Silencing CaTPS1 Increases the Sensitivity to Low Temperature and Salt Stresses in Pepper

Round 1

Reviewer 1 Report

The authors obtained quite interesting results on the role of trehalose metabolism in stress protection. However, there are many unclear places in the work that require editing and clarification. In addition, the value of the work is reduced by the lack of direct measurements of the level of damage under the studied stress effects, which is only partially compensated by an indirect marker (chlorophyll level). Some wording of the conclusions also needs clarification.

 

L16 Please explain "CK, POD, CAT, APX". Explain the pTRV2-CaTPS1, CK and PTRV2-00 variants. There should be no undeciphered abbreviations in the abstract.

L 32-37 Provide data on the mechanism of the effect of trehalose on resistance to stress.

L 43 Edit the end of the sentence "TPS gene families have been identified in many species, such as 53 TPSs genes in 43 cotton [12], 23 in soybean [13], 8 in potato [14], and 11 in pepper, Arabidopsis thaliana and rice respectively, [9,15]."

L 43-52 Explain why it is necessary for the gene to have TPSase activity but not TPPase activity.

L 55-57 "The transformation of the StTPS1 gene in potatoes enhanced photosynthesis in transgenic plants and consequently improved the resistance to abiotic stress [21]." It is unclear what happens to this potato gene in transgenic plants.

L 65-66 "The expression of CsTPS1, CsTPS9 and 65 CsTPPA were highest under at least one abiotic stress, and it was speculated that these three genes might play a key role in the abiotic stress response [24]." Specify a plant.

L 71 Decipher the abbreviation VIGS at the first use in the main text.

L 72 Explain how the genes StSSH2, StWTF, StUGT, StBHP, and StFLTP and all other genes mentioned in this paragraph are related to trehalose metabolism or stress resistance.

L 84 "response to abiotic stress of CaTPS1 in pepper has seldom been reported". Please provide references and details of these works in order to appreciate the novelty of the proposed manuscript.

L 93 Were the plants grown in soil or hydroponically?

L 133 "normal plants and silent plants (the CK (Blank control), TRV2-00, and 133 TRV2-CaTPS1 plants)": not clear what CK is - are these wild type plants?

L 146 "Take the treated fresh leaves and stick them from the back tissue of the leaves with scotch tape." Specify the method that was meant.

L 149 "stomata area and stomata perimeter". In reference [43] there is no calculation of the perimeter. I can assume that the authors measured the length and width of the stomata, and then calculated the area and "perimeter" of the stomata using the well-known mathematical formulas of the area and circumference of the ellipse. If this is true, then this should be written in the methodology.

L 156-158 Decipher POD, CAT, APX at the first mention in the text.

L 136 Specify the method of NaCl treatment - watering the soil, adding to the nutrient medium?

L 170 "CaTPS1 expression rising by 50% relative to CK". Specify which plants were used in this experiment and what do you compare them with, what does CK mean here?

"3.3. Stomatal Structure of CaTPS1 Silenced Plants under Cold and Salt Stresses"

What is the point of using 2 indicators (stomata area and perimeter), apparently calculated from the same initial values of stomata length and width?

L.279 "which may indicate that the stomata have become smaller after abiotic stress".

Usually, stomata (like most other plant cell types) grow to a certain size and can no longer change size due to the presence of a rigid cell wall. Accordingly, the conclusions of L273-280 are erroneous. Your results may be due to 1) inhibition of stomatal growth in young leaves under stress (unlikely for a period of 3 days, but needs to be checked), 2) an increase in the proportion of closed stomata under stress. Closing the stomata reduces their width and, accordingly, the area, and possibly the perimeter (due to the compression of the side walls; of course, if the real perimeter is measured, not the width). The length of the stomata can be influenced only by the first reason, which can be used for preliminary differentiation of these reasons. Try plotting the same graphs using the length and width of the stomata. Perhaps this will help to find out why cold stress has a stronger effect on the area and weaker on the perimeter. However, most likely, the data on stomata in any case can be considered only as preliminary and should be excluded from the manuscript: For the correct disclosure of the first reason, longer experiments are needed, and the second reason is to take into account the number of open and closed stomata.

