A, Venkateshwari V, Rajasekharan R. Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions. Plant Physiol. 2012 May;159(1):95-104. doi: 10.1104/pp.112.197194. Epub 2012 Mar 20. PMID: 22434039; PMCID: PMC3375988. (2) Niu R, Chen F, Liu C, Duan X. Composition and Rheological Properties of Peanut Oil Bodies from Aqueous Enzymatic Extraction. J Oleo Sci. 2021 Mar 4;70(3):375-383. doi: 10.5650/jos.ess20247. Epub 2021 Feb 12. PMID: 33583919.

Comments 4: Lines 203-205: The legends for Figures 5 - 8 have to be rewritten as the two sentences are unnecessarily separated by line spaces.

Response4: Thank you for your suggestion. We have rewritten the legend of Figure 5-8 in the revised manuscript, removing unnecessary spaces.

Comments 5: The conclusions made in lines 214-16 are a bit misleading.

Response5: Thank you for your suggestions. We reorganized this part in the revised MS as followed. To evaluate the cold tolerance of Cyperus esculentus, over-expression of CeOle4 at 4℃ (low-temperature) alone would not lead to conclusion that cold tolerance is attained by overexpression of the gene. Other biochemical analysis (water content, glycerol, total sugar, trehalose and sorbitol content etc) are required along the course of low-temperature treatment times. This component is missing but very essential if any validated report is intended to be made.

Comments 6: Clarification: (Figure 7) Seven (OE1-7) transgenic lines of Camelina sativa were used to?

Response 6: Thank you for your suggestions. OE1-7 were Ole4 gene CDS transgenic lines. We have clarified the specific meaning of the OE representation in the Figure 8 legend in Page 9, lines 339-340.

Comments 7: Why agronomic trait of transgenic Camelina sativa seeds were considered?

Response 7: Thank you for your question. Transgenic Camelina sativa mustard is an oil model plant, which is easy to transform and can be heterologous expressed. The function of this gene was preliminarily determined whether it is related to oil metabolism. The transfer of oil body protein genes affects the growth and development of seeds by influencing the accumulation of oil and cell structure inside the seeds. Specifically, the transfer of oil body protein genes can promote the accumulation of oil and fat inside seeds, which helps to increase the weight and volume of seeds. The accumulation of oil not only increases the nutritional value of seeds, but also directly affects their physical size. In addition, the transfer of this gene may also affect the growth and division rate of cells, further promoting seed enlargement. Therefore, introducing the oil body protein gene into seeds through transgenic technology can effectively increase the volume and weight of seeds, thereby improving their yield and quality. Progress has been made in the research of key genes for high-yield soybean, which may enable synergistic regulation of seed size, oil content, and protein content.

Reference: Wang, S., Liu, S., Wang, J., Yokosho, K., Zhou, B., Yu, Y.C., Liu, Z., Frommer, W.B., Ma, J.F., Chen, L.Q. and Guan, Y., 2020. Simultaneous changes in seed size, oil content and protein content driven by selection of SWEET homologues during soybean domestication. National Science Review, 7(11), pp.1776-1786.

Comments 8: How do you reconcile the role of TAG as part of cell membrane to improve membrane fluidity (Lines 284-86) and increase in intracellular TAG for enhancement of cold tolerance (Lines 293-94)? It indicates the presence of TAG in both plasma membrane and intracellular space has cold tolerance effect. How could it be?

Response 8: Thank you for your suggestion. Some scholars have demonstrated that the content of TAG in plants can regulate their tolerance to cold stress. Meanwhile, TAG content can regulate the fluidity of cell membrane. We referred to the corresponding literature.

Reference: Arisz SA, Heo JY, Koevoets IT, Zhao T, van Egmond P, Meyer AJ, Zeng W, Niu X, Wang B, Mitchell-Olds T, Schranz ME. DIACYLGLYCEROL ACYLTRANSFERASE1 contributes to freezing tolerance. Plant Physiology. 2018 Aug 1;177(4):1410-24.

Comments 9: Lines 320: The main finding and conclusion seem the CeOl4 may regulate seed coat color changes and cold tolerance. Which plant organs (seeds, roots or leaves) of the plant are more important to investigate agronomic traits that confer cold tolerance? This is critically important as it determines the validity of the experiments executed for heterologous expression in different expression lines as well.

Japanese scholars have found that as the accumulation of pigments on the seed coat increases, the cold tolerance may become stronger.

Response 9: Thank you for your suggestion. Some scholars have found that as the accumulation of pigments on the seed coat increases, the cold tolerance may become stronger. We referred to the corresponding literature.

Reference: Yamaguchi, N., Suzuki, C., Yamashita, Y., & Senda, M. (2021). A pubescence color gene enhances tolerance to cold-induced seed cracking in yellow soybean. Breeding science, 71(4), 467-473.

