Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated the effect of salt stress and supplementation of mannose on ascorbic acid (AsA) acculuation. The results showed that salt stress increased the AsA levels in tomato and supplemented mannose also increased the level at immature stage. The manuscript should be carefully revised before it could be considered for publication.

 

In each Figure, statistical analysis should be conducted.

In the text and Figures (Fig 2 and 5), the use of ascorbic acid or vitamin C should be consistent in the whole manuscript.

In the Abstract, the current research gap and research purpose should be clearly stated.

Lines 11-16 when describing the results, the authors are suggested to clearly describe whether the comparison was between salt stress and control, or between different fruit development stages. The same issues exist in the text.

Skin refers to exocarp? It’s better to describe clearly.

Line 52 What do “strong properties” mean?

In “Materials and Methods”, English words should be used to start the setences. e.g. Lines 85, 87, 94, 98, 100, etc. Check the gramar for these sentences: Lines 87-88, 94-95.

When citing Figures, the number details should be provided. e.g. use Figure 2A, rather than Figure 2. Check the Results and Discussion sections.

Fig 2: what tissues were these measured in? Skin and seed (Line 149)? Seed and pericarp (Line 420). I am confused. Also in Line 276, which data in Fig 2 was meausred in the pericarp?

Results and Discussion: no need to repeat the information that has been introduced in Introduction.

Line 173: while or when?

Line 181: what is “seed function”?

Line 194: AsA was measured in pericarpt tissues (epicarp and endocarp). epicarp refers to exocarp/skin? Mesocarp was not included?

Fig 4 title: use “breaker” rather than “B”.

Fig 5A: the numbering A is missing. Fig 7 should be deleted, since it is excatly the same as Fig 5, the data should be presented only once.

Fig 5B, and Fig 6C and D: What does the diagonal line “mesocarp/endocarp” mean? Or? And? This should be clearly stated.

Fig 5-8: The full “control” in the x axis should be used.

Discussion: Line 240-244 This paragraph does not provide useful information. Line 245-265 Most of the information has been provided in Introduction section. Line 241: Where is the tissue hardness data? It is suggested to revise the Discussion section, different information should form a coherent whole with logical connections.

Conclusions: The main results should be summarized.

 

 

 

 

 

 

Comments on the Quality of English Language

The language shoud be polished. 

Author Response

Dear Reviewer 1,

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript titled “Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato” (horticulturae-3537821). As indicated in the following responses, we have considered all these comments in preparing this resubmitted manuscript. Detailed responses to each comment are listed below.

 

 

Reviewer 1

 

The authors investigated the effect of salt stress and supplementation of mannose on ascorbic acid (AsA) acculuation. The results showed that salt stress increased the AsA levels in tomato and supplemented mannose also increased the level at immature stage. The manuscript should be carefully revised before it could be considered for publication.

 

Thank you for your review. We have thoroughly revised our manuscript, focusing on the discussion points you raised, and had a native English speaker proofread the manuscript to ensure clarity for "Horticulturae" readers.

 

 

In each Figure, statistical analysis should be conducted.

 

According to the reviewer’s comments, we added the sentence for statistical analysis in the method sections and revised Figure 2, 5, and 7.

 

(Line 158)

2.7. Statistical analysis

The data were expressed as the mean values ± SD taken from 4–9 independent biological experiments. The experimental data of the samples were statistically analyzed through one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using Statistica 13.1 software (StatSoft, Inc., Tulsa, OK, USA). The results with a P-value ≤ 0.05 and a P-value ≤ 0.01 were considered statistically significant.

 

 

In the text and Figures (Fig 2 and 5), the use of ascorbic acid or vitamin C should be consistent in the whole manuscript.

 

We have corrected this error by changing all "vitamin C" words to "ascorbic acid" based on the reviewer's advice.

 

 

In the Abstract, the current research gap and research purpose should be clearly stated.

Lines 11-16 when describing the results, the authors are suggested to clearly describe whether the comparison was between salt stress and control, or between different fruit development stages. The same issues exist in the text.

 

According to the reviewer’s advice, we have revised the abstract as follows.

 

(Line 8)

Abstract: Salt stress causes osmotic stress and ion toxicity, often inhibiting plant growth and metabolism. However, salt-stressed tomato plants accumulate ascorbic acid, resulting in fruits with high commercial value. However, it was not well understood how mannose, the material for the synthesis of ascorbic acid, and its metabolism are affected under salt stress conditions. In this study, we found that tomatoes grown under salinity stress had increased levels of ascorbic acid, which correlated with decreased levels of mannan in the skin and seeds. Expression analysis of the ascorbic acid synthase gene showed increased expression in early ripening stages under salt stress. In addition, the expression of cellulose synthase-like A(CSLA), genes involved in mannan metabolism, increased significantly during mid-ripening in the control condition. Since ascorbic acid and mannan share mannose as a precursor, they are likely to compete for it. This suggests that salt-stressed tomatoes may be deficient in both ascorbic acid and mannose, thereby affecting mannan synthesis. To investigate this trade-off, we developed a culture system with added mannose. The results showed that in salt-stressed tomatoes supplemented with mannose, ascorbic acid levels in unripe green peels reached those of fully ripe fruit, highlighting the influence of mannose availability on ascorbic acid accumulation.