 

An almost identical phrase is repeated 3 times:

The expression of CsTPS1, CsTPS9 and CsTPPA were highest under at least one abiotic stress, and it was speculated that these three genes might play a key role in the abiotic stress response [24]. L65-67

CsTPS1, CsTPS9 and CsTPPA had the highest expression in response to at least one abiotic stress, and it is speculated that these three genes may be involved in responding to abiotic stresses [24]. L260-261

The expression of CsTPS1, CsTPS9 and CsTPPA were the highest in response to at least one abiotic stress, which is consistent with the above results [24]. L271-272

 

 

L296-312. Interpretation of data on antioxidant enzymes may not be so unambiguous. These enzymes not only destroy toxic oxygen species (resistance enhancement), but can also be markers of ill health as they are induced by oxidative stress. Antioxidant enzymes in resistant plants may not respond well to exposure because they are not damaged by stress. To clarify the situation, it is necessary to measure the content of reactive oxygen species.

Author Response

Dear Editor and reviewer：

Thank you very much for the letter and the comments. We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don’t hesitate to let me know.

 Sincerely,

 Bingqiang Wei

Detailed responses to reviewer’s comments and Editor’s advice.

The authors obtained quite interesting results on the role of trehalose metabolism in stress protection. However, there are many unclear places in the work that require editing and clarification. In addition, the value of the work is reduced by the lack of direct measurements of the level of damage under the studied stress effects, which is only partially compensated by an indirect marker (chlorophyll level). Some wording of the conclusions also needs clarification.

L16 Please explain "CK, POD, CAT, APX". Explain the pTRV2-CaTPS1, CK and PTRV2-00 variants. There should be no undeciphered abbreviations in the abstract.

Re: They have been explained separately as follows: peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), CaTPS1-silenced plants (pTRV2-CaTPS1), the non-VIGS plants (CK) and Negative control/ empty vector (PTRV2-00). All references to CK in the abstract refer to normal plants without VIGS treatment to serve as a blank control for the VIGS experiment. The undeciphered abbreviations in the abstract have been added in full.,

L 32-37 Provide data on the mechanism of the effect of trehalose on resistance to stress.

Re: Data on the mechanism of the resistance effect of trehalose have been added to the text. The mechanism of the effect of trehalose on resistance to stress is as follows: “Trehalose is considered to be a potential osmostatic protective agent for plants, mainly in the following ways to enhance plant tolerance: First, trehalose absorbs nutrients by improving the integrity of membrane and the water relationship of plants, reducing electrolyte leakage and lipid oxidation; Second, trehalose improves the gas exchange characteristics, protects the photosynthetic mechanism from the oxidative damage caused by salinity, and causes the change of ultrastructure in plants. Third, the antioxidant activity of plants and the expression of stress-responsive proteins and genes were improved. Fourthly, it participates in the signal connection with signal molecules and plant hormones.”

L 43 Edit the end of the sentence "TPS gene families have been identified in many species, such as 53 TPSs genes in 43 cotton [12], 23 in soybean [13], 8 in potato [14], and 11 in pepper, Arabidopsis thaliana and rice respectively, [9,15]."

Re: The TPS gene family has been found in many species. For example, in cotton, there is a gene family containing 53 TPSs members [12], 23 TPSs genes were found in soybeans [13], and 8 TPSs genes were found in potatoes [14], Eleven TPSs genes have been found in pepper, Arabidopsis and rice [9,15].

L 43-52 Explain why it is necessary for the gene to have TPSase activity but not TPPase activity.

Re: Whether the gene has TPSase activity rather than TPPase activity was verified by functional complementation experiments in yeast. The available findings suggest that active TPS in plants such as ATTPS1 and OSTPS1 have additional TPP-like structural domains, which may lead to a more stable complex. It also suggests that plant TPS complexes may control T6P levels rather than rapidly producing trehalose. It has also been shown that SCTPS1, which has only a TPS structural domain, interacts with other subunits of the brewer's yeast TPS complex, confirming the importance of TPS enzyme activity for complex formation. Thus, it is speculated that TPS1 may be an important component of the TPS complex. The reason for TPS1 having TPSase activity rather than TPPase activity may be that his TPP structural domain is not sufficiently complete, but the exact reason remains to be verified.

L 55-57 "The transformation of the StTPS1 gene in potatoes enhanced photosynthesis in transgenic plants and consequently improved the resistance to abiotic stress [21]." It is unclear what happens to this potato gene in transgenic plants.

Re: We have reviewed the literature to verify that it is indeed unclear what happens to this potato gene in transgenic plants, and that the existing studies have been carried out by transferring the TPS gene from yeast into potato for stress-related experiments and observing the phenotypes and related indicators that lead to improved resistance to stress in potato.

The sentence has been amended to “Transfer of the yeast TPS gene into potato revealed that during drought treatment, the transgenic plants had delayed wilting and higher stomatal conductance, net photosynthetic rate, and CO₂ assimilation rate than wild-type plants, resulting in increased resistance to abiotic stresses.”

