The Sink–Source Relationship Regulated Camellia oleifera Flower Bud Differentiation by Influencing Endogenous Hormones and Photosynthetic Characteristics

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Your comments are very helpful to the further improvement of our manuscript Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We have given our response in the point-by-point response letter

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Can be improved

We have given our response in the point-by-point response letter

Are the methods adequately described?

Must be improved

We have given our response in the point-by-point response letter

Are the results clearly presented?

Can be improved

We have given our response in the point-by-point response letter

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

My general comment is related to experimental design. Authors mainly used correlation analysis for revealing ways of regulation of flower bud differentiation by sink-source relationships. I agree that it is effective method; however, other approaches can be potentially used for additional checking their conclusions. For example, plants can be treated by exogenous hormones. I understand that any investigation is limited; however, these ways of investigation should, at least, be discussed in the work, if it is possible.

Response 1:

Thank you for pointing this out. We agree with this comment. We have supplemented the effects of growth regulators or chemicals on the flower bud differentiation in the Discussion 4.2. This change can be found – page 11 and lines 400-404

Comments 2:

Section 2.3: What stages of growth had plants during these measurements?

Response 2:

Thank you for pointing this out. The plant was before flower bud development when the endogenous hormones were measured.

Comments 3:

P. 4, line 157: “… and each sample was repeated three times.” Were biological (different plants) or chemical (different samples from one plant) repetitions?

Response 3:

Thank you for pointing this out. ‘each sample was repeated three times.’ refers to chemical (different samples from the same shoot or bud on the same shoot) repetitions. However, prior to chemical repetition, we performed six biological repeats, which have been added to the text. This change can be found – page 4 and lines153-156

Comments 4:

Section 2.5: Were single leaves or groups of leaves measured? If single leaves, what leaves were used for measurements? Were similar leaves used in the different experiments (with similar position in the branch)? If group of leaves, how were measurements normalized per area? Were samples attached to plants or not? If they were not attached, it can disrupt photosynthetic parameters. Thus, many experimental details of photosynthetic measurements should be completely described.

Response 4:

Thank you for pointing this out. In this experiment, we used mature leaves at similar position in the shoot, and the leaves were not in vitro. More experiment details have been supplemented in Part 2.5.

Comments 5:

Section 2.5: Additionally, there are absent technical details of measuring procedure for photosynthetic CO2 assimilation measurements. What was actinic light used (spectral characteristics, intensity)? What duration of illumination by the actinic light before the CO2 assimilation measurement? Were CO2 and H2O content and temperature in measuring chamber controlled? What were values of these parameters? These details can strongly influence results of photosynthetic measurements and, therefore, should be clarified.

Response 5:

Thank you for pointing this out. The red and blue light was set to 1500μmol m^-2·s⁻¹. The leaf temperature was set to 25 ℃, and relative humidity 60-70%. The carbon dioxide concentration in the reference room was stable at about 380 μmol mol^-1. The duration of illumination by the actinic light is 30 minutes before the CO₂ assimilation measurement. More technical details of measuring procedure have been supplemented in Part 2.5.

Comments 6:

Figures 2-4: Only one experimental point (plants with same age?) seems to be investigated in these figures. It is not fully clear: what were values used for correlation analysis? Or were correlation coefficients calculated on basis of individual plants (all parameters were simultaneously measured in individual plants)? It should be clarified.

Response 6:

Thank you for pointing this out. Figure 2：flower bud differentiation rate was calculated in the late flower bud differentiation (late June). Figure 3 and Figures 4 have been incorporated into one graph. Endogenous hormone content was calculated at the preflower bud differentiation stage (mid-April). The correlation between flower bud differentiation rate of twelve new shoots (each type contains six new shoots) randomly selected and the endogenous hormones comment of leaves or buds of twelve new shoots (each type contains six new shoots) was analyzed. The correlation between flower bud differentiation rate of twelve new shoots (each type contains six new shoots) randomly selected and the Soluble sugar (or starch) of leaves or flower buds of twelve new shoots (each type contains six new shoots) was analyzed. It has been clarified in section 2.7.

Comments 7:

Additionally, other parameters seem to be investigated on three stages of development (see, e.g., Tables 2 and 3, Figure 5). Why hormone content was not investigated on these three stages. It should be clarified.

