Changes in the Differentiation Program of Birch Cambial Derivatives following Trunk Girdling

Round 1

Reviewer 1 Report

The title of the article is Changes in the Differentiation Programme of Birch Cambial Derivatives under Conditions of Increased Photoassimilate Level. This paper explored the changes in the differentiation of birch derivatives with increasing levels of photoassimilation. The overall writing of the article is complete and systematic, novel, and the overall discussion is sufficient. The presentation of the graphs and tables is complete and conditioning. The article overall is of good quality, but there are still some areas for improvement.

 

 

Minor revisions:

1. Line 60 suggest that in the literature review link, macro references can be used to introduce the impact on the overall social ecology in the context of increased photoassimilation levels, rather than just some plants.

2. Lines 78-79. There is very little introduction to the selection of specific regions and tree species, and the important research significance of the selection of this region and tree species should be explained.

3. Lines 83-86 introduce the previous conclusions, not the last paragraph of the INTRODUCTION link. The last paragraph focuses on the experimental scheme and significance of this paper.

4. Lines 91-99. Selection of birch species, 32-20-12, followed by sampling, girdling and controled. For the selection of 20 and 12, whether it is a random choice or there is a certain basis, it needs to be explained.

5. Lines 100-108. For the application of proper nouns, the explanation of proper vocabulary such as AG1 and DAG should be supplemented, so that scholars in different fields can get a better understanding.

6. The conclusion link can be discussed in points to present more conditioning.

Author Response

Reviewer 1

 

Comments and Suggestions for Authors

The title of the article is Changes in the Differentiation Programme of Birch Cambial Derivatives under Conditions of Increased Photoassimilate Level. This paper explored the changes in the differentiation of birch derivatives with increasing levels of photoassimilation. The overall writing of the article is complete and systematic, novel, and the overall discussion is sufficient. The presentation of the graphs and tables is complete and conditioning. The article overall is of good quality, but there are still some areas for improvement.

We are grateful to the respected reviewer for his interest and appreciation of our work. Thanks for highlighting areas of improvement.

Minor revisions:

1. Line 60 suggest that in the literature review link, macro references can be used to introduce the impact on the overall social ecology in the context of increased photoassimilation levels, rather than just some plants.

We added to the text: «See the reviews [13,15–17] and references there in»

2. Lines 78-79. There is very little introduction to the selection of specific regions and tree species, and the important research significance of the selection of this region and tree species should be explained.

We changed in the text: «The research was carried out in the territory of the Republic of Karelia, northwestern Russia. The study of cambium responses to changes in available assimilate levels is relevant to this region, because the variable climatic conditions during the growing season may lead here to the phloem transport restriction in the trees on the ongoing photosynthesis background [39]. We conducted an experiment on the trunk girdling of Betula pendula Roth, one of the economically and ecologically important tree species in Northern Europe. The selection of this species is also explained by the following reasons: (1) the methods for determining enzyme activity have been previously adapted for silver birch [40–43]; (2) the silver birch genome was sequenced in 2017 [44]. We study changes that occurred in xylem and phloem structure, activity of sucrose-degrading enzymes and antioxidant system (AOS) enzymes, as well as determine the expression of genes involved in metabolism and transport of sugars and auxin. It is known that the zone immediately above the girdle is where sugars are accumulated the most [24]. Prolonged disturbance of phloem transport leads to a further increase of sugars level above the trunk and in the crown of girdled trees [36,45]. Therefore, samples were taken at two heights above the girdle. Comparative analysis of tissues at different distances from the girdle at different time points after girdling allowed us to study the mechanisms of excess sugars utilization in the birch cambial zone at different levels of photoassimilate excess».

3. Lines 83-86 introduce the previous conclusions, not the last paragraph of the INTRODUCTION link. The last paragraph focuses on the experimental scheme and significance of this paper.

We changed. See the above paragraph.

4. Lines 91-99. Selection of birch species, 32-20-12, followed by sampling, girdling and controled. For the selection of 20 and 12, whether it is a random choice or there is a certain basis, it needs to be explained.

We added to the text: «For each sampling, we selected 8 closely growing trees so that they were in the most similar conditions. At the same time, it was decided to include more trees within the experimental group to account for individual reactions».

5. Lines 100-108. For the application of proper nouns, the explanation of proper vocabulary such as AG1 and DAG should be supplemented, so that scholars in different fields can get a better understanding.

We added abbreviations the first time they appear in each of the three sections: the abstract; the main text; the first figure or table as per the journal's rules.

6. The conclusion link can be discussed in points to present more conditioning.

We have rewritten the Conclusion section to present the results as bullets:

The main results of our experiment can be summarised:

(1) the compromise between xylogenesis and phloemogenesis as described in the literature can be controlled by the level of incoming photoassimilates;

(2) an increase in the level of photoassimilates causes an increase in the proportion of parenchyma in the composition of the conducting tissues. The potential mechanisms involved in switching the cell differentiation program can be the active supply of apoplast sugars and the inactivation of auxin;

(3) the formation of thick sclereid cell walls may be one of the mechanisms involved in sugar level regulating in the cambial zone.

