Genome-Wide Identification and Characterization of Tomato Acyl-CoA Oxidase Family Genes ACX

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article deals with the identification and functional characterization of tomato acyl-CoA oxidase family genes ACX. Six members of ACX genes were identified in the tomato genome by wide screening. and the sequences of the encoded proteins revealed differences in their physicochemical properties suggesting differences in their functions. Analysis of the gene organization showed, as expected, conserved motifs, and diverse cis-acting sequences in their putative promoters exhibiting elements for responses to hormones, environmental factors such as temperature, light or to stress conditions (water deficit and salinity), as well as some related to developmental processes. Analysis of ACX structure and organization showed differences in structure, in the number of motifs, introns and exons. Encoded ACX proteins also exhibited differences in physicochemical properties, cellular localization (peroxisomes, chloroplasts, mitochondria, nuclei, vacuole, endoplasmic reticulum, cytosol and plasma membrane), and in motifs. ACX genes were located in chromosomes 04, 08, and 10. A phylogenetic tree was constructed using Arabidopsis thaliana, Glycine max, Oryza sativa, Solanum lycopersicum and Zea mays genomes and the analysis revealed the evolutionary relationships between 27 ACX genes. Analysis of gene duplication and collinearity of SlACX genes was also performed showing collinearity between some SlACX and some of A. thaliana and Solanum tuberosum members. qRT-PCR assays showed differential expression profiles in diverse organs and tissues, being SlACX5 and SlACX6 highly expressed in unopened buds and fully opened flowers. Experiments under hormone (ABA and methyljasmonate) or stress conditions (UV, cold, heat, H2O2, PEG treatment, and salinity) revealed differences in responses among all ACX genes. In general this manuscript was well written and well organized. It is a kind of an explorative article previously reported for a wide range of genes in different plant species to give an idea of gene families and their possible functions and their participation in different processes or to give ideas on the evolutionary changes they have suffered. Although this article is not of high originality, it can contribute to the knowledge and understanding of the functions or roles that ACX genes may play in tomato. Some corrections/comments/observations have been highlighted in the text that the authors must accomplish in the revised/corrected manuscript version. In Materials an Methods section it is necessary to describe in a detailed way all the conditions of plant growth, and all treatments (hormone concentrations, time of exposure, and all stresses conditions) as well as the number of replicates and experiments performed. It is recommended to read carefully the Instructions for Authors of horticulturae to prepare in a uniform style ALL the references.

Comments for author File: Comments.pdf

Author Response

Comments 1: Cis (Italicized in ALL cases.)

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have made revisions to the entire text, such as lines 18, 68 and so on.

Comments 2: Gene abbreviations must be italicized in ALL cases in the text an References.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the entire text to keep the genes italic, for example, lines73, 96, 97 and so on.

Comments 3: Why starting this word with capital letter?. A uniform style in ALL heading should be maintained. Take as an example the heading 2.1 style for uniformity in the subsequent headings.

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made revisions to the entire text to maintain a uniform format, such as lines 74 and 79 and so on.

Comments 4: A description of ALL conditions of hormones and environmental factors treatments must be included in Materials and Methods section.

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, We have made supplements, specifically as follows: Uniform 'Micro-Tom' tomato seeds were surface-sterilized with 1% sodium hypochlorite for 10 min and rinsed thoroughly with sterile water. The seeds were then germinated aseptically in a shaker (25 °C, 180 rpm) for three days with daily water changes. Subsequently, the germinated seeds were sown in soil and maintained in a growth chamber under controlled conditions (light: 250 μmol·m⁻²·s⁻¹, 16/8 h light/dark at 26±2/20±2 °C, 60 % RH). After two weeks, seedlings were transplanted to a hydroponic system, and uniform 21-day-old plants were selected for experimentation.

To analyze the expression patterns of SlRBOH genes during vegetative growth, we collected roots, stems, and leaves from untreated 21-day-old seedlings. For the SlACX genes expression analysis during reproductive growth, samples were taken from 55-day-old plants at the flowering stage, including roots, stems, leaves, flowers, as well as corresponding green fruits (25 days after pollination) and mature fruits (45 days after pollination), with one fruit collected per plant. Each sample type consisted of three biological replicates, each comprising a pool of tissues from eight plants.