 

 

 

Skin refers to exocarp? It’s better to describe clearly.

 

We have corrected the error “exocarp” to “skin”.

 

 

Line 52 What do “strong properties” mean?

 

We have corrected the error “strong properties” to “strong mechanical properties”.

 

 

In “Materials and Methods”, English words should be used to start the setences. e.g. Lines 85, 87, 94, 98, 100, etc. Check the gramar for these sentences: Lines 87-88, 94-95.

 

According to the reviewer’s comments, we changed the sentences in “Materials and Methods” as follows.

 

(Line 101)

2.2. Extraction of cell walls

Three seeds and 100 mg fresh weight of each tissue were frozen and crushed with a mortar and pestle, and 1.5 ml of 80% EtOH was added. The mixture was centrifuged at 15,000 rpm for 1 minute and the supernatant was removed. Pellets were washed three times with water, three times with 1 ml of methanol: chloroform (MC, 1∶1), and three times with 1 ml of acetone (Wako Pure Chemical Industries, Ltd.). The sample was dried, weighed and recorded as the amount of cell wall. The extracted cell walls were transferred to a screw-capped test tube (ST-13M, Nichiden Rika Glass), and 250 µl of 2 M TFA was added using a Pasteur pipette. The tube was then heated at 121°C for 2 hours (EYELA MG-2200) to perform hydrolysis. Then 300 µl of isopropanol was added, and the tube was dried at room temperature using a spray evaporator. The pellet was mixed with 100 l of distilled water, agitated, and centrifuged, and the TFA-soluble fraction was extracted from the supernatant. The TFA soluble fraction contains pectin and hemicellulose, so the amount of mannose can be measured.

2.3. Analysis of mannan and cell wall sugar components by gas chromatography

The TFA soluble fraction sample (30 µl) of seed and pericarp was lyophilized overnight. 250 µl of HCl/MeOH was added, the tube was sealed and heated at 80°C for 15 hours (EYELA MG-2200). To the sample, 100 µL of t-butyl alcohol was added, and the tube was dried at room temperature with a spray evaporator. The samples were then treated with 100 µL of pyridine, 100 µL of hexamethyldiphenylsilane and 50 µL of trimethylchlorosilane. The tubes were sealed and the mixture was heated at 80°C for 20 minutes using an EYELA MG-2200. These TMS reagents were gently removed using a spray evaporator and the mixture was dissolved in 1 mL of hexane and centrifuged. The supernatant was carefully transferred to a new test tube and dried again using a spray evaporator. The mixture was dissolved in 5 μL of hexane, and 1 μL of the solution was analyzed by gas chromatography (GC-2010 Plus series, Shimadzu Corporation, Kyoto, Japan) using a DB-1 column. The amount of sugar was measured by the anthrone sulfate method for neutral sugars and the carbazole sulfate method for acidic sugars.

2.4. Expression analysis of mannan synthase gene and ascorbic acid synthase gene in tomato fruit under salinity conditions

Each tissue from the fruit at each stage of maturity (100 mg) was frozen and crushed using a mortar and pestle. Total RNA was extracted from each tissue using RNeasy® Plant Mini Kit (QIAGEN), and cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN) (Gene Amp® PCR System 9700). Go-Taq qPCR Master Mix was used for expression analysis. Gotaq® 5 µl, DNase-free water 4 µl, 10 µM forward and reverse primer (Table S1) 0.2 µl, CXR reference dye 0.1 µl, and template 0.5 µl were mixed, and PCR was performed under the following conditions (Applied Biosystems 7300 Fast Real-Time PCR System): 95℃ 2 min → (95℃ 5 sec, 63℃ 10 sec, 72℃ 31 sec) × 50 cycles → dissociation step.

 

 

When citing Figures, the number details should be provided. e.g. use Figure 2A, rather than Figure 2. Check the Results and Discussion sections.

 

Many thanks for your advice. We revised the citing Figure in “Results” and “Discussion” as follows.

 

(Line 171)

The amount of mannose in the seeds (Fig. 2A) and pericarp (Fig. 2C) was measured by analyzing the sugars that make up the cell walls using a biochemical method based on gas chromatography. As a result, the amount of mannose in the pericarp did not increase during fruit ripening under control conditions, whereas the mannose content in the fruits under salt stress conditions increased as they ripened to red (Fig. 2C). On the other hand, in the seeds, the amount of mannose was lower at all stages of ripening compared to control conditions (Fig. 2A). Even at the R stage, which is the final red ripened fruit, it contained only about half the amount of mannose. The amount of ascorbic acid was also measured in the pericarpand seeds. As for ascorbic acid, the content of ascorbic acid was higher under salt stress conditions compared to the control conditions, and an increase of about 20% was observed in each tissue (Fig. B, D). From the above, it was found that the amount of mannose and ascorbic acid increased in pericarp the amount of mannose decreased and the amount of ascorbic acid increased in seeds under salt stress conditions.