The cold tolerance experiment of plants will continue to be carried out in the future when the experimental conditions permit, and the application will be considered. Here, according to your suggestion, we changed our conclusion: CEOLE4 can promote lipid synthesis, improve the stability of oil at low temperature, and change the seed phenotype.

Comments 1: This is critical statements, hence you need to justify this claim, it needs strong support of this statement.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have added literature in the revised manuscript to support of this statement, this change can be found page 1, paragraph 2, and line 42.

Comments 2: This should be reflected on the results and discussion- complemented with data gathered?

Response 2: Agree. We have supplemented the methodology accordingly results in the revised manuscript. We have added 2.6 Cloning and Vector Construction of CeOle4 Genes in Tiger Nut, 2.9 TAG and DAG content detection, 2.12 Observation of Phenotype of Transgenic Camelina Sativa, and 2.11 CeOle4 Overexpressed Transgenic Camelina Sativa and expression analysis to our method to fully present our experimental process and demonstrate our results, this change can bu found in page2-5.

Comments 3: Your methodology should be fitted with the objectives you have stated in your introduction? Does it really complement with the hypotheisi you are trying to investigate, try to be more specific with your method in order to come up with a quality dasta that will support your potential answer to your hypotheisis?

Response 3: Agree. We have reorganized the methodological section based on the content of the article to make the connection between the method and the result in the revised version. We have supplemented the experimental methods, experimental data, and supplementary data files in the methodology to support the results and hypotheses of our article.

Comments 4: Include also the condition of the study sites, what are the statistical design, how many plots, area and other important parameters/condition that the study conducted?

Response 4: We acknowledge your question. The growth period of Cyperus esculentus is 120 days. During the entire development phase, we conducted Cyperus esculentus cultivation with multiple different planting plots and control repetitions. The biological replicates of the experiment were performed three times. Sampling was carried out at different developmental stages of the oil body accumulation site, which is the tuber tissue of Cyperus esculentus. Nile red staining was used to visualize the oil body structure in tissue sections, and fluorescence microscopy was employed to observe the stained tuber sections, assessing the extent of oil body accumulation in the tubers. After 120 days, the Cyperus esculentus tubers were fully matured and reached the harvest period, at which point the observation of oil body accumulation was concluded.

Comments 5: It is better if you could use references in here regarding the methodology or such modifications from previous published articles.

Response 5: Thank you for your suggestion, we cited relevant reference as following.

Reference: Sun D, Quan W, Wang D, Cui J, Wang T, Lin M, Wang Y, Wang N, Dong Y, Li X, Liu W, Wang F. Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz. Int J Mol Sci. 2022 Nov 22;23(23):14550. doi: 10.3390/ijms232314550. PMID: 36498878; PMCID: PMC9738755.

Comments 6: Any reference for this method?

Response 6: Thank you for your question. In this part, the Nile Red staining experimental method was performed and referenced as followed. We have added in revise manuscript.

Reference: Escorcia W, Ruter DL, Nhan J, Curran SP. Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining. J Vis Exp. 2018 Mar 5;(133):57352. doi: 10.3791/57352. PMID: 29553519; PMCID: PMC5931440.

Comments 7: Para-phrase this, make it clearer such as what tissue? Where chamber etc.?

Response 7: Thank you for your question. We revised this part in the revised version Page 2, line 92.

Comments 8: Is this original method or it can be cited from previous published papers?

Response 8: Thank you for your suggestion. We cited reference as followed and inserted it in the revised MS.

Reference: Tang J, Yang X, Xiao C, Li J, Chen Y, Li R, Li S, Lü S, Hu H. GDSL lipase occluded stomatal pore 1 is required for wax biosynthesis and stomatal cuticular ledge formation. New Phytol. 2020 Dec;228(6):1880-1896. doi: 10.1111/nph.16741. Epub 2020 Jul 24. PMID: 32542680.

Comments 9: Explain clearl, your vector is from? Condition and other details or procedure for transformation, simple but easy to understand.

Response 9:Thank you for your advice. We added vector information in Page 4 lines 157-162, and procedure of transformation in Page 5 lines 213-225 and provide specific literature.

Comments 10: What is this primers?

Response 10: Thank you for your suggestion. The primer sequence is provided in the modified version in in Page 5 lines 218-220. Furthermore, the other primers mentioned in the article are provided table S2 in the supplementary documents.

Comments 11: Why until t3 generation?

Response 11: Thank you for your question. In plant phenotyping experiments, selecting T3 generation plants is crucial to ensure genetic stability and reliability. T3 plants have typically gone through several generations of screening and breeding, so the transgene or mutation traits in the genome are stably inherited, which allows for a more accurate reflection of the plant's phenotypic characteristics. This helps to minimize experimental errors due to genomic instability or heterogeneity.