L 65-66 "The expression of CsTPS1, CsTPS9 and 65 CsTPPA were highest under at least one abiotic stress, and it was speculated that these three genes might play a key role in the abiotic stress response [24]." Specify a plant.

Re: The sentence has been changed to “CsTPS1, CsTPS9 and CsTPPA were most highly expressed under at least one abiotic stress, and it is speculated that these three genes may play a key role in the response of tea plants (Camellia sinensis (L.)) to abiotic stresses”

L 71 Decipher the abbreviation VIGS at the first use in the main text.

Re: Virus-induced gene silencing (VIGS) has been noted in the main text.

L 72 Explain how the genes StSSH2, StWTF, StUGT, StBHP, and StFLTP and all other genes mentioned in this paragraph are related to trehalose metabolism or stress resistance.

Re: These genes are associated with senescence in potato and their relationship with trehalose is currently reported in the literature. This paragraph focuses on the widespread use of VIGS and then gives different examples to demonstrate that it, and trehalose or resistance, are not directly related.

L 84 "response to abiotic stress of CaTPS1 in pepper has seldom been reported". Please provide references and details of these works in order to appreciate the novelty of the proposed manuscript.

Re: A review of relevant reports in the last decade by keyword TPS in Web of Science, PubMeb, Foreing resource Retrieval system，X-MOL, Springer and other platforms revealed that the role of CaTPS1 in pepper under low temperature and salt stress has not been reported.

L 93 Were the plants grown in soil or hydroponically?

Re: The plants were grown in the matrix of the nutrition bowl. The details are as follows: Pick the pepper seeds with full grain and wrap them with gauze, soak them in hot water at 55°C for 20 minutes, disinfect them with 20% trisodium phosphate for 20 minutes, germinate at 28°C for 72 hours, and sow them in the nutrition bowl (vermiculite: Nutrient soil = 3:1), incubated in a light incubator.

L 133 "normal plants and silent plants (the CK (Blank control), TRV2-00, and  TRV2-CaTPS1 plants)": not clear what CK is - are these wild type plants?

Re: Normal plants are "Qiangfeng 101" of pepper material without VIGS treatment and were mainly used to explore the expression pattern of CaTPS1 under different time periods of low temperature and salt stress.

Silenced plants (CK (blank control), pTRV2-00 and pTRV2-CaTPS1 plants)" refers to the treatment material "Qiangfeng 101" used in the VIGS silencing experiment, where CK, pTRV2-00 and pTRV2-CaTPS1 were the non-VIGS plants (CK), Negative control/ empty vector (PTRV2-00) and CaTPS1-silenced plants (pTRV2-CaTPS1) respectively.

L 146 "Take the treated fresh leaves and stick them from the back tissue of the leaves with scotch tape." Specify the method that was meant.

Re: The method is the transparent tape adhesion method, which is an experiment done with reference to this literature, and the reference is " Chen Baihong, Li Xinsheng, Cao Ziyi, Yao Qingrong. A method for observing stomata by adhering transparent tape to the leaf epidermis[J]. Plant Physiology Letters,2004(02):215-218. (In Chinese)".

L 149 "stomata area and stomata perimeter". In reference [43] there is no calculation of the perimeter. I can assume that the authors measured the length and width of the stomata, and then calculated the area and "perimeter" of the stomata using the well-known mathematical formulas of the area and circumference of the ellipse. If this is true, then this should be written in the methodology.

Re: The stomatal properties were determined using the Motic Images advanced image analysis processing system to determine the longitudinal and transverse diameters and perimeters of the stomata, and the area was calculated using the formulae in reference [43].

L 156-158 Decipher POD, CAT, APX at the first mention in the text.

Re: Peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX)has been noted in the main text.

L 136 Specify the method of NaCl treatment - watering the soil, adding to the nutrient medium?

Re: Initially 100mM NaCl was used, then the NaCl concentration in the Hoagland nutrient solution was gradually increased to 150, 200 and 300mM at 12-hour intervals poured into the matrix of the plants to be treated to prevent salt shock from the high NaCl.

L 170 "CaTPS1 expression rising by 50% relative to CK". Specify which plants were used in this experiment and what do you compare them with, what does CK mean here?

Re: The plant material used in this experiment was "Qiangfeng 101", which is indicated in these results. As follow: “To explore the expression pattern of the CaTPS1 gene under abiotic stress, we analyzed the expression levels of this gene in the pepper material "Qiangfeng 101" after treatment with low temperature and NaCl.”

CK is the low temperature treatment for 0 h and has been changed to 0 h to prevent confusion with the experiment below.