 

(Line 280)

Conversely, in the pericarp, the main edible part of the fruit, both mannan and ascorbic acid levels increased under salinity stress conditions (Fig. 2B, D). Despite the overall mannose deficiency in salinity-stressed tomato fruits, the pericarp maintained active synthesis of both ascorbic acid and mannan.

 

 

Fig 2: what tissues were these measured in? Skin and seed (Line 149)? Seed and pericarp (Line 420). I am confused. Also in Line 276, which data in Fig 2 was meausred in the pericarp?

 

We have corrected the error “skin” to “pericarp”.

 

 

Results and Discussion: no need to repeat the information that has been introduced in Introduction.

 

We have deleted the repeat section in discussion and revised the discussion as follows.

 

(Line 261)

4. Discussion

In this study, we measured mannose and ascorbic acid levels in tomato fruits under salinity stress and analyzed the expression of mannan synthase and ascorbic acid synthase genes. Additionally, we investigated how adding mannose affects ascorbic acid levels under salinity stress.

Expression analysis of mannan synthase genes and ascorbic acid synthesis genes in seeds revealed that salinity stress reduced the expression of mannan synthase CSLA (Fig. 3). This suggests that under salinity stress, GDP-mannose is preferentially allocated to ascorbic acid synthesis, thereby reducing the expression of mannan synthesis genes. Gene expression data further indicate that seed mannan levels decrease under salinity stress (Fig. 2A), resulting in poor seed germination. This supports the hypothesis that there is competition between ascorbic acid and mannan synthesis for GDP-mannose. Mannan, a major cell wall component in tomato seeds, is particularly abundant in the endosperm. Seed mannan of tomato probably originates from the endosperm. This mannan has two functions: (i) to form a crystalline or cross-linked structure that binds to cellulose, and (ii) to act as a storage polysaccharide in seed endosperm and vegetative tissues. When mannose was supplied externally, previously non-germinating salt-stressed seeds showed partial germination (Fig. 4), reinforcing the idea that seed germination capacity is closely linked to mannan availability.

 

 

 

 

Line 173: while or when?

 

We have corrected the error “while” to “when”.

 

 

Line 181: what is “seed function”?

 

We have corrected the error “seed function” to “seed germination ability”.

 

 

Line 194: AsA was measured in pericarpt tissues (epicarp and endocarp). epicarp refers to exocarp/skin? Mesocarp was not included?

 

Many thanks for your advice. We revised the sentences as follows.

 

(Line 215)

3.4. Measurement of ascorbic acid content under salt stress conditions with added mannose

It has been shown that the ascorbic acid content in tomato fruit is increased by salt stress. In this study, ascorbic acid content was measured in skin and mesocarp / endocarp tissues. As a result, it was found that ascorbic acid in the exocarp at M stage, which is the early ripening stage, was significantly increased under salt stress conditions as compared to the control. NaCl induced AsA accumulation by 174% in comparison to control (Fig. 5A). Although no significant difference was observed in mesocarp / endocarp, ascorbic acid in M stage showed a tendency to increase by about 39% under salt stress conditions as in skin (Fig. 5B). The experiment in Fig. 4 suggested that saline culture caused a deficiency of ascorbic acid and mannose, which is a material for mannan, so we constructed a culture system in which mannose was added in addition to saline culture. The ascorbic acid content of tomato fruits grown under salinity stress conditions with mannose was measured in each tissue and compared with the ascorbic acid content of tomato fruits under control and salinity stress conditions. As a result, in tomato fruits grown under salinity stress conditions with mannose, ascorbic acid was significantly increased by about 322% at the M stage of the skin compared to the control condition and showed a tendency to increase compared to salinity stress conditions (Fig. 5A). In addition, ascorbic acid at the M stage of mesocarp / endocarp showed a tendency to increase compared to the other two conditions (Fig. 5B). However, the amount of ascorbic acid accumulated at the R stage was similar in all conditions and tissues.

 

 

 

 

Fig 4 title: use “breaker” rather than “B”.

 

We have corrected this error.

 

 

Fig 5A: the numbering A is missing.

 

Thank you. We have added A in Figure 5.

 

 

Fig 7 should be deleted, since it is excatly the same as Fig 5, the data should be presented only once.

 

We have removed Figure 7 and corrected the errors in the discussion of this old Figure 7.

 

 

Fig 5B, and Fig 6C and D: What does the diagonal line “mesocarp/endocarp” mean? Or? And? This should be clearly stated.

 

We have revised Figure legends of Fig.5 and Fig.6 as follows.

 

(Line 490)

Figure 5. Ascorbic acid (AsA) content per 1 g fresh weight of tomato fruit grown under control, and mannose (1 mM) with salinity conditions. Ascorbic acids were extracted from tomato fruit skin and mesocarp/endocarp. (A): Skin, (B): Mesocarp and endocarp (Mesocarp/endocarp). Ripening stage: mature green (M), red ripe(R). Error bars indicate the SD (n=9). Different letters in each panel indicate significant differences at P < 0.05 (Tukey’s test).

 

(Line 511)

Figure 6. Ascorbic acid biosynthesis-related gene expression patterns during fruit ripening. Gene expression was analyzed by qRT-PCR in tomato. GDP-Mannose Epimerase (GME) -1 and 2 are involved in ascor biosynthesis. (A), (B): Skin, (C), (D): Mesocarp and endocarp (Mesocarp/endocarp). Ripening stages were as follows: M, mature green; B, breaker; T, turning; R, red ripe. Error bars indicate the SD [n = 5(Control) and n = 4 (NaCl)].