Reference: (1) Noman M, Jameel A, Qiang WD, Ahmad N, Liu WC, Wang FW, et al. Overexpression of GmCAMTA12 Enhanced Drought Tolerance in Arabidopsis and Soybean. International journal of molecular sciences. 2019;20(19). Epub 2019/10/02. doi: 10.3390/ijms20194849. PubMed PMID: 31569565; PubMed Central PMCID: PMCPMC6801534.

(2) Raffan S, Sparks C, Huttly A, Hyde L, Martignago D, Mead A, et al. Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2. Plant biotechnology journal. 2021;19(8):1602-13. Epub 2021/02/28. doi: 10.1111/pbi.13573. PubMed PMID: 33638281; PubMed Central PMCID: PMCPMC8384593.

Comments 12: This should be properly discuss in your methodology.

Big question on statistical design and lay out? How many trials were being conducted, using of what specific equipment for this morphological changes of lipid droplet accumulation, how about if you increase more days prior to observation?

Response 12: Thank you for your suggestion. We have explained and added methodology in the revised MS. The growth period of Cyperus esculentus is 120 days. During the entire development phase, we conducted Cyperus esculentus cultivation with multiple different planting plots and control repetitions. The biological replicates of the experiment were performed three times. Sampling was carried out at different developmental stages of the oil body accumulation site, which is the tuber tissue of Cyperus esculentus. Nile red staining was used to visualize the oil body structure in tissue sections, and fluorescence microscopy was employed to observe the stained tuber sections, assessing the extent of oil body accumulation in the tubers. After 120 days, the Cyperus esculentus tubers were fully matured and reached the harvest period, at which point the observation of oil body accumulation was concluded.

Comments 13:How about controlled treatment as basis for comparison?

Response13: Thank you for your suggestion. 35 DAS is the earliest sampling stage, which is used as a reference stage for gene expression changes. The calculation of gene expression does not standardize the 35DAS stage relatively. 2− ΔCT method standardization was used to compare the gene expression levels of other developing stages in parallel. We supplement it in our revised methodology in line 123.

Comments 14: ok

Response14: Thank you for your recognition of this content.

Comments 15:alright

Response15: Thank you for your recognition of this content.

Comments 16: is this trials for what? Any justification why 5 hours after?

Response16: Yeast cell is a mature eukaryotic receptor material for gene function study. Previously, we have used yeast to verify the function of plant stress resistance genes. In this paper, CeOLE4 gene was induced and expressed in yeast cells to observe the effect on the expression of oil body. Although the yeast cells have the expression of oil body, but the transgenic yeast of oil-body expression was significantly increased after induction with CeOLE4 gene, which confirmed the function of CeOLE4 on increasing oil body content. At the same time, the high oil body protein expression time of yeast cells was confirmed and used for later cold tolerance experiments.

“Any justification why 5 hours after?” is very good question. There may indeed be a significant increase in expression level between 4h and 8h. Here, 5h was not included in the experimental design. In the future, more time points can be set in this interval to accurately confirm the change in expression level.

Comments 17: Include only figures with strong evidences in order to justify the claims, do not include anymore figures which are not (nothing to compare o no expression at all?

Response17: The images indeed reflect a significant decrease in the stability of oil body at low temperatures. Compared to the initial period, the later images show a reduction in the number of oil bodies and instability in their structure. It provided evidence for the observed phenomena however the decreased fluorescence intensity makes the images less clear.

Comments 18: Is this with an interaction with DAS, how many trials were being used in this data, average of? And what did this legend for? (blacl gray etc.)

Response18: Thank you for your suggestions. Here we analyzed a dataset from a study on transgenic Camelina sativa seeds, focusing on the content of triacylglycerol (TAG) and diacylglycerol (DAG). It is not relevant with DAS. We analyzed a large number of transgenic strains, conducting the biological experiments in triplicate. The results, shown in Figure 8, indicate that the average levels of TAG and DAG are significantly higher in the overexpressing strains compared to the control strains. We supplemented it in the revised MS.

Comments 19: It should have strong details on the methodology in order to be more conclusive with the results, try to integrate in your methodology and it will be complemented with the results that you have in this figure.

Response19: Thank you very much for your suggestion. Our previous method did indeed lack this section. We have added 2.6 Cloning and Vector Construction of CeOle4 Genes in Tiger Nut, 2.9 TAG and DAG content detection, 2.12 Observation of Phenotype of Transgenic Camelina Sativa, and 2.11 CeOle4 Overexpressed Transgenic Camelina Sativa and expression analysis to our method to fully present our experimental process and demonstrate our results.