"3.3. Stomatal Structure of CaTPS1 Silenced Plants under Cold and Salt Stresses"

What is the point of using 2 indicators (stomata area and perimeter), apparently calculated from the same initial values of stomata length and width?L.279 "which may indicate that the stomata have become smaller after abiotic stress".

Usually, stomata (like most other plant cell types) grow to a certain size and can no longer change size due to the presence of a rigid cell wall. Accordingly, the conclusions of L273-280 are erroneous. Your results may be due to 1) inhibition of stomatal growth in young leaves under stress (unlikely for a period of 3 days, but needs to be checked), 2) an increase in the proportion of closed stomata under stress. Closing the stomata reduces their width and, accordingly, the area, and possibly the perimeter (due to the compression of the side walls; of course, if the real perimeter is measured, not the width). The length of the stomata can be influenced only by the first reason, which can be used for preliminary differentiation of these reasons. Try plotting the same graphs using the length and width of the stomata. Perhaps this will help to find out why cold stress has a stronger effect on the area and weaker on the perimeter. However, most likely, the data on stomata in any case can be considered only as preliminary and should be excluded from the manuscript: For the correct disclosure of the first reason, longer experiments are needed, and the second reason is to take into account the number of open and closed stomata.

Re: Following your advice and reminder, and having reviewed the relevant literature, we have found that this part of the stomatal experiment has not been considered sufficiently, and the conclusions obtained so far do not provide a reliable basis for stomatal changes after stress. This section has therefore been removed from the manuscript.

An almost identical phrase is repeated 3 times:

The expression of CsTPS1, CsTPS9 and CsTPPA were highest under at least one abiotic stress, and it was speculated that these three genes might play a key role in the abiotic stress response [24]. L65-67

Re: The sentence has been changed to “CsTPS1, CsTPS9 and CsTPPA were most highly expressed under at least one abiotic stress, and it is speculated that these three genes may play a key role in the response of tea plants to abiotic stresses”

CsTPS1, CsTPS9 and CsTPPA had the highest expression in response to at least one abiotic stress, and it is speculated that these three genes may be involved in responding to abiotic stresses [24]. L260-261

Re: The sentence has been replaced with “CsTPS1, CsTPS9 and CsTPPA were the most highly expressed in response to at least one abiotic stress and hypothesized that these three genes may be involved in the abiotic stress pathway in tea tree.”

The expression of CsTPS1, CsTPS9 and CsTPPA were the highest in response to at least one abiotic stress, which is consistent with the above results [24]. L271-272

Re: The sentence has been replaced by “There was higher expression of 

CsTPS1, CsTPS9 and CsTPPA in abiotic stress, which is consistent with the above results”

L296-312. Interpretation of data on antioxidant enzymes may not be so unambiguous. These enzymes not only destroy toxic oxygen species (resistance enhancement) but can also be markers of ill health as they are induced by oxidative stress. Antioxidant enzymes in resistant plants may not respond well to exposure because they are not damaged by stress. To clarify the situation, it is necessary to measure the content of reactive oxygen species.

Re: The culture environment and other conditions of CK, pTRV2-00 and pTRV2-CaTPS1 plants were consistent. Since the seedlings after VIGS treatment were cultured in a climate box at 22°C during the day and 18°C at night, the control group and salt stress may have some low temperature stress. The slightly lower temperature of the culture environment resulted in mild stress, resulting in significantly higher antioxidant enzyme activity in the CK group than in the TPS group under normal conditions (similar to that in the low temperature stress group).

Antioxidant enzymes are important substances to scavenge ROS, and the change of their content can indirectly measure the degree of damage caused by ROS to plants. CAT, POD and APX can reduce the production rate of superoxide anion radicals and the content of hydrogen peroxide. As the repair time is only seven days, we assume that the samples made in the experiment have been used up, and the time is too late to make up the ROS-related experimental data. Based on your suggestions, we will study the issue of coercion in the future to ensure the completeness of the experiment. Thank you very much for your valuable comments.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The functional characterization of homologous genes in crops is very important, however, the manuscript is highly descriptive and the impact is lost. The authors describe the potential contribution of this study to agriculture.

An explanation of CaTPS1 is also required. Is this the first report of CaTPS1?

 

The other comments are below.

 

1. The direct evidence that CaTPS1 catalyzes G6P should be required using recombinant enzyme protein.

In addition, a phylogenetic analysis of CaTPS1 would be also required.

 

2. It is unclear whether plant growth will be affected by the silencing of CaTPS1.

 

3. The expression pattern of CaTPS1 under salt stress is quite different between Fig. 1B and Fig. 2B. Is there any problem in the reproductivity of this experiment?