 

 

Fig 5-8: The full “control” in the x axis should be used.

 

We have corrected this error.

 

 

Discussion: Line 240-244 This paragraph does not provide useful information. Line 245-265 Most of the information has been provided in Introduction section. Line 241: Where is the tissue hardness data? It is suggested to revise the Discussion section, different information should form a coherent whole with logical connections.

 

We have deleted the repeat section in discussion and revised the discussion as follows.

 

(Line 261)

4. Discussion

In this study, we measured mannose and ascorbic acid levels in tomato fruits under salinity stress and analyzed the expression of mannan synthase and ascorbic acid synthase genes. Additionally, we investigated how adding mannose affects ascorbic acid levels under salinity stress.

Expression analysis of mannan synthase genes and ascorbic acid synthesis genes in seeds revealed that salinity stress reduced the expression of mannan synthase CSLA (Fig. 3). This suggests that under salinity stress, GDP-mannose is preferentially allocated to ascorbic acid synthesis, thereby reducing the expression of mannan synthesis genes. Gene expression data further indicate that seed mannan levels decrease under salinity stress (Fig. 2A), resulting in poor seed germination. This supports the hypothesis that there is competition between ascorbic acid and mannan synthesis for GDP-mannose. Mannan, a major cell wall component in tomato seeds, is particularly abundant in the endosperm. Seed mannan of tomato probably originates from the endosperm. This mannan has two functions: (i) to form a crystalline or cross-linked structure that binds to cellulose, and (ii) to act as a storage polysaccharide in seed endosperm and vegetative tissues. When mannose was supplied externally, previously non-germinating salt-stressed seeds showed partial germination (Fig. 4), reinforcing the idea that seed germination capacity is closely linked to mannan availability.

 

 

Conclusions: The main results should be summarized.

 

According to reviewer’s comments, we changed the “Conclusions” as follows.

 

(Line 317)

5. Conclusions

This study showed increased levels of ascorbic acid in tomatoes grown under salinity stress, which correlated with reduced mannan content in skin and seeds. Expression analysis revealed higher expression of the ascorbic acid synthase gene during early ripening stages under salt stress. In addition, genes related to mannan metabolism showed increased expression during mid-ripening. Since ascorbic acid and mannan both use mannose as a precursor, their synthesis competes under salt stress, potentially leading to deficiencies in both. To investigate this, a mannose-supplemented culture system was developed. In conclusion, tomatoes supplemented with mannose under salt stress conditions exhibited increased ascorbic acid synthesis at the immature M stage compared to control or salt-stressed tomatoes alone. This suggests that tomatoes with higher ascorbic acid content could be produced for markets where fruit is harvested and shipped at the immature M stage. These results provide insights for optimizing salt-stressed tomato breeding to improve nutritional quality and marketability.

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study of the influence of abiotic stress on plants is a relevant area of ​​research. The authors of the article touched upon a rather interesting topic - the accumulation of vitamin C under salt stress in tomatoes. I consider the article relevant and suitable for the subject of the journal Hоrticulturаe.

The applied methods are adequate and modern. However, there are some remarks that need to be noted.

1. Line 78 - The sentence "salt stress by tissue" is not correctly constructed.
2. Line 83 - "roe"?
3. In the Methods section for reagents, the manufacturer's company and country should also be indicated.
4. Line 113 - What fetal tissue or tissues were taken for analysis?
5. Line 121 - for clarity, it is better to collect the primers in a table.
6. In the Methods chapter, "Plant material and growing conditions" should be indicated.
7. Here we can also place Fig. 1, which the authors unsuccessfully placed in the Introduction.
8. It is also necessary to add a chapter describing statistical data processing.
9. In Fig. 3 the inscription at the top in black letters is not legible.

Based on the mentioned above, I think that this article can by recommended for publication in the Horticulturae after revision.     

 

Author Response

Dear Reviewer 2,

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript titled “Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato” (horticulturae-3537821). As indicated in the following responses, we have considered all these comments in preparing this resubmitted manuscript. Detailed responses to each comment are listed below.

 

 

Reviewer 2

 

The study of the influence of abiotic stress on plants is a relevant area of research. The authors of the article touched upon a rather interesting topic - the accumulation of vitamin C under salt stress in tomatoes. I consider the article relevant and suitable for the subject of the journal Hоrticulturаe.

 

We would like to thank reviewer 2 for the very positive evaluation of our paper.

 

 

The applied methods are adequate and modern. However, there are some remarks that need to be noted.

1. Line 78 - The sentence "salt stress by tissue" is not correctly constructed.

 

We have deleted “ by tissue”.

 

 

2. Line 83 - "roe"?

 

We have corrected the errors as follows.

 

(Line 102)

Three seeds and 100 mg fresh weight of each tissue were frozen and crushed with a mortar and pestle,

 

 

3. In the Methods section for reagents, the manufacturer's company and country should also be indicated.

 

We have added the the manufacturer's company and country in the Methods section.

