Changes in Root Anatomy of Peanut (Arachis hypogaea L.) under Different Durations of Early Season Drought

Round  1

Reviewer 1 Report

The authors studied the root anatomy of the peanut plant and measured several traits including vessel number, vessel diameter and vessel area in the first and second-order lateral roots. There are several concerns regarding the methodology, presented data and conclusion of this manuscript. It is not clear that in each replication how many plants were analyzed? Moreover, in most cases, the differences between treatments for the measured traits are not significant. The data derived from this experiment have high variation and probably high coefficient of variation (C.V) indicating the inaccuracy of the experiment. Furthermore, authors only selected a drought-tolerant variety and as a result, they could not detect any significant difference after drought exposure. Overall, the data are poorly presented and interpreted. In several cases the data are not in agreement with the presented graphs and the conclusion of this study is not supported by the presented data.

Major points:

How many plants were analyzed in each replication for each treatment?

Page 9 lines 12-15: This sentence is only correct for data in 2013. According to figure 6 right panel (2014) vessel area only in smaller vessels increased and reduction in bigger vessels is not significant.

Page 10 line 1-2: “Diameter of smaller xylem vessels had smaller size under drought stress compared to under non-stress treatment”. This sentence is not matched with the respective graph. I failed to see any reduction in the size of the smaller xylem vessels after drought treatment.

What is the purpose of showing figures 1 and 2? What kind of information is prepared for readers regarding these figures?

Figure 5 is not explained. Although there is no significant difference between treatments, authors need to state the obtained results.

Page 9 lines 1-2: It is not clear what the author’s point is? Please re-write this sentence.

The manuscript needs to be corrected by a native English proofreader to increase the readability.

Page 16 lines 4-7: The presented data do not support this sentence.

Minor points:

Please be consistent for the scientific name. For instance, see chili on page 2 lines 24 and 26.

Page 4 line 23: Please use subscript for (Ca(H2PO4)2H2O).

The authors need to re-check the manuscript text since there are several typos. To give just an example see Page 7 line 24: Fig.2, Fig 3.

Author Response

Subject: Submission of Agronomy-472193

Dear Editor

We appreciate the valuable suggestions on further improving the manuscript of “Changes in root anatomy of peanut under different durations of early season drought” in the light of reviewer’s comments. We agree with comments and suggestions, thus the manuscript has been revised as editor’s and reviewer’s suggestions. The details of the revision are given below.

            - Thank you for your kind information.

            - The reviewer’s comments were highly insightful and enabled us to greatly improve the quality of our manuscript. In the follow pages are our point by point response to each of the comments.

Comments and Suggestions for Authors (Reviewer 1)

The authors studied the root anatomy of the peanut plant and measured several traits including vessel number, vessel diameter and vessel area in the first and second-order lateral roots. There are several concerns regarding the methodology, presented data and conclusion of this manuscript. It is not clear that in each replication how many plants were analyzed? Moreover, in most cases, the differences between treatments for the measured traits are not significant. The data derived from this experiment have high variation and probably high coefficient of variation (C.V) indicating the inaccuracy of the experiment. Furthermore, authors only selected a drought-tolerant variety and as a result, they could not detect any significant difference after drought exposure. Overall, the data are poorly presented and interpreted. In several cases the data are not in agreement with the presented graphs and the conclusion of this study is not supported by the presented data.

Base on the previous reports, most of information based on structural analyses of primary roots in dicotyledons, and of both primary seminal and adventitious roots in monocotyledons. The best of our knowledge, only for a few cereals, lateral root structures have been analyzed in detail. The structure of lateral roots is very little known, their different types and categories, or their different structure-related functions. For dicotyledons, available information on the drought with different order lateral roots is scarce. The knowledge about the peanut different lateral root orders based on developmental changes only [14].

We were also focus on the first order 

Author Response File: Author Response.pdf

Reviewer 2 Report

The reviewed work concerns the interesting and important topic of plant resistance to drought and one of the many aspects / mechanisms of this resistance, which is the adjustment of the root structure to stress conditions.In the title (and not only in keywords), the Latin name peanut (Arachis hypogaea) should be given.In the introduction (it is only in the discussion) there is no reference to the most important elements of these changes, such as ingrowths and phi-thickenings. The authors should especially refer to the knowledge about the occurrence of these structures in the roots of plants of the Fabaceae family, to which Arachis hypogaea belongs.It is worth to refer and cite chapter 2 „Role of Phi Cells Under Abiotic Stress in Plants” z książki Root Engineering, Soil Biology 40. Morte and A. Varma (eds.), DOI 10.1007/978-3-642-54276-3_2, ©Springer-Verlag Berlin Heidelberg 2014 by Fernandez-Garcia, Lopez-Berenguer and Olmos.Tested for first order roots and second order roots, vessel parameters: number, diameter, area, and microscopic observations of the structures present in cross sections can provide valuable information on plant adaptation to stress.However, this requires a very transparent clear overview and comparison of the results obtained with each other.In addition, the structures visible in the pictures should be accurately, exactly visualized and precisely described.In connection with this, the authors must make major changes to the presentation, description and interpretation of the results.In the pictures there are numerous and regularly placed- arranged in one - two layers cortical cells - the opposite of the intercellular spaces localized adjacent to the endodermis - designated as the peri-endodermal layer. Only single ingrowths are visible in other places - this should be noted and interpreted.These structures can indeed fulfill other than mechanical functions.On the other hand, so-called phi-thickenings, which can occur in hypodermal or innercortical layers, are not actually visible in the presented pictures. They could be seen at much higher magnification and using an electron microscopeBoth the photos-microscopic images and the Figures with the vessels parameters should be presented in such a way that you can compare the first and second order roots.The main reservations refer to the irrelevance of results, the lack of the letter designations for statistical significance, incorrect and incomplete signatures and the lack of explanations regarding the method of staining root preparations.The legibility of all signatures must be improved, starting with Fig.1b,Figures 2 and 3 and Figures 7 and 8 - it is necessary to explain in the title of the graphs how how the cells were dyed on red and why staining has occurred in these places. The captions on the remaining charts are too small, unreadable and otherwise not properly placed.On the Y axis, it has to be numbers [Fig. 4 & Fig. 9], diameters (μm) [Fig. 5 & Fig. 10], area (μm2) [Fig. 6 & Fig. 11], in the header: normal vessels [a1, b1], biger vessels (size> ...) [a2, b2], smaller vessels (size <...) [a3, b3], the words first order (not 1 order ) order LR and second (not 2 order) order LR should be placed once over six charts and inscriptions 2013 and 2014 on three relevant charts. First order and second order graphs must be compiled in one panel - next to each other or one after the other. It is necessary to apply the statistical significance letters calculated in the ANOVA analysis.Unfortunately, due to the low significance of the results, we cannot talk about differences but about the upward trend of parameters along with DAE. Calculate the correlation coefficients between the parameters (number, diameter, area) and DAE.What guided the authors by selecting plants from different periods (corresponding to different weather conditions) in 2013 and 2014. The convergence of results from 2013 and 2014 should be interpreted despite differences in weather conditions.