For chemical and osmotic stress treatments, seedlings were transferred to a half-strength nutrient solution containing one of the following: 200 mM NaCl, 100 μM ABA, 100 μM MeJA, or 20 % (w/v) PEG 6000. For the oxidative stress treatment, seedlings were exposed to a nutrient solution supplemented with 10% (v/v) hydrogen peroxide. For temperature and light stress, seedlings were subjected to either 4°C (cold) or 40 °C (heat) in incubators. A dark treatment group was placed in complete darkness, while a UV stress group was exposed to UV-C radiation (253.7 nm) under otherwise control conditions. The aerial tissues of seedlings from each treatment group, including 0 hour control, were harvested after 0, 6, 12, and 24 hours of exposure. Each sample, representing one biological replicate, was pooled from eight seedlings. All harvested tissues were immediately frozen in liquid nitrogen and stored at -80 °C. For every treatment and time point, three independent biological replicates were collected.

Comments 5: Which gene was used as the iinternal control?.

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, SlActin (NC015447.3) was used as a reference gene in this study.

Comments 6: It is recommended to describe results in past tense in ALL cases to maintain style uniformity along the document.

Response 6: Thank you for pointing this out. We agree with this comment. Therefore, We have made revisions to the full text, for example. Six members of the SlACX gene family were finally obtained and named SlACX1-SlACX6 respectively according to their chromosomal positions. The ORF of SlACX1 was the largest, and that of SlACX6 was the smallest. The same was true for the number of amino acids and molecular weight. The pI distribution of SlACX1-SlACX6 ranged from 6.31 (SlACX3) to 8.74 (SlACX1). The protein domain positions owned by the members of the SlACX gene family were all different, but there was overlap (Table 2). 

Comments 7: It is not clear which criteria were used for this classification on the basis of gene structure. For example, in Class I there is no a clear resemblance between both genes. A similar observation can be made for Class III members.

Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we further analyzed the conserved motifs of all SlACX proteins. The analysis results show that although there are differences in the gene structures (intron-exons) of the internal members of Class I and Class III, they respectively share a set of highly specific conserved motifs. The composition and arrangement order of these motifs are highly conserved within the class, but show significant differences between classes. This discovery provides key structural evidence at the functional domain level that goes beyond phylogenetic relationships for current classification.

Comments 8: Describe the putative functions of such motifs.

Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we have already supplemented it in the text, specifically as follows, From the motifs sequences in Table 5, it can be known that motifs 1, 2, 3, 4, and 7 may jointly constitute the FAD (flavin adenine dinucleotide) binding domain and catalytic core of the ACX enzyme. Motif 5, 8 May be involved in the formation of substrate (acyl-coA) binding channels, responsible for recognizing and binding to aliphatic acyl chains of different lengths. Motif 6, 9, and 10 are involved in maintaining the tertiary structure of proteins, mediating protein-protein interactions, or undergoing post-transcriptional modifications (such as phosphorylation), thereby precisely regulating enzyme activity.

Comments 9: Figur 5-B change to Figure 5-B.

Response 9: Thank you for pointing this out. We agree with this comment. Therefore, we have changed Figur 5-B to Figure 5-B.

Comments 10:Were ALL these experiments performed in plants or fruits or where?. Describe all the experimental conditions in Materials and Methods.

Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we have supplemented the relevant content (lines 96-121).

Comments 11:Which is the rationale for this statement?