                                                              

 

 

4. Line 113 - What fetal tissue or tissues were taken for analysis?

 

We have revised the sentences as follows.

 

(Line 115)

2.3. Analysis of mannan and cell wall sugar components by gas chromatography

The TFA soluble fraction sample (30 µl) of seed and pericarp was lyophilized overnight.

 

 

5. Line 121 – for clarity, it is better to collect the primers in a table.

 

We have added the supplemental table (Table S1) for primers.

 

 

6. In the Methods chapter, “Plant material and growing conditions” should be indicated.

 

We have added the “Plant material and growing conditions” in Methods chapter as follows.

 

(Line 83)

2.1. Plant materials and growth conditions

Tomato seeds (Solanum lycopersicum cv. 'Micro-Tom') were germinated at room temperature on moist paper. After one week, seedlings were transplanted into 5x5 cm rockwool pots and grown in a growth chamber (TOMY CL-301) under 16 h of light and 8 h of dark at 25 °C for 6 weeks. Plants were grown in plastic trays with a constant volume of 2.0 L. A commercial nutrient solution (Otsuka A; Otsuka Chemical Co. Ltd., Osaka, Japan) with an electrical conductivity (EC) of 2.0 dS m^-1 was used. Seven weeks after germination, when the first truss flowered, half of the plants were transferred to a saline nutrient solution with an EC of 15.0 dS m^-1, which was achieved by adding NaCl (equivalent to 160 mM NaCl), which was reported to be the appropriate concentration for long-term cultivation under salinity conditions [10]. Plants were maintained in a nutrient solution with an electrical conductivity of 2.0 dS m^-1 (0 mM NaCl) as a control. Plants were exposed to two different levels of electrical conductivity (EC) to adapt to salinity stress. Plants were exposed to two different salinity levels, EC 9.0 dS m^-1 (80 mM NaCl) and EC 12.0 dS m^-1 (120 mM NaCl), for four days each before being transferred to a solution with EC 15.0 dS m^-1. Tomato fruits at the corresponding developmental stages were also collected: M (30 days post anthesis (DPA)), B (35 DPA), T (37 DPA), R (45 DPA)(Fig. 1).

 

 

7. Here we can also place Fig. 1, which the authors unsuccessfully placed in the Introduction.

 

Please see our response to reviewer comments 6.

 

 

8. It is also necessary to add a chapter describing statistical data processing.

 

According to the reviewer’s comments, we added the sentence for statistical analysis in the method sections and revised Figure 2, 5, and 7.

 

(Line 158)

2.7. Statistical analysis

The data were expressed as the mean values ± SD taken from 4–9 independent biological experiments. The experimental data of the samples were statistically analyzed through one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using Statistica 13.1 software (StatSoft, Inc., Tulsa, OK, USA). The results with a P-value ≤ 0.05 and a P-value ≤ 0.01 were considered statistically significant.

 

 

9. In Fig. 3 the inscription at the top in black letters is not legible.

 

We have revised the Fig. 3.

 

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors!

The article is relevant for the field and presented in a well-structured manner.

The references are mostly old publications, but I think they are relevant. It does not include an excessive number of self-citations. Please, put the reference into square brackets (lines 167, 204, 207, 209). You should add the year of the articles of Ye et al (line 210), Fry` group  (line 298). I didn`t find the work of Ye et al. in the references, so add it there.

The manuscript is interesting, scientifically sound and the experimental design is appropriate to test the hypothesis.

The manuscript’s results are reproducible based on the details given in the methods section.

The data are interpreted appropriately and consistently throughout the manuscript. The Figures properly show the data, but they are not easy to understand. I have some recommendations to make the figures better:

• What do (C ) and (D) figures mean (Figure 2)? You should change the font of the names of the genes (Figure 3).
• Change Cont to Control (Figures 5, 6,7, 8). I think you should rephrase the caption of Figure 5 to  “The influence of  mM  NaCl alone and in combination with 1 mM mannose to ascorbic acid (AsA) content in tomato fruit.  Ripening stage: mature green (M), red ripe(R). Error bars indicate the SD (n=9).” The numbers on the y-axis are difficult to see (Figure 5). The Figures 5 and 7 are the same. Delete one of them.
• Delete one of the sentences “Skin (A, B), Mesocarp and endocarp (C, D)” (Figure 6, lines 480-481). Sentences are repeated.
• I think you should rephrase the caption of Figure 8 to “The effect of 160 mM NaCl alone and in combination with 1 mM mannose to mannose content in tomato fruit. Ripening stage: mature green (M), red ripe (R). Error bars indicate the SD (n=9).”
• Check the numbers of Figures in the text. You begin to describe the results on Figure 4 (line 219), and then refer toFigure 7 (lines 227, 229).

The conclusions are consistent with the evidence and arguments presented.

You should add different letters (or asterisks) to your Figures to indicate statistical differences at P≤0.05. Write information about statistics in Methods section.

 

Comments on the Quality of English Language

Dear Authors!