Response 7:

Thank you for pointing this out. Endogenous hormones control the process of flower bud differentiation and the ratio of flower bud differentiation, particularly the change of hormone level prior to flower bud differentiation. As a result, hormones are frequently sprayed before flower bud differentiation in orchards to manage the ratio of flower buds and leaf buds. The distribution features of different types of sink and source, as well as their impact on fruit development and quality, can be reflected in changes in soluble sugar and starch in different organs at different developmental stages. At different stages of development, the ability of the source to accumulate carbon and convey it to the sink will change. The ability photosynthetic carbon assimilation of different types of new shoots at different developmental stages is reflected in the net photosynthetic rate and chlorophyll concentration. In summary, it is necessary to carry out research on the changes of soluble sugar, starch, net photosynthetic rate and chlorophyll content in different types of shoots at different developmental stages.

Comments 8:

Table 4: Why correlation coefficients between flower bud differentiation rate and soluble sugar/starch were high on the preflower bud differentiation stage, only?

Response 8:

Thank you for pointing this out. Nutrients accumulated before flowering directly affect the quantity and quality of flowers. For example, the soluble sugar content in the terminal buds and adjacent leaves of apples changed significantly at preflower bud differentiation stage; in the process of mango flowering, the starch content of 'Nanduomei' with high flowering rate was higher than that of 'Yexiang' with low flowering rate at preflower bud differentiation stage (Xing L B, et al; Gao T Y, et al).

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Your comments are very helpful to the further improvement of our manuscript Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

We have given our response in the point-by-point response letter

Are all the cited references relevant to the research?

Must be improved

We have given our response in the point-by-point response letter

Is the research design appropriate?

Must be improved

We have given our response in the point-by-point response letter

Are the methods adequately described?

Must be improved

We have given our response in the point-by-point response letter

Are the results clearly presented?

Must be improved

We have given our response in the point-by-point response letter

Are the conclusions supported by the results?

Must be improved

We have given our response in the point-by-point response letter

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

the part M&M, which in many sections poorly describing the procedure for sampling and analyzing

Response 1:

Thank you for pointing this out. We agree with this comment. In the part M&M，more details have been supplemented. For example, the collection method of test materials is described in detail in Section 2.3, and the setting of experimental instrument parameters was added in Section 2.5.

Comments 2:

insufficient captions are not helpful for understanding

Response 2:

Thank you for pointing this out. The supplementary explanations of the tables and pictures in the text have been supplemented. This change can be found – Figure 1-5, and Table 1-3.

Comments 3:

The quality of the Figs and Tables are below standard. (e.g., Fig. 3 and 4 should be combined).

Response 3:

Thank you for pointing this out. The quality of the Figs and Tables has been improved. Fig. 3 and 4 have been integrated together. This change can be found – Figure 3-4, and Table 2.

Comments 4:

The correlation analysis and the direct comparing of different plant hormones, as IAA, zeatin, abscisic acid, and GA3 is really questionable.

Response 4:

Thank you for pointing this out. The correlation between flower bud differentiation rate of twelve new shoots (each type contains six new shoots) randomly selected and the endogenous hormones comment of leaves or buds of twelve new shoots (each type contains six new shoots) was analyzed. The direct comparing of different plant hormones refers to the comparison of two types of new shoots.

Comments 5:

Using isotopic dilution method was done, for 13C accumulation and allocation, but the right interpretation of these interesting results, except for “increasing” or “decreasing”, could not found in the manuscript.

Response 5:

Thank you for pointing this out. In section 3.5, we added some interesting findings. – page 9 and lines 332-339.

Comments 6:

Section 2.3 ‘Two new shoots of T1 and T2 were selected at random in each direction’, also according to the level, lower, middle and upper region of a tree?

Response 6:

Thank you for pointing this out. Two new shoots of T1 and T2 were randomly selected in each direction, and were not accorded to the level, lower, middle and upper region of a tree.

Comments 7:

Section 2.3 ‘The contents of endogenous hormones were determined using the high-performance liquid chromatography (HPLC) method with some modifications (28)’, This reference is not available in English, except the Abstract; method description is obligatory!

Response 7:

Thank you for pointing this out. We agree with this comment. The method description of HPLC has been supplemented in the manuscript. This change can be found – page 4 and lines 161-181.

Comments 8:

Section 2.3 ‘zeatin (ZT)’ cis or trans-?

Response 8:

Thank you for pointing this out. zeatin refers to trans-zeatin. This change can be found – page 4 and line 160.

Comments 9:

Section 2.4. ‘Determination of nutrients’ carbohydrates / saccharides

Response 9:

Thank you for pointing this out. The title of section 2.4 was modified as ‘Determination of carbohydrates’

Comments 10:

Section 2.5 the net photosynthetic rate and chlorophyll content must be improved

Response 10:

Thank you for pointing this out. More experimental details of the net photosynthetic rate and chlorophyll content have been supplemented in Part 2.5.