Response 11: Thank you for pointing this out. We agree with this comment. Specifically, SlACX1/SlACX5 were continuously induced under ABA signals, while SlACX6 demonstrated a rapid and intense transient response to MeJA treatment. The specificity and temporal differences of this expression suggest that they may mediate specific metabolic flow reprogramming downstream of different stress signals. It is known that ACX is the rate-limiting enzyme of fatty acid β -oxidation, and its activity directly regulates the fatty acid degradation flux and the flow direction of the carbon skeleton. Therefore, we infer that the upregulation of SlACX1/SlACX5 may mainly use the energy (ATP) and carbon sources produced by fatty acid degradation for basic stress responses such as osmotic regulation; The rapid activation of SlACX6 is more likely to preferentially provide precursors for the biosynthesis of stress-related secondary metabolites such as jasmonic acid (which itself is a precursor for JA synthesis). The differences in expression patterns are precisely the direct clues that may lead to their differentiated regulation of the two downstream pathways of "energy metabolism" and "secondary synthesis".

Comments 12:This article title style is different from the previous one because in this case ALL word start with capital letters while the previous one not. Check Instructions for Authors to maintain a uniform style in ALL references.

Response 12: Thank you for pointing this out. We agree with this comment. Therefore, We have checked and revised the entire text to ensure the uniformity of the format. The revised parts have been marked in red.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study described in this paper focuses on ACX genes in tomato; structure, function, organization, phylogeny, and projected involvement in whole plant responses. The paper is mostly well-written and presented but the reviewer offers the following suggestions for improvement:

Abstract

Line 19) “…with cis-acting elements…” delete; redundant.

Keywords

These terms should be presented in alphabetical order.

Introduction

Line 32) “…the best model plant for plant research.” Suggest change to “…one of the best model plant species for research.”

Line 45) “…IAA, JA and SA…” these acronyms should be fully defined the first time they are introduced (see JA definition in line 65, later in the manuscript).

Materials & Methods

Line 62) “Download the genome data from the Solanum lycopersicum…” change to “Genomic reference data for S. lycopersicum were obtained from…”.

Line 65) “…genome data…” change to “…and genome data…”.

Lines 65-71) Starting with “Download the ACX family…” this content should be revised according to acceptable English grammar.

Lines 85-86) “…Arabidopsis thaliana, Soybean and Zea mays L…” change to “…A.

thaliana, G. max, and Zea mays L.”

Lines 91-92) “…were statistically analyzed…” suggest that the general methods be described in more detail in the present manuscript. The information in lines 104-107 should be presented here. 

Line 96) “…and its purity and integrity…” change to “…the purity and integrity of…”.

Lines 97-98) More details on the methods used for reverse transcription, including references, are needed.

Results

Line 110) “After screening, six…” change to “Six…”.

Lines 111-112) “Analyzing their physicochemical properties, it can be known that the ORF…” change to “The orf…”.

Line 115) “…owned by…” change to “…of…”.

Lines 118-120) “It can be known from Table 3 that the secondary structure of the SlACX family members consists of Alpha helix, Beta turn and Random coil, and Alpha helix accounts for the largest proportion, followed by Random coil, and Beta turn is the least.” Change to “The secondary structure of the SlACX family member peptides consisted of Alpha helix, Beta turn and Random coil, and Alpha helix accounted for the largest proportion, followed by Random coil, and Beta turn is the least prevalent (Table 3).”.

Line 124) “Subcellular localization analysis of the SlACX1-SlACX6 genes was conducted…” change to “The subcellular localization of SlACX1-SlACX6 genes was determined…”.

Line 128) “…genes…” change to “…SlACX family genes…”.

Line 133) “Among them,…” Class I…” change to “Class I…”.

Lines 139-140) “To clarify the chromosomal location distribution of the SlACXs gene family, this study conducted chromosomal location analysis on the SlACXs gene family members.” Delete.

Lines 147-149) This section is improperly formatted.  

Line 148) “…SlACX1-SlACX6 genes are classified into three groups…” The authors should consider combine this section with the previous section. This will avert confusion by addressing gene structures and classification systems in a more unified and consistent manner.

Figures 1 and 3) These should be harmonized. The three main groups depicted in Fig. 3 should be labeled using the criteria presented earlier in the M&M section (Class I, Class II, etc.).

Table 5) This graphic should be cited in the text.

Lines 175-179) “This section is improperly formatted.”