The manuscript is not very clear. My recommendations:

• Decipher MI (line 58), TFA (line 91), DW (line 94), TMS(line 103), CXR (line 118), EC (line 134)
• Probably, you should delete the words by tissue (line 78), and change the sentence to “We then investigated the metabolic regulation of cell wall polysaccharides and AsA in tomato fruit tissues under salt stress conditions,…”
• I didn`t understand the combinations and concentrations of NaCl and mannose (line 135-137). Probably, the table would be more understandable.
• I didn`t understand the sentence “From the above, it was found that the amount of mannose and ascorbic acid increased in skin the amount of mannose decreased and the amount of ascorbic acid increased in seeds under salt stress conditions” (lines 159-161). Rephrase it.
• You write about higher concentration (line 187). What substance were you describing? Probably, you mean “…after the increase of mannose concentration, seed germination further accelerated”.
• Probably, you should divide the sentence on lines 195-197 to 2 smaller sentences. For example, “As a result, it was found that ascorbic acid in the exocarp at M stage, which is the early ripening stage, was significantly increased under salt stress conditions as compared to the control. NaCl induced AsA accumulation by 174% in comparison to control (Fig. 5A).”
• You should add the words the expression of gene in the sentences: ”As a result, in the exocarp, the expression of GME1 gene was significantly increased by about 434% at the M stage, about 185% at the B stage, and the expression of GME2 gene was significantly increased by about 185% at the M stage under salt stress conditions compared to the control (Fig. 6 A, B). In the endocarp, the expression of GME1 gene was significantly increased by about 88% at the B stage and the expression of GME2 gene was significantly increased by about 81% at the T stage under  salt stress conditions compared to the control (Fig. 6C, D)” (lines 211-216).
• Divide the sentence on lines 240-244 into smaller sentences.
• Write references to the sentence: “Similarly, in legumes such as locust bean and guar, galactomannans are abundant in seed cell walls, and in tomato, seed mannan probably originates from the endosperm” (line 262-263).
• Rephrase the sentence on line 293. For example, “… that ascorbic acid accumulation under salinity stress reflects its active participation in ROS scavenging.

Author Response

Dear Reviewer 3,

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript titled “Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato” (horticulturae-3537821). As indicated in the following responses, we have considered all these comments in preparing this resubmitted manuscript. Detailed responses to each comment are listed below.

 

 

Reviewer 3

 

Dear Authors!

The article is relevant for the field and presented in a well-structured manner.

The references are mostly old publications, but I think they are relevant. It does not include an excessive number of self-citations. Please, put the reference into square brackets (lines 167, 204, 207, 209). You should add the year of the articles of Ye et al (line 210), Fry` group  (line 298). I didn`t find the work of Ye et al. in the references, so add it there.

 

We have corrected the errors.

Ye et al. was also our mistakes. We have corrected the errors “Ye et al.” to “Yin et al.”. (Ref. 10)

 

 

The manuscript is interesting, scientifically sound and the experimental design is appropriate to test the hypothesis.

The manuscript’s results are reproducible based on the details given in the methods section.

The data are interpreted appropriately and consistently throughout the manuscript. The Figures properly show the data, but they are not easy to understand. I have some recommendations to make the figures better:

• What do (C ) and (D) figures mean (Figure 2)? You should change the font of the names of the genes (Figure 3).

 

Thank you for your comments. We have revised the figure legend of Figure 2 and 3 as follows.

Figure 2. Measurements of amounts of mannose and ascorbic acid of red ripe tomato fruit grown under control and saline conditions (160 mM). (A), (B): Seed, and (C), D): Pericarp. (A), (C): Mannose amounts. (B), (D): Ascorbic acid amounts. Ripening stages were as follows: M, mature green; B, breaker; T, turning; R, red ripe. Error bars indicate the SD [n = 5(Control) and n = 4 (NaCl)]. Different letters in each panel indicate significant differences at P < 0.05 (Tukey’s test).

Figure 3. Gene expression patterns related to mannan and ascorbic acid biosynthesis during fruit ripening. Gene expression was analyzed by qRT-PCR in tomato seeds. Cellulose synthase-like A (CSLA) involved in mannan biosynthesis and GDP-mannose epimerase (GME) involved in ascorbic acid biosynthesis. Expression levels were compared with Elongation factor 1α in the same assay. Ripening stages were as follows: M, mature green; B, breaker; T, turning; R, red ripe. Values are means ±SD (n=25).

 

 

• Change Cont to Control (Figures 5, 6,7, 8). I think you should rephrase the caption of Figure 5 to“The influence of  mM  NaCl alone and in combination with 1 mM mannose to ascorbic acid (AsA) content in tomato fruit.  Ripening stage: mature green (M), red ripe(R). Error bars indicate the SD (n=9).” The numbers on the y-axis are difficult to see (Figure 5). The Figures 5 and 7 are the same. Delete one of them.

 

We have corrected the errors in Figures 5, 6, 7, 8.

We have changed the figure legends of Figure 5 as follows.

Figure 5. The influence of 160 mM NaCl alone and in combination with 1 mM mannose to ascorbic acid (AsA) content in tomato fruit. Ascorbic acids were extracted from tomato fruit skin and mesocarp/endocarp. (A): Skin, (B): Mesocarp and endocarp (Mesocarp/endocarp). Ripening stage: mature green (M), red ripe(R). Error bars indicate the SD (n=9). Different letters in each panel indicate significant differences at P < 0.05 (Tukey’s test).