Comments 11:

Section 2.6 and Section 3.5: ‘13C’ was not superscripted

Response 11:

Thank you for pointing this out. 13 in ‘13C’ has been superscripted.

Comments 12:

Section 2.6: ‘killed’ scientific writing style

Response 12:

Thank you for pointing this out. ‘All samples were killed at 105 °C for 15 min’ changed to ‘All samples were deactivated at 105 °C for 15 min’. This change can be found – page 5 and line 218

Comments 13:

Section 2.6 ‘13C isotope determination, including the δ value of 13C and the percentage of total carbon.’ Include standard for 13C and calculations for 13C

Response 13:

Thank you for pointing this out. We have included standard for 13C and calculations for 13C as follows:

The natural abundance of 13C in unlabeled samples is represented by δ 13C. The abundance of artificial 13C labeled samples is represented by Atom%. The amount of carbon in each organ of the plant is represented by Ci. The amount of 13C in each organ of the plant is represented by 13Ci. Analysis of 13CO2 was performed using the method de-scribed by Wen (16).

δ 13C (‰) = (Rs / R-1), Rs = 13C/12C unlabeled samples; R is the standard ratio of carbon isotope, R=0.1112372.

Atom%13C= (δ 13C + 1000) × R / [(δ 13C + 1000) × R + 1000] ×100.

Ci (g) = Dry matter weight (g) × C%.

13Ci (mg) = Ci × [(Atom%13C) labeled abundance - (Atom%13C) unlabeled abundance] ×1000.

This change can be found – page 6 and lines 225-235.

Comments 14:

Section 3.1: need add the ‘at late Flower bud differentiation’ after ‘Flower bud differentiation rate of new shoots in different sink source relationships’

Response 14:

Thank you for pointing this out. ‘Flower bud differentiation rate of new shoots in different sink source relationships’ changed to ‘Flower bud differentiation rate of new shoots in different sink source relationships at late Flower bud differentiation’ at Section 3.1.

Comments 15:

Section 3.1: control, not meaningful。

Response 15:

Thank you for pointing this out. The sink of T1 included buds and one fruit, while the sink of T2 only included buds. Photosynthetic assimilates produced by leaves (the main source) were distributed to buds and fruits in T1, but only to buds in T2, so the flower bud differentiation rate of T2 was higher than that of T1. In the orchard, if there are more fruits in the current year, it is necessary to properly thin the fruits, so that the assimilates are more distributed to the buds, forming more flower buds, and ensuring the increase of fruit yield in the next year. In summary, studying the flower bud differentiation rate of the above two new shoots can guide the problem of ' flower thinning or fruit thinning ' in production, which has positive significance for production.

Comments 16:

Figure 2: Caption must be improved. According to Fig. 1, T1:1/ (1+4) x100=20%

Response 16:

Thank you for pointing this out. we have improved the caption. Fig.1 shows that different types of new shoots with one fruit (T1) and new shoots without fruit (T2). The flower bud differentiation rate refers to the ratio of flower buds and leaf buds germinated from same new shoots with one fruit or new shoots without fruit.

Comments 17:

Section 3.2: deleted the ‘in different sink-source relationship’.

Response 17:

Thank you for pointing this out. We have deleted the ‘in different sink-source relationships’ at Section 3.2.

Comments 18:

Section 3.2: This is clearly NOT meaningful, to combine metabolites of such different pathways. Sure, in a wider context these plant hormones are members of a hole network...

Response 18:

Thank you for pointing this out. We measured the content of four hormones, for comparison between T1 and T2 types of new shoots, and not combine metabolites of such different pathways. The results of flower bud differentiation rate of new shoots with different types of source-sink relationship showed that the flower bud differentiation rate of T2 was significantly higher than that of T1. The hormone content at the preflower bud differentiation stage was measured to study which hormones were involved in flower bud differentiation, or which hormones could cause the difference of flower bud differentiation rate between the two types of new shoots? The results (Figure 3) showed that the contents of GA3, ABA and ZT were significantly different in both types of new shoots, indicating that they were all involved in flower bud differentiation. Moreover, the content of GA3 and ZT in T2 was significantly higher than that in T1, indicated that they were the key factors to improve the flower bud differentiation rate. In the orchard, GA3 and ZT can be appropriately sprayed to increase the rate of flower bud differentiation. It is of positive significance to production.

Comments 19:

Figure 4: ‘Endogenous hormone content in leaves of different types of new shoots’ four leaves？

Response 19:

Thank you for pointing this out. Four leaves as a biological repetition on each new shoot were mixed and sampled to determine the content of Endogenous hormone. Details have been in the section 2.3.