Line 179) “…(Figur 5-B)…” change to “…(Fig. 5B)…”.

Lines 184-185) “As shown in the figure 6, the promoter of the SlACXs gene contains various functional elements, such as LTR, MBS, ABRE, TGA-element, P-box and so on.” Change to “The promoter of the SlACXs gene contained various functional elements, such as LTR, MBS, ABRE, TGA-element, and P-box (Fig. 6).”.

Lines 196-197) “To examine the expression of the SlACX gene in different states of the same organs and in different organs, In this study,…” Delete.

Figure 10) This graphic is not cited in the text.

Lines 249-250) “…and was vegetative until 12 h…” It is unclear what the authors intend to convey with the descriptor “vegetative”.

Line 262) “…lead the adaptive adjustment…” It is unclear what the authors intend to convey with the descriptor “adaptive adjustment”.

Discussion

Line 324) “…its response…” change to “…the response by SlACX…”.

Line 337) “…a high expression…” it seems that the authors are also reporting that the response to high temperatures is immediate; if so change to “…immediately a high level of expression…”.

Line 346) “…is the core hub…” suggest change to “…is a core hub…”.

Line 351) “This has…” suggest change to “These results have…”.

Conclusion(s)

Line 359) “…and adverse stress…” suggest change to “…and diverse stress factors…”.

Author Response

Comments 1: Line 19) “…with cis-acting elements…” delete; redundant.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have deleted it. Specifically: Analysis of gene structure and conserved motifs reveals that SlACXs were highly conserved in evolution, but the cis-acting elements in the promoter region were rich and diverse, suggesting that it may integrate multiple signaling pathways.

Comments 2: These terms should be presented in alphabetical order.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have modified the key words, specifically as follows: Acyl-CoA Oxidase; Expression analysis; Gene family; Tomato.

Comments 3: Line 32) “…the best model plant for plant research.” Suggest change to “…one of the best model plant species for research.”

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made the modifications, specifically as follows: Tomato (Solanum lycopersicum), one of the most important crops in the world, is also one of the best model plant species for plant research.

Comments 4: Line 45) “…IAA, JA and SA…” these acronyms should be fully defined the first time they are introduced (see JA definition in line 65, later in the manuscript).

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have supplemented the full names of IAA, JA and SA, specifically IAA (Auxin), JA (Jasmonic Acid) and SA (Salicylic Acid).

Comments 5: Line 62) “Download the genome data from the Solanum lycopersicum…” change to “Genomic reference data for S. lycopersicum were obtained from…”.

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have already changed to “Genomic reference data for s. lycopersicum were obtained from https://sol-genomics.sgn.cornell.edu”.

Comments 6: Line 65) “…genome data…” change to “…and genome data…”.

Response 6: Thank you for pointing this out. We agree with this comment. Therefore, we have already changed “genome data” to “and genome data”.

Comments 7: Lines 65-71) Starting with “Download the ACX family…” this content should be revised according to acceptable English grammar.

Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we have made the modifications, specifically as follows. The ACX family sequences (PF01756) are downloaded from the Pfam database (http://pfam.xfam.org/), a comparative analysis of ACX gene family members is performed using HMMER 3.0 with Hmmsearch, and the identified candidates are validated via the SMART online platform (https: //smart.embl-heidelberg.de/) to confirm and finalize the SlACX gene family members.

Comments 8: Lines 85-86) “…Arabidopsis thaliana, Soybean and Zea mays L…” change to “…A.

thaliana, G. max, and Zea mays L.”

Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “Arabidopsis thaliana, Soybean and Zea mays L”to“A.thaliana, G. max, and Zea mays L.”

Comments 9: Lines 91-92) “…were statistically analyzed…” suggest that the general methods be described in more detail in the present manuscript. The information in lines 104-107 should be presented here.

Response 9: Thank you for pointing this out. We agree with this comment. Therefore, In this study, only the quantity of different response elements was statistically analyzed.

Comments 10:Line 96) “…and its purity and integrity…” change to “…the purity and integrity of…”.

Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we have changed“and its purity and integrity”to“the purity and integrity of.

Comments 11:Lines 97-98) More details on the methods used for reverse transcription, including references, are needed.

Response 11: Thank you for pointing this out. We agree with this comment. Therefore, We have made relevant supplements, specifically as follows. Subsequently, high-quality total RNA (1 µg) was reverse-transcribed into first-strand complementary DNA (cDNA) using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions, to provide a stable template for subsequent gene expression analysis [15].

Comments 12: Line 110) “After screening, six…” change to “Six…”.

Response 12: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “After screening, six…” to “Six…”. Specifically as follows: Six members of the SlACX gene family were finally obtained and named SlACX1-SlACX6 respectively according to their chromosomal positions.

Comments 13: Lines 111-112) “Analyzing their physicochemical properties, it can be known that the ORF…” change to “The orf…”.

Response 13: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “Analyzing their physicochemical properties, it can be known that the ORF…” to “The orf…”. Specifically as follows: The ORF of SlACX1 was the largest, and that of SlACX6 was the smallest.

Comments 14: Lines 118-120) “It can be known from Table 3 that the secondary structure of the SlACX family members consists of Alpha helix, Beta turn and Random coil, and Alpha helix accounts for the largest proportion, followed by Random coil, and Beta turn is the least.” Change to “The secondary structure of the SlACX family member peptides consisted of Alpha helix, Beta turn and Random coil, and Alpha helix accounted for the largest proportion, followed by Random coil, and Beta turn is the least prevalent (Table 3).”.

Response 14: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “It can be known from Table 3 that the secondary structure of the SlACX family members consists of Alpha helix, Beta turn and Random coil, and Alpha helix accounts for the largest proportion, followed by Random coil, and Beta turn is the least.” to “The secondary structure of the SlACX family member peptides consisted of Alpha helix, Beta turn and Random coil, and Alpha helix accounted for the largest proportion, followed by Random coil, and Beta turn is the least prevalent (Table 3).”.

Comments 15: Line 124) “Subcellular localization analysis of the SlACX1-SlACX6 genes was conducted…” change to “The subcellular localization of SlACX1-SlACX6 genes was determined…”.

Response 15: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “Subcellular localization analysis of the SlACX1-SlACX6 genes was conducted…” to “The subcellular localization of SlACX1-SlACX6 genes was determined…”.

Comments 16:Line 128) “…genes…” change to “…SlACX family genes…”.

Response 16: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “…genes…”to“…SlACX family genes…. Specifically：The SlACX family genes structure analysis results show that the SlACX1-SlACX6 genes can be classified into three groups (Class Ⅰ, Class Ⅱ, and Class Ⅲ) based on evolutionary tree kinship.

Comments 17: Line 133) “Among them,…” Class I…” change to “Class I…”.

Response 17: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “Among them,…” Class I…” to “Class I…”.

Comments 18: Lines 139-140) “To clarify the chromosomal location distribution of the SlACXs gene family, this study conducted chromosomal location analysis on the SlACXs gene family members.” Delete.

Response 18: Thank you for pointing this out. We agree with this comment. Therefore, we have deleted.

Comments 19: Lines 147-149) This section is improperly formatted.

Response 19: Thank you for pointing this out. We agree with this comment. Therefore, we have made format modifications. For details, please refer to the article.

Comments 20: Line 148) “…SlACX1-SlACX6 genes are classified into three groups…” The authors should consider combine this section with the previous section. This will avert confusion by addressing gene structures and classification systems in a more unified and consistent manner.

Response 20: Thank you for pointing this out. We agree with this comment. Therefore, Combining the elaboration of gene classification and structural analysis helps to present the systematic characteristics of the SlACX family more clearly and coherently. It uniformly explains the evolutionary correlations and structural similarities and differences of Class I, Class II and Class III genes from three levels: phylogenetic relationship, gene structure (exon-intron) and conserved motif. The focus is on explaining the conservation of various internal genes in terms of motif composition and arrangement sequence, as well as the differentiation rules among classes in terms of gene structure complexity, so as to comprehensively clarify the classification basis and structure-function association within the framework of evolution and avoid content dispersion. Based on this, we will adjust the structure of the manuscript to enhance logical coherence and consistency in discussion.