 

4. The authors need to compare the expression level of CaTPS1 under cold and high salt conditions between 24 h and 36 h (in Figure 1 or 2).

 

5. The authors need a discussion about the difference between CK and pTRV2-00(Negative control). For example, Fig. 6C showed that the APX activity is significantly reduced in pTRV2-00 compared to CK.

 

6. Some abbreviations (including Latin plant names) need clarification.

 

 

7. Please add the biosynthetic pathway of trehalose as a new figure or supplementary figure.

Author Response

Dear Editor and reviewer：

Thank you very much for the letter and the comments. We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don’t hesitate to let me know.

 Sincerely,

 Bingqiang Wei

Detailed responses to reviewer’s comments and Editor’s advice.

The functional characterization of homologous genes in crops is very important, however, the manuscript is highly descriptive and the impact is lost. The authors describe the potential contribution of this study to agriculture. An explanation of CaTPS1 is also required. Is this the first report of CaTPS1?

Re: This study explored the function of CaTPS1 gene in pepper cold and salt stress by VIGS, which is beneficial to understand the biological function of CaTPS1 and contribute to the study of the molecular mechanism of abiotic stress in pepper, thus providing new genes for the molecular breeding of pepper low temperature and salt stress.

CaTPS1 (Capana07g002060) was identified in reference for "Identification of the CaTPS gene family in pepper and expression analysis of CaTPS1. Acta Horticulturae Sinica 2016, 43 (8), 1504-1512. (In Chinese)".

A review of relevant reports in the last decade by keyword TPS in Web of Science, PubMeb, Foreing resource Retrieval system，X-MOL, Springer and other platforms revealed that the role of CaTPS1 in pepper under low temperature and salt stress has not been reported.

The other comments are below.

1. The direct evidence that CaTPS1 catalyzes G6P should be required using recombinant enzyme protein. In addition, a phylogenetic analysis of CaTPS1 would be also required.

Re:We reviewed the literature and found that TPS1 catalyzes G6P. Whether CaTPS1 catalyzes G6P remains to be verified. Our laboratory has no experie-nce in making protein at present, and we will do this later.

A phylogenetic analysis of CaTPS1 has been added to the manuscript and it is closely related to potato, tomato and tobacco, and more distantly related to pumpkin.

2. It is unclear whether plant growth will be affected by the silencing of CaTPS1.

Re: Based on our comparison of plant height and stem thickness at 15, 30 and 45 days after infiltration, we found that plant growth was affected by CaTPS1 silencing.

3. The expression pattern of CaTPS1 under salt stress is quite different between Fig. 1B and Fig. 2B. Is there any problem in the reproductivity of this experiment?

Re: The tests in Figures 1B and 2B have been redone and some of the results in Figure 1B were found to be incorrect and have been corrected.

4. The authors need to compare the expression level of CaTPS1 under cold and high salt conditions between 24 h and 36 h (in Figure 1 or 2).

Re: The experiments in Figure 1 have been redone under time-permitting conditions, and in turn it was found that CaTPS1 expression was higher at 36 hours than at 24 hours, regardless of whether the conditions were low or high salt.

5. The authors need a discussion about the difference between CK and pTRV2-00 (Negative control). For example, Fig. 6C showed that the APX activity is significantly reduced in pTRV2-00 compared to CK.

Re: The difference between CK (blank control) and pTRV2-00 (negative control) without injection of TRV2 vectors was mainly to rule out detection errors caused by the viral vector itself. Although some indicators in pTRV2-00 were significantly lower than those in CK, pTRV2-CaTPS1 was also significantly lower than those in pTRV2-00, indicating that TRV2 virus was responsible for these indicators. (Plant height, stem diameter, chlorophyll content, POD, APX enzyme activity) The impact of is greater, but the change of each indicator after CaTPS1 is silenced is not affected.

Some abbreviations (including Latin plant names) need clarification.

Re: Abbreviations not explained in the manuscript have been elaborated, see manuscript markings for details

6. Please add the biosynthetic pathway of trehalose as a new figure or supplementary figure.

Re: A diagram of the biosynthetic pathway of trehalose has been added, as follows:

Dear Editor and reviewer：

Thank you very much for the letter and the comments. We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don’t hesitate to let me know.

 Sincerely,

 Bingqiang Wei

Detailed responses to reviewer’s comments and Editor’s advice.

The functional characterization of homologous genes in crops is very important, however, the manuscript is highly descriptive and the impact is lost. The authors describe the potential contribution of this study to agriculture. An explanation of CaTPS1 is also required. Is this the first report of CaTPS1?