We have removed the Figure 7.

 

 

 

• Delete one of the sentences “Skin (A, B), Mesocarp and endocarp (C, D)” (Figure 6, lines 480-481). Sentences are repeated.

 

We have corrected the errors. Many thanks.

 

 

• I think you should rephrase the caption of Figure 8 to “The effect of 160 mM NaCl alone and in combination with 1 mM mannose to mannose content in tomato fruit. Ripening stage: mature green (M), red ripe (R). Error bars indicate the SD (n=9).”

 

Thank you so much. We have changed the caption of Figure 8 (New Figure 7) as follows.

 

Figure 7. The effect of 160 mM NaCl alone and in combination with 1 mM mannose to mannose content in tomato fruit. Ripening stage: mature green (M), red ripe (R). Error bars indicate the SD (n=9). Different letters in each panel indicate significant differences at P < 0.05 (Tukey’s test).

 

 

• Check the numbers of Figures in the text. You begin to describe the results on Figure 4 (line 219), and then refer toFigure 7 (lines 227, 229).

 

Thank you so much. We check the numbers of Figures in Results sections.

 

 

The conclusions are consistent with the evidence and arguments presented.

You should add different letters (or asterisks) to your Figures to indicate statistical differences at P≤0.05. Write information about statistics in Methods section.

 

According to the reviewer’s comments, we added the sentence for statistical analysis in the method sections and revised Figure 2, 5, and 7.

 

(Line 158)

2.7. Statistical analysis

The data were expressed as the mean values ± SD taken from 4–9 independent biological experiments. The experimental data of the samples were statistically analyzed through one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using Statistica 13.1 software (StatSoft, Inc., Tulsa, OK, USA). The results with a P-value ≤ 0.05 and a P-value ≤ 0.01 were considered statistically significant.

 

 

Comments on the Quality of English Language

Dear Authors!

The manuscript is not very clear. My recommendations:

• Decipher MI (line 58), TFA (line 91), DW (line 94), TMS(line 103), CXR (line 118), EC (line 134)

 

We have corrected the errors,

“MI pathway” to “myo-inositol pathway”, (Line 59)

“2 M TFA” to “2 M trifluoroacetic acid (TFA)” (Line 109)

“DW” to “distilled water” (Line 112)

“TMS” to “trimethylsilyl reaction” (Line 122)

“CXR” to “Carboxy-X-Rhodamine (CXR)” (Line 137)

“EC” to “Electrical Conductivity (EC)”. (Line 145)

 

• Probably, you should delete the words by tissue (line 78), and change the sentence to “We then investigated the metabolic regulation of cell wall polysaccharides and AsA in tomato fruit tissues under salt stress conditions,…”

 

We have deleted “ by tissue”.

 

 

• I didn`t understand the combinations and concentrations of NaCl and mannose (line 135-137). Probably, the table would be more understandable.

 

We considered adding a table, but that would have created more confusion, so we decided to add the following text:

(Line 149)

Finally, the culture solution concentration was 160 mM NaCl and 1 mM mannose.

 

 

• I didn`t understand the sentence “From the above, it was found that the amount of mannose and ascorbic acid increased in skin the amount of mannose decreased and the amount of ascorbic acid increased in seeds under salt stress conditions” (lines 159-161). Rephrase it.

 

We have changed the sentences as follows.

 

(Line 181)

From the above, it was found that under salt stress, the amount of mannose and ascorbic acid both increased in pericarp, whereas the amount of mannose decreased and the amount of ascorbic acid increased in seeds.

 

 

• You write about higher concentration (line 187). What substance were you describing? Probably, you mean “…after the increase of mannose concentration, seed germination further accelerated”.

 

Thank you so much your advice. We have changed the sentences as follows.

 

(Line 207)

However, when mannose was added externally, the germination rate of control seeds reached 100% on the sixth day when distilled water was used, and after the increase of mannose concentration, seed germination further accelerated (Fig. 4).

 

 

• Probably, you should divide the sentence on lines 195-197 to 2 smaller sentences. For example, “As a result, it was found that ascorbic acid in the exocarp at M stage, which is the early ripening stage, was significantly increased under salt stress conditions as compared to the control. NaCl induced AsA accumulation by 174% in comparison to control (Fig. 5A).”

 

Thank you so much your advice. We have changed the sentences as follows.

 

(Line 218)

As a result, it was found that ascorbic acid in the exocarp at M stage, which is the early ripening stage, was significantly increased under salt stress conditions as compared to the control. NaCl induced AsA accumulation by 174% in comparison to control (Fig. 5A).

 

 

• You should add the words the expression of gene in the sentences: ”As a result, in the exocarp, the expression of GME1 gene was significantly increased by about 434% at the M stage, about 185% at the B stage, and the expression of GME2 gene was significantly increased by about 185% at the M stage under salt stress conditions compared to the control (Fig. 6 A, B). In the endocarp, the expression of GME1 gene was significantly increased by about 88% at the B stage and the expression of GME2 gene was significantly increased by about 81% at the T stage undersalt stress conditions compared to the control (Fig. 6C, D)” (lines 211-216).