Reviewer 2 Report

The authors have evaluated anatomical changes in the phloem and xylem of girdled silver birch trees.  Additionally, they measure activities of selected enzymes predicted to be relevant to tissue- and girdling-specific responses.  Finally, they measure expression of selected genes also predicted to be relevant to tissue- and girdling-specific responses.  Overall, the girdling treatment led to suppression of xylem development and stimulation of phloem development.  Although there are other girdling studies represented in the literature, it appears that this study stands out for its careful attention to and detailed description of changes in the phloem.

 

Strengths

The micrographs documenting girdling-induced changes are of very high quality.  The authors have provided quantitative data regarding changes in vascular cell types and they have done a good job of presenting these data in a manner that is informative and easy to understand. The reported activities of enzymes and genes chosen for measurements enrich the understanding of the anatomical observations, especially when considered in the context of existing knowledge of how changes in sucrose concentration affect xylem and phloem development.

 

Questions and issues for authors’ response

1.    It’s not clear to this reviewer how the sampling was conducted. Were the same trees sampled multiple times, e.g., at different times (10, 20, 30 DAG)?  Please elaborate on this point in the methods section.

2.    At line 265, the authors state, “All data in the diagrams appear as M ± SD.”  Yet there are many data sets here that do not show error bars.  Please clarify.

3.    Related to number 2, in some cases the authors make reference to significant differences, e.g., line 336, “Girdling significantly affected the activity of sucrose metabolising enzymes in stem tissues”  or line 382, “Significant differences between control and girdled trees were detected in the expression level of the gene encoding the protein inhibitor of CWInv activity (CIF).” where no statistical analysis is presented.  Such data should be analyzed for significance or the term “significant” should be omitted from the text.

4.    The authors should present sequences in the supplemental section that confirm the identity of the PCR products used for quantitative RT-PCR.  Sizing of bands is not sufficient.

5.    At line 457, the authors write, “As a result, in both levels of girdled trees, the cambium actively produces phloem cells which differentiate into sieve tubes”.  This statement is misleading given that while some sieve tube development did occur in girdled trees, it was reduced compared to that in control trees (Figure 5a).

6.    At line 476, the authors write, “In our study, the level of sugars in 10DAG in the phloem apoplast in AG1 level appeared to be very high”.  This gives the impression that the authors measured sugars for this work, which they did not. Please rewrite.

7.    A minor point: in a few cases the authors use “got”, e.g., line 30, “The results got are important for understanding…”.  Please refrain from using “got” in this way.  Use “obtained” instead, for example.

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

The authors have evaluated anatomical changes in the phloem and xylem of girdled silver birch trees.  Additionally, they measure activities of selected enzymes predicted to be relevant to tissue- and girdling-specific responses.  Finally, they measure expression of selected genes also predicted to be relevant to tissue- and girdling-specific responses.  Overall, the girdling treatment led to suppression of xylem development and stimulation of phloem development.  Although there are other girdling studies represented in the literature, it appears that this study stands out for its careful attention to and detailed description of changes in the phloem.

Strengths

The micrographs documenting girdling-induced changes are of very high quality.  The authors have provided quantitative data regarding changes in vascular cell types and they have done a good job of presenting these data in a manner that is informative and easy to understand. The reported activities of enzymes and genes chosen for measurements enrich the understanding of the anatomical observations, especially when considered in the context of existing knowledge of how changes in sucrose concentration affect xylem and phloem development.

We are grateful to the respected reviewer for his interest and appreciation of our work. Thanks for highlighting areas of improvement.

Questions and issues for authors’ response

1. It’s not clear to this reviewer how the sampling was conducted. Were the same trees sampled multiple times, e.g., at different times (10, 20, 30 DAG)?  Please elaborate on this point in the methods section.

We added to the text: «At each date, samples were taken from eight other trees, as during the sampling process, the trees were subjected to serious injuries».

2. At line 265, the authors state, “All data in the diagrams appear as M ± SD.”  Yet there are many data sets here that do not show error bars.  Please clarify.

We added to the text: «In Figures 3, 5c,d, Supplementary Figure S1, the data in the diagrams are presented as M ± SD». The lines 130-133 explain the absence of error bars in other cases.

3. Related to number 2, in some cases the authors make reference to significant differences, e.g., line 336, “Girdling significantly affected the activity of sucrose metabolising enzymes in stem tissues”  or line 382, “Significant differences between control and girdled trees were detected in the expression level of the gene encoding the protein inhibitor of CWInv activity (CIF).” where no statistical analysis is presented.  Such data should be analyzed for significance or the term “significant” should be omitted from the text.

We have excluded that.