Re: This study explored the function of CaTPS1 gene in pepper cold and salt stress by VIGS, which is beneficial to understand the biological function of CaTPS1 and contribute to the study of the molecular mechanism of abiotic stress in pepper, thus providing new genes for the molecular breeding of pepper low temperature and salt stress.

CaTPS1 (Capana07g002060) was identified in reference for "Identification of the CaTPS gene family in pepper and expression analysis of CaTPS1. Acta Horticulturae Sinica 2016, 43 (8), 1504-1512. (In Chinese)".

A review of relevant reports in the last decade by keyword TPS in Web of Science, PubMeb, Foreing resource Retrieval system，X-MOL, Springer and other platforms revealed that the role of CaTPS1 in pepper under low temperature and salt stress has not been reported.

The other comments are below.

1. The direct evidence that CaTPS1 catalyzes G6P should be required using recombinant enzyme protein. In addition, a phylogenetic analysis of CaTPS1 would be also required.

Re:We reviewed the literature and found that TPS1 catalyzes G6P. Whether CaTPS1 catalyzes G6P remains to be verified. Our laboratory has no experie-nce in making protein at present, and we will do this later.

A phylogenetic analysis of CaTPS1 has been added to the manuscript and it is closely related to potato, tomato and tobacco, and more distantly related to pumpkin.

2. It is unclear whether plant growth will be affected by the silencing of CaTPS1.

Re: Based on our comparison of plant height and stem thickness at 15, 30 and 45 days after infiltration, we found that plant growth was affected by CaTPS1 silencing.

3. The expression pattern of CaTPS1 under salt stress is quite different between Fig. 1B and Fig. 2B. Is there any problem in the reproductivity of this experiment?

Re: The tests in Figures 1B and 2B have been redone and some of the results in Figure 1B were found to be incorrect and have been corrected.

4. The authors need to compare the expression level of CaTPS1 under cold and high salt conditions between 24 h and 36 h (in Figure 1 or 2).

Re: The experiments in Figure 1 have been redone under time-permitting conditions, and in turn it was found that CaTPS1 expression was higher at 36 hours than at 24 hours, regardless of whether the conditions were low or high salt.

5. The authors need a discussion about the difference between CK and pTRV2-00 (Negative control). For example, Fig. 6C showed that the APX activity is significantly reduced in pTRV2-00 compared to CK.

Re: The difference between CK (blank control) and pTRV2-00 (negative control) without injection of TRV2 vectors was mainly to rule out detection errors caused by the viral vector itself. Although some indicators in pTRV2-00 were significantly lower than those in CK, pTRV2-CaTPS1 was also significantly lower than those in pTRV2-00, indicating that TRV2 virus was responsible for these indicators. (Plant height, stem diameter, chlorophyll content, POD, APX enzyme activity) The impact of is greater, but the change of each indicator after CaTPS1 is silenced is not affected.

Some abbreviations (including Latin plant names) need clarification.

Re: Abbreviations not explained in the manuscript have been elaborated, see manuscript markings for details

6. Please add the biosynthetic pathway of trehalose as a new figure or supplementary figure.

Re: A diagram of the biosynthetic pathway of trehalose has been added, as follows:

 Supplementary Figure 1 The main pathway of trehalose metabolism

The ‘TPS-TPP’ pathway of trehalose metabolism involved two key enzymes, trehalose-6-phosphate synthase (TPS) and Trehalose phosphate phosphatase (TPP), and this process can be divided into two steps. Firstly, TPS catalyzes the substrate, uridine diphosphate-glucose (UDP-Glc) and glucose-6-phosphate (Glc-6-p), to synthesize Trehalose-6-phosphate (T6P). Secondly, T6P is dephosphorylated by TPP to form trehalose However, trehalose is eventually hydrolyzed by trehalase into two molecules of glucose[1-5].

References

1. Gancedo, C.; Flores, C. L. The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res 2004, 4 (4-5), 351-9.
2. Avonce, N.; Mendoza-Vargas, A.;  Morett, E.; Iturriaga, G. Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 2006, 6, 109.
3. Vandesteene, L.; Lopez-Galvis, L.;  Vanneste, K.;  Feil, R.;  Maere, S.;  Lammens, W.;  Rolland, F.;  Lunn, J. E.;  Avonce, N.;  Beeckman, T.; Van Dijck, P. Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis. Plant Physiol 2012, 160 (2), 884-96.
4. Paul, M. J.; Oszvald, M.;  Jesus, C.;  Rajulu, C.; Griffiths, C. A. Increasing crop yield and resilience with trehalose 6-phosphate: targeting a feast-famine mechanism in cereals for better source-sink optimization. J Exp Bot 2017, 68 (16), 4455-4462.
5. Pohane, A. A.; Carr, C. R.;  Garhyan, J.;  Swarts, B. M.; Siegrist, M. S. Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope. Mbio 2021, 12 (1).