 

Thank you so much your advice. We have changed the sentences as follows.

 

(Line 236)

3.5. Expression analysis of ascorbic acid synthase gene in skin and pericarp.

Several pathways have been proposed for the biosynthesis of ascorbic acid in plants. Among them, the first pathway of ascorbic acid synthesis in higher plants was the D-mannose/L-galactose pathway [4]. The GDP-mannose 3',5'-epimerase (GME) gene, which functions in this pathway, is an enzyme gene that converts GDP-D-mannose to GDP-L-galactose and is the most highly conserved ascorbic acid biosynthesis-related gene among higher plants [5], so it has been extensively studied. GME exists as a single-copy gene in many plants, but two genes, GME1 and GME2, are known to exist in tomato [6]. The primers used for the expression analysis of each gene were designed based on Yin et al[10]. The expression analysis of GME1 and GME2 was then performed by real-time PCR. As a result, in the skin, the expression of GME1 gene was significantly increased by about 434% at the M stage, about 185% at the B stage, and the expression of GME2 gene was significantly increased by about 185% at the M stage under salt stress conditions compared to the control (Fig. 6 A, B). In the endocarp, the expression of GME1 gene was significantly increased by about 88% at the B stage and the expression of GME2 gene was significantly increased by about 81% at the T stage under  salt stress conditions compared to the control (Fig. 6C, D)

 

 

• Divide the sentence on lines 240-244 into smaller sentences.

 

We have changed the sentences as follows.

(Line 262)

In this study, we measured mannose and ascorbic acid levels in tomato fruits under salinity stress and analyzed the expression of mannan synthase and ascorbic acid synthase genes. Additionally, we investigated how adding mannose affects ascorbic acid levels under salinity stress.

 

 

• Write references to the sentence: “Similarly, in legumes such as locust bean and guar, galactomannans are abundant in seed cell walls, and in tomato, seed mannan probably originates from the endosperm” (line 262-263).

 

Based on comments from other reviewers, we have removed these sentences.

 

 

• Rephrase the sentence on line 293. For example, “… that ascorbic acid accumulation under salinity stress reflects its active participation in ROS scavenging.

 

We have changed the sentences as follows.

 

(Line 297)

Furthermore, no significant differences in hydrogen peroxide levels were observed between control and salinity-stressed tomato fruits [28,29], suggesting that ascorbic acid accumulation under salinity stress reflects its active participation in ROS scavenging.

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved, and it can be considered for publication.

Author Response

Dear Reviewer 1,

 

Comments: Reviewer 1

The manuscript has been improved, and it can be considered for publication.

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript.

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors of the article for correcting all my comments and answering all my questions.

I have no new comments, except that the color combination in Figures 5 and 7 (red/green) is not very good for colorblind people, I recommend changing the colors.

I wish you further success in science.

Author Response

Dear Reviewer 2,

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript titled “Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato” (horticulturae-3537821). As indicated in the following responses, we have considered all these comments in preparing this resubmitted manuscript. Detailed responses to each comment are listed below.

 

 

Comments: Reviewer 2

 

I thank the authors of the article for correcting all my comments and answering all my questions.

I have no new comments, except that the color combination in Figures 5 and 7 (red/green) is not very good for colorblind people, I recommend changing the colors.

I wish you further success in science.

 

Thank you so much for your helpful comments. We have changed the color combination in Figure 5 and 7 (black/gray).

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors!

This is my second review to this manuscript. It became more understandable.

I have only two recommendations:

1) Probably, you mean ml in the phrase “… The pellet was mixed with 100 l” (line 112).

2) You should write  the names of genes in italics (lines 237-252, 288, etc).

Author Response

Dear Reviewer 3,

 

We are grateful to the editor and reviewers for their constructive and helpful comments, which greatly helped us improve our manuscript titled “Salt Stress-Induced Ascorbic Acid Accumulation and Its Trade-Off with Mannan Content in Tomato” (horticulturae-3537821). As indicated in the following responses, we have considered all these comments in preparing this resubmitted manuscript. Detailed responses to each comment are listed below.

 

 

Comments: Reviewer 3

 

Dear Authors!

This is my second review to this manuscript. It became more understandable.

I have only two recommendations:

• Probably, you mean ml in the phrase “… The pellet was mixed with 100 l” (line 112).

Many thanks. We have corrected the error. (line112)

 

• You should write  the names of genes in italics (lines 237-252, 288, etc).

We have corrected the errors. (lines 15, 194-199, 237-252, 267, 288, 455-456, 512)

 

We believe our manuscript now meets standards for publication. I am looking forward to hearing from you soon.

Sincerely yours,

 

Hiroaki Iwai

 

Tokai University

Department of Biology, School of Biological Sciences,

Sapporo, Hokkaido 005-8601, Japan

iwai.hiroaki.gb@tokai.ac.jp

 

University of Tsukuba, 

Institute of Life and Environmental Sciences, 

Tsukuba, Ibaraki 305-8572, Japan

iwai.hiroaki.gb@u.tsukuba.ac.jp

 

Author Response File: Author Response.pdf