Comments 20:

Table 2 and Table3: Soluble sugar and starch content refers to the dry weight conditions?

Response 20:

Thank you for pointing this out. Soluble sugar and starch content refers to the soluble sugar and starch content of samples under the condition of dry weight. This change can be found – page 8.

Comments 21:

Figure 6 ‘Accumulation and allocation of 13C assimilates (mg·g−1) of different types of new shoots in different sink-source relationships at different stages. (A) Shoot. (B) Leaf. (C) Flower bud. (D) Fruit.’ is not meaningful.

Response 21:

Thank you for pointing this out. Camellia oleifera is a flower and fruit contemporaneous plant. Nutrient competition between fruit and flower bud is crucial for the yield of the current year and next year. By studying the accumulation and distribution of carbon assimilates of different sink-source types in various organs during fruit development, it can provide a theoretical basis for flower and fruit thinning and achieve stable yield on orchard.

4. Response to Comments on the Quality of English Language

Point 1: Moderate editing of English language required.

Response 1:

Native English speakers have modified the manuscript.

1. Summary

Thank you very much for taking the time to review this manuscript. Your comments are very helpful to the further improvement of our manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Does the introduction provide sufficient background and include all relevant references?

Are all the cited references relevant to the research?

Is the research design appropriate?

Are the methods adequately described?

Are the results clearly presented?

Are the conclusions supported by the results?

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

What’s about “flower buds”, for both T1 and T2; see Fig. 5 C!

Response 1:

Thank you for pointing this out. We are very sorry for not describing clearly that stage Ⅰ, Ⅱ and Ⅲ measured the accumulation of 13C assimilates in buds, because it could not distinguish flower buds. However, the accumulation of 13C assimilates in flower buds was measured in stage Ⅳ.

Comments 2:

Line 237 ‘date’ mean, standard deviation (sd), standard error - see Table 2

Response 2:

Thank you for pointing this out. We have shown that the date refers to the mean ±standard error (SE) in all figures or tables.

Comments 3:

Section 2.7 ‘t test’ should be adapted for Figs and Tables. Line 241 ‘comment’ changed ‘content’

Response 3:

Thank you for pointing this out. We agree with this comment.’ ‘t test’ has been consistent in the manuscription. We have replaced ‘comment’ with ‘content’.

Comments 4:

Figure 2, ‘T-test’ or ‘t-test’ should be used (control needed for Figs and Tables)

Response 4:

Thank you for pointing this out. ‘t-test’ have been used.

Comments 5:

Table 3 ‘p’ italic?

Response 5:

Thank you for pointing this out. The format of ‘p’ has been changed to italic.

Comments 6:

Line 308 ‘The same applies below.’?

Response 6:

Thank you for pointing this out. ‘The same applies below.’ have been deleted.

Comments 7:

Line 321 ‘the differences’ between T1 and T2?

Response 7:

Thank you for pointing this out. We have added ‘the differences between T1 and T2’ in the manuscript.

Comments 8:

Lines 331-332, ‘In general, the distribution of assimilates in leaves, buds, shoot and fruit at different types of new shoots in different sink-source relationships.’ Sentence not complete

Response 8:

Thank you for pointing this out. We have supplemented the sentences ‘In general, the distribution of assimilates in leaves, buds, shoot and fruit was different at different types of new shoots in different sink-source relationships’.

Comments 9:

Lines 353-360, This sentence needs to be rewritten before acceptance and publication!

Response 9:

Thank you for pointing this out. We have rewritten the sentence of lines 353-360.

Comments 10:

see line 225, unit dry weight

Response 10:

Thank you for pointing this out. We have modified the unit of the Figure 5.

Comments 11:

Lines 372-376 Very long sentence; should be revised.

Response 11:

Thank you for pointing this out. We have shortened the sentences.

Comments 12:

Lines 395-398 This hypothesis is/can be supported by the lower 13C-content in T1 leaves, which always markedly lower (Fig. 5B) than the 13C-content in T2 leaves. The reason is the stronger dilution by unlabeled 12C, by higher net photosynthesis and chlorophyll content, shown in Fig. 4.

Response 12:

Thank you for pointing this out. We have rewritten the sentence of lines 395-398.

Comments 13:

Line 376 ‘speculate’ change ‘assume’

Response 13:

Thank you for pointing this out. We have replaced ‘speculate’ with ‘assume’

Comments 14:

line 423-427 should be reconsidered and improved.

Response 14:

Thank you for pointing this out. Line 423-427 have been reconsidered and improved.

4. Response to Comments on the Quality of English Language

Point 1: Some corrections needed.

Response 1:

We have made some corrections on the Quality of Quality of English Language.