 

 Supplementary Figure 1 The main pathway of trehalose metabolism

The ‘TPS-TPP’ pathway of trehalose metabolism involved two key enzymes, trehalose-6-phosphate synthase (TPS) and Trehalose phosphate phosphatase (TPP), and this process can be divided into two steps. Firstly, TPS catalyzes the substrate, uridine diphosphate-glucose (UDP-Glc) and glucose-6-phosphate (Glc-6-p), to synthesize Trehalose-6-phosphate (T6P). Secondly, T6P is dephosphorylated by TPP to form trehalose However, trehalose is eventually hydrolyzed by trehalase into two molecules of glucose[1-5].

References

1. Gancedo, C.; Flores, C. L. The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. FEMS Yeast Res 2004, 4 (4-5), 351-9.
2. Avonce, N.; Mendoza-Vargas, A.;  Morett, E.; Iturriaga, G. Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 2006, 6, 109.
3. Vandesteene, L.; Lopez-Galvis, L.;  Vanneste, K.;  Feil, R.;  Maere, S.;  Lammens, W.;  Rolland, F.;  Lunn, J. E.;  Avonce, N.;  Beeckman, T.; Van Dijck, P. Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis. Plant Physiol 2012, 160 (2), 884-96.
4. Paul, M. J.; Oszvald, M.;  Jesus, C.;  Rajulu, C.; Griffiths, C. A. Increasing crop yield and resilience with trehalose 6-phosphate: targeting a feast-famine mechanism in cereals for better source-sink optimization. J Exp Bot 2017, 68 (16), 4455-4462.
5. Pohane, A. A.; Carr, C. R.;  Garhyan, J.;  Swarts, B. M.; Siegrist, M. S. Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope. Mbio 2021, 12 (1).

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

OK

Author Response

Dear Editor and reviewer：

Thank you very much for the letter and the comments. We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don’t hesitate to let me know.

Sincerely,

Bingqiang Wei

Detailed responses to reviewer’s comments and Editor’s advice.

The manuscript has been revised, but there are several concerns that remain.

1. The authors should describe the previous study about CaTPS genes in Capsicum annuum in the introduction section

Re: Previous studies on CaTPS genes in pepper have been described in detail in the introduction section as follows: A total of 11 pepper TPS genes were identified in the whole genome database of pepper, except CaTPS3, which contains 1 TPS and 2 TPP structural domains, while the rest contain 1 TPS and 1 TPP structural domain, respectively. the expression of CaTPS1 in leaves is about 5-6 times higher than that in roots and stems, indicating that the expression of CaTPS1 has obvious tissue specificity.

The only information on CaTPS available from the literature is reference 9, which is "Identification of the CaTPS gene family in pepper and expression analysis of CaTPS1. Acta Horticulturae Sinica 2016 , 43 (8), 1504-1512. (In Chinese)". No other literature was reviewed in.

2. Phylogenetic tree is not effective. If the authors will be not able to show direct evidence that CaTPS1 catalyzes G6P, they should describe the sequence similarity between CaTPS1 and other TPS proteins that had been characterized. It would be better if the phylogenetic tree include TPSs that are described in the introduction section such as AtTPS. The first paragraph of the result section is quite strange. It is never important information that CaTPS1 is distantly related to Pumpkin.

Re: Based on your comments, we describe the sequence similarity between CaTPS1 and other TPS proteins (AtTPS1, OsTPS1, ZmTPS1) that have been characterized. This is modified as follows:

From Figure 1, The length of the TPS structural domain domain of TPS1 in these four species is much larger than that of TPP. The TPS and TPP structural domains of CaTPS1 and AtTPS1, OsTPS1, ZmTPS1 are all relatively conserved, and the sequence similarity is basically greater than or equal to 75%. It can be concluded that CaTPS1 may have the protein function of catalyzing G6pd like AtTPS1, OsTPS1 and ZmTPS1.

Figure 1. Alignment of pepper (CaTPS1), Rice (OsTPS1), Arabidopsis (AtTPS1) and maize (ZmTPS1) protein sequences. The TPS domain is underlined in blue and TPP domain in green. Amino acid residues with 100% similarity in all sequences are shaded in dark blue, while letters with greater than or equal to 75% similarity are shaded in pink and greater than or equal to 50% similarity in blue.