Comments 21: Figures 1 and 3) These should be harmonized. The three main groups depicted in Fig. 3 should be labeled using the criteria presented earlier in the M&M section (Class I, Class II, etc.).

Response 21: Thank you for pointing this out. We agree with this comment. Therefore, the annotations in the charts should be strictly consistent with the classification criteria defined in the "Materials and Methods" section, which is the cornerstone for ensuring the scientific rigor of the paper. We will immediately re-label the three main evolutionary branches in Figure 3 according to the classification criteria based on phylogenetic relationships (Class I, Class II, and Class III) you mentioned to ensure that they are completely consistent with the classification system of the entire text.

Comments 22: Table 5) This graphic should be cited in the text.

Response 22: Thank you for pointing this out. We agree with this comment. Therefore, We have already cited Table 5 in the text.

Comments 23: Lines 147-149) This section is improperly formatted.

Response 23: Thank you for pointing this out. We agree with this comment. Therefore, we have made format modifications. For details, please refer to the article.

Comments 24:Line 179) “…(Figur 5-B)…” change to “…(Fig. 5B)…”.

Response 24 Thank you for pointing this out. We agree with this comment. Therefore, we have changed “(Figur 5-B)…”to“…(Fig. 5B).

Comments 25: Lines 184-185) “As shown in the figure 6, the promoter of the SlACXs gene contains various functional elements, such as LTR, MBS, ABRE, TGA-element, P-box and so on.” Change to “The promoter of the SlACXs gene contained various functional elements, such as LTR, MBS, ABRE, TGA-element, and P-box (Fig. 6).”.

Response 25: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “As shown in the figure 6, the promoter of the SlACXs gene contains various functional elements, such as LTR, MBS, ABRE, TGA-element, P-box and so on.” to “The promoter of the SlACXs gene contained various functional elements, such as LTR, MBS, ABRE, TGA-element, and P-box (Fig. 6).”

Comments 26: Lines 196-197) “To examine the expression of the SlACX gene in different states of the same organs and in different organs, In this study,…” Delete.

Response 26: Thank you for pointing this out. We agree with this comment. Therefore, we have deleted.

Comments 27: Figure 10) This graphic is not cited in the text.

Response 27: Thank you for pointing this out. We agree with this comment. Therefore, we have cited Figure 10 in the text.

Comments 28: Lines 249-250) “…and was vegetative until 12 h…” It is unclear what the authors intend to convey with the descriptor “vegetative”.

Response 28: Thank you for pointing this out. We agree with this comment. Therefore, We have made modifications, specifically as follows, Under oxidative stress, SlACX1 showed specific high expression at 6 h.

Comments 29: Line 262) “…lead the adaptive adjustment…” It is unclear what the authors intend to convey with the descriptor “adaptive adjustment”.

Response 29: Thank you for pointing this out. We agree with this comment. Therefore, We deleted this sentence because the expression was unclear.

Comments 30: Line 324) “…its response…” change to “…the response by SlACX…”.

Response 30: Thank you for pointing this out. We agree with this comment. Therefore, We have changed“…its response…”change to “…the response by SlACX…”.

Comments 31: Line 337) “…a high expression…” it seems that the authors are also reporting that the response to high temperatures is immediate; if so change to “…immediately a high level of expression…”.

Response 31: Thank you for pointing this out. We agree with this comment. Therefore, we have made the modifications, specifically as follows: High-temperature stress, on the other hand, immediately a high level of expression of SlACXs,

Comments 32: Line 346) “…is the core hub…” suggest change to “…is a core hub…”.

Response 32: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “…is the core hub…” suggest change to “…is a core hub…”.

Comments 33: Line 351) “This has…” suggest change to “These results have…”.

Response 33: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “This has…” suggest change to “These results have…”.