4. The authors should present sequences in the supplemental section that confirm the identity of the PCR products used for quantitative RT-PCR.  Sizing of bands is not sufficient.

We thank you for your comment and we will try to take it into account in future investigations. PCR products sequencing for the genes for which data are presented in this article has not been performed. We considered that determination the specificity of PCR products from melting curves and acrylamide gel electrophoresis data can be enough, since the B. pendula genome has been completely sequenced out to date, and primers for RT-PCR were designed based on known sequences. While doing the investigation we always try to follow the MIQE guidelines described in Johnson et al., 2013, therefore we provide a primer sequence so each researcher can repeat the experiment.

5. At line 457, the authors write, “As a result, in both levels of girdled trees, the cambium actively produces phloem cells which differentiate into sieve tubes”.  This statement is misleading given that while some sieve tube development did occur in girdled trees, it was reduced compared to that in control trees (Figure 5a).

We changed the sentence: «As a result, in both levels of girdled trees, the cambium actively produces phloem cells».

6. At line 476, the authors write, “In our study, the level of sugars in 10DAG in the phloem apoplast in AG1 level appeared to be very high”.  This gives the impression that the authors measured sugars for this work, which they did not. Please rewrite.

We have changed the wording: «In our study, the level of sugars 10DAG in the phloem apoplast in AG1 level expected to be very high». 

7. A minor point: in a few cases the authors use “got”, e.g., line 30, “The results got are important for understanding…”.  Please refrain from using “got” in this way.  Use “obtained” instead, for example.

We have changed that.

Reviewer 3 Report

The authors Serkova et al. have analyzed the potential mechanisms that regulate the tree trunk radial growth at different levels (from genes to tissue level). Results indicate that increased sucrose concentration in the cambial zone causes activation of phloemogenesis. The authors further found that high levels of sugars stimulate differentiation of cambial derivatives into parenchyma cells. This observation is novel and relevant to advance scientific understanding of how cambium responses to the photoassimilate distribution changes. The results are specifically relevant to estimation of tree productivity and survival under changing environmental conditions. The paper is well written, the conclusions are generally supported by the results and the figures are of high quality.

 

Author Response

We are grateful to the respected reviewer for the high appreciation of our work.

Reviewer 4 Report

I suggest some changes

Introduction:

Several hypotheses of what is expected to be found in response to increased sucrose could be
incorporated for all levels of analysis (anatomical an biochemical analysis levels).
In this way, the organization of the discussion would be better understood.

Results:

In Fig 1 and 2 identify with arrows the different cell types of xylem and phloem
(vessels, fibrotracheids, parenchyma, sieve tubes, sclereids).

Fig. 3. Make the graphs with the same scale (in um)
Fig 3, in panel b the statistical significance of the of xylem control is wrong. According to
the supplementary data, all trees indicate significant differences between AG1 and AG35. Why was tree C2 chosen for display? Trees C1 and C3 give inverse results between AG1 and AG3.
This should at least be mentioned in the text….

Discussion:

The integration of the different results and scales of analysis is very well achieved

 

 

Author Response

Reviewer 4

Comments and Suggestions for Authors

I suggest some changes

Introduction:
Several hypotheses of what is expected to be found in response to increased sucrose could be
incorporated for all levels of analysis (anatomical an biochemical analysis levels).
In this way, the organization of the discussion would be better understood.

 

We added to the text: «Based on previous investigations on Karelian birch (a form of silver birch with an abnormal structure of conductive tissues) [46,47], we formulated the following working hypothesis: an increase in available sugars level in the cambial zone of silver birch with non-figured wood will lead to an intensive entry of hexoses into cells, which in turn stimulates the differentiation of cambial derivatives into parenchyma cells».

Results:
In Fig 1 and 2 identify with arrows the different cell types of xylem and phloem
(vessels, fibrotracheids, parenchyma, sieve tubes, sclereids).

 

We added the signs for different cell types in Figures 1 and 2. The letters in the figures are: vs - vessels, ft - fibrous tracheids, rp - ray parenchyma, ap - axial parenchyma, st - sieve tubes, sc - sclereids.

Fig.3. Make the graphs with the same scale (in um)

Fig 3, in panel b the statistical significance of the of xylem control is wrong. According to
the supplementary data, all trees indicate significant differences between AG1 and AG35. Why was tree C2 chosen for display? Trees C1 and C3 give inverse results between AG1 and AG3.
This should at least be mentioned in the text….

 

We have made graphs in the same scale.

We greatly regret the error made in the differences between the trees. We corrected: "In control trees, the width of phloem increments in levels AG1 and AG35 did not differ significantly. There were small but statistically significant differences in xylem increments at AG1 and AG35 levels.”

 

Tree C2 was randomly selected.

Discussion:

The integration of the different results and scales of analysis is very well achieved

We are grateful to the respected reviewer for his interest and appreciation of our work. Thanks for highlighting areas of improvement.