3. On page 6, several texts are unreadable due to overlapping diagrams.

Re: The overlapping charts have been removed and the text is clearer.

4. Please improve the revised manuscript as it is very difficult to read.

Re: It's a lot of changes, and the format of the reworked manuscript is text format. Insert new documents in Word with EndNote, and the numbering starts again from 1, so the order of all references is not appropriate. Therefore, the previous reference was deleted and reinserted. This way, more things are revised, and it's harder to read the revised manuscript. I found that saving as a PDF version, the revised manuscript is clearer and clearer. The PDF version of the revised manuscript has also been uploaded. Please check it. Thank you.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript has been revised, but there are several concerns that remain.

 

1. The authors should describe the previous study about CaTPS genes in Capsicum annuum in the introduction section.

 

2. Phylogenetic tree is not effective. If the authors will be not able to show direct evidence that CaTPS1 catalyzes G6P, they should describe the sequence similarity between CaTPS1 and other TPS proteins that had been characterized. It would be better if the phylogenetic tree include TPSs that are described in the introduction section such as AtTPS.

The first paragraph of the result section is quite strange. It is never important information that CaTPS1 is distantly related to Pumpkin.

 

3. On page 6, several texts are unreadable due to overlapping diagrams.

 

Please improve the revised manuscript as it is very difficult to read.

Author Response

Dear Editor and reviewer：

Thank you very much for the letter and the comments. We have checked the manuscript and revised it according to the comments. We submit here the revised manuscript as well as a list of changes. If you have any question about this paper, please don’t hesitate to let me know.

Sincerely,

Bingqiang Wei

Detailed responses to reviewer’s comments and Editor’s advice.

The manuscript has been revised, but there are several concerns that remain.

1. The authors should describe the previous study about CaTPS genes in Capsicum annuum in the introduction section

Re: Previous studies on CaTPS genes in pepper have been described in detail in the introduction section as follows: A total of 11 pepper TPS genes were identified in the whole genome database of pepper, except CaTPS3, which contains 1 TPS and 2 TPP structural domains, while the rest contain 1 TPS and 1 TPP structural domain, respectively. the expression of CaTPS1 in leaves is about 5-6 times higher than that in roots and stems, indicating that the expression of CaTPS1 has obvious tissue specificity.

The only information on CaTPS available from the literature is reference 9, which is "Identification of the CaTPS gene family in pepper and expression analysis of CaTPS1. Acta Horticulturae Sinica 2016 , 43 (8), 1504-1512. (In Chinese)". No other literature was reviewed in.

2. Phylogenetic tree is not effective. If the authors will be not able to show direct evidence that CaTPS1 catalyzes G6P, they should describe the sequence similarity between CaTPS1 and other TPS proteins that had been characterized. It would be better if the phylogenetic tree include TPSs that are described in the introduction section such as AtTPS. The first paragraph of the result section is quite strange. It is never important information that CaTPS1 is distantly related to Pumpkin.

Re: Based on your comments, we describe the sequence similarity between CaTPS1 and other TPS proteins (AtTPS1, OsTPS1, ZmTPS1) that have been characterized. This is modified as follows:

From Figure 1, The length of the TPS structural domain domain of TPS1 in these four species is much larger than that of TPP. The TPS and TPP structural domains of CaTPS1 and AtTPS1, OsTPS1, ZmTPS1 are all relatively conserved, and the sequence similarity is basically greater than or equal to 75%. It can be concluded that CaTPS1 may have the protein function of catalyzing G6pd like AtTPS1, OsTPS1 and ZmTPS1.

Figure 1. Alignment of pepper (CaTPS1), Rice (OsTPS1), Arabidopsis (AtTPS1) and maize (ZmTPS1) protein sequences. The TPS domain is underlined in blue and TPP domain in green. Amino acid residues with 100% similarity in all sequences are shaded in dark blue, while letters with greater than or equal to 75% similarity are shaded in pink and greater than or equal to 50% similarity in blue.

3. On page 6, several texts are unreadable due to overlapping diagrams.

Re: The overlapping charts have been removed and the text is clearer.

4. Please improve the revised manuscript as it is very difficult to read.

Re: It's a lot of changes, and the format of the reworked manuscript is text format. Insert new documents in Word with EndNote, and the numbering starts again from 1, so the order of all references is not appropriate. Therefore, the previous reference was deleted and reinserted. This way, more things are revised, and it's harder to read the revised manuscript. I found that saving as a PDF version, the revised manuscript is clearer and clearer. The PDF version of the revised manuscript has also been uploaded. Please check it. Thank you.

 

Author Response File: Author Response.pdf