Comments 34: Line 359) “…and adverse stress…” suggest change to “…and diverse stress factors…”.

Response 34: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “…and adverse stress…”  to “…and diverse stress factors…”.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

The research presented addresses molecular aspects relevant to its field of study, as it identifies acyl-CoA oxidase gene isoforms in the tomato genome, which play an important role in plant metabolic processes. It presents a detailed characterization of the gene structure and its relative expression, as well as the evolutionary relationship within the genus and between different genera of economically important plants with respect to the model plant, A. thaliana.

Aspects requiring improvement and clarification in the manuscript are identified.
The qPCR assays require controls and raw data to verify their validity. Therefore, I do not believe these experiments can be included in the research without them. Without them, the research would be merely in silico, which would diminish the significance and importance of this information.

Comments for author File: Comments.pdf

Author Response

Comments 1: Define the type of Characterization.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, in this study, "Characterization" (property description) mainly refers to the systematic computational analysis of the tomato ACX gene family through bioinformatics methods. Its connotation includes the following aspects: First, there is the description of basic characteristics, such as determining the chromosomal localization of genes, exon-intron structure, composition of conserved protein motifs, and physicochemical and biochemical properties (such as molecular weight, isoelectric point); Secondly, there is the analysis of evolutionary characteristics, including the construction of a phylogenetic tree to clarify the evolutionary kinship and classification among members, and conducting collinearity analysis with other species to infer their evolutionary history. The last aspect is the prediction of functional characteristics, such as analyzing the cation-acting elements in the promoter regions of genes to infer their potential expression regulatory patterns, and preliminarily infering the biological processes they may be involved in based on public expression profile data or homologous gene information. In conclusion, the "Characterization" in this paper aims to provide a comprehensive theoretical prediction and hypothetical basis for subsequent experimental verification from multiple dimensions such as genomic structure, evolutionary relationships, and regulatory mechanisms.

Comments 2: Repetitive. Modify!

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have changed in line 36.

Comments 3:State which organism this form of the gene corresponds to.

Response 3: Thank you for pointing this out. We agree with this comment. GhACX stands for Gossypium hirsutum Linn.

Comments 4: Same, state organisms.

Response 4: Thank you for pointing this out. We agree with this comment. AtACX stands for Arabidopsisthaliana.

Comments 5: I suggest placing the objective here, at the end of the introduction, rather than the findings, which are already included in the abstract.

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, placing the research objective clearly at the end of the introduction can more clearly outline the research context and framework of the entire text, enabling readers to accurately grasp the core tasks and exploration directions of this study before delving into the specific results.

Comments 6: The introduction does not provide a justification for comparing tomato genes with those of these plant genera. I recommend mentioning this to justify these methodological activities.

Response 6: Thank you for pointing this out. We agree with this comment. Therefore, We made some supplements. Species such as tomatoes and grapes all belong to the rose group in the middle of the true dicotyledonous plants in evolution, and there is a high degree of genomic collinearity conservation among them. Meanwhile, the functions of the ACX gene family of these species have been well annotated. Therefore, choosing these representative species for cross-phylogenetic scale comparative analysis helps to more accurately infer the evolutionary relationship, functional differentiation process and conservation characteristics of the SlACX gene in tomatoes in an evolutionary context, thereby providing a theoretical framework for subsequent functional research. This supplement makes the choice of comparative analysis more scientifically logical.

Comments 7: This formula requires data from endogenous genes, which are not adjustable under experimental conditions.

Response 7: Thank you for pointing this out. We agree with this comment. Therefore, We have supplemented the reference genes. All Ct value data used to calculate the relative expression levels (such as using the 2-^ΔΔCt method) were obtained from the same qPCR reaction plate under exactly the same thermal cycling conditions, thus ensuring the rigor of formula application and the validity of data comparison. We believe this clarification can address your concerns.

Comments 8:What were the endogenous genes?

Response 8: Thank you for pointing this out. We agree with this comment. Therefore, SlActin (NC015447.3) was used as a reference gene in this study.

 

 

Author Response File: Author Response.pdf