Author Response

Subject: Submission of Agronomy-472193

Dear Editor

We appreciate the valuable suggestions on further improving the manuscript of “Changes in root anatomy of peanut under different durations of early season drought” in the light of reviewer’s comments. We agree with comments and suggestions, thus the manuscript has been revised as editor’s and reviewer’s suggestions. The details of the revision are given below.

            - Thank you for your kind information.

            - The reviewer’s comments were highly insightful and enabled us to greatly improve the quality of our manuscript. In the follow pages are our point by point response to each of the comments.

Comments and Suggestions for Authors (Reviewer 2)

The reviewed work concerns the interesting and important topic of plant resistance to drought and one of the many aspects/mechanisms of this resistance, which is the adjustment of the root structure to stress conditions. In the title (and not only in keywords), the Latin name peanut (Arachis hypogaea) should be given.

We put the Latin name peanut (Arachis hypogaea L.) in to the title and deleted in the keywords part to avoid redundancy

In the introduction (it is only in the discussion) there is no reference to the most important elements of these changes, such as ingrowths and phi-thickenings. The authors should especially refer to the knowledge about the occurrence of these structures in the roots of plants of the Fabaceae family, to which Arachis hypogaea belongs.

We reviewed on the Fabaceae family which is related to Arachis hypogaea L.

We put more information on introduction part:

The sentences “Cell-wall ingrowths or phi-thickening have been reported in loquat (Eriobotrya japonica Lindl.) root [11], apple (Pyrus malus) [12], geranium (Pelargonium hortorum) roots [12] Sibipiruna (Caesalpinia peltophoroides) [13] with solute movement (salt stress) [12], water logging [13], and drought stress [11]. Although the effect of early season drought on ingrowths and phi-thickenings has not been investigated in peanut and further investigations are necessary to understand phi-thickenings. The response of phi-thickening might be related to the transport processes in the peanut root.” in page 2 lines 31-34 and in page 3 lines 1-3.

Materials and methods part:

The cell-wall ingrowths were compared drought and well watered treatments in cortical layers in both first-order and second-order lateral roots (page 7 lines 6-8).

The results part:

The cell-wall ingrowths in the first order lateral roots were detected in the cortical cells under both well-watered and drought stress treatments (Fig. 3). The cell-wall ingrowths were localized at the opposite side of the intercellular spaces adjacent to the endodermis except in under drought at 28 DAE (Fig. 3 b2). The cell-wall ingrowths were found in two positions which were opposite side of the intercellular spaces and cell-cell conjungtion. The 1-2 layers of this cell were found and indicated as peri-endodermal layer. (page 14 lines 6-12).

Cell-wall ingrowths appeared in the cortical cells of the second order lateral root under both conditions (Fig. 8). The 1-2 layers of cell-wall ingrowths were found in peri-endodermal layer. (page 20 lines 6-8).

Discussion part:

It is worth to refer and cite chapter 2 Role of Phi Cells Under Abiotic Stress in Plants” z książki Root Engineering, Soil Biology 40. Morte and A. Varma (eds.), DOI 10.1007/978-3-642-54276-3_2, ©Springer-Verlag Berlin Heidelberg 2014 by Fernandez-Garcia, Lopez-Berenguer and Olmos.

We added this supporting information on page 22 lines 13-20.

In Ferna´ndez-Garcı´a,  Lo´pez-Berenguer, and Olmos book chapter on the role of Phi Cells under abiotic stress the authors noted that phi thickening is not the exception in the root anatomy.  They noted that the literature has described 16 different families, covering more than 100 species, which present the phi thickening in the roots. While most of the species described presenting phi thickenings are dicotyledons, they noted one exception, the presence of phi thickenings in the rhizodermis of Zea mays (a monocotyledon). In this chapter the authors note that Van Tieghem (1888) classified Phi thickening “into three types based on their root cell location: type I, the most frequently found, phi cell layer is located in contact with the endodermis. Type II phi cell layer is located in contact with the epidermis and Type III phi cell layers are located in the inner cortical cells, but not in contact with either the epidermis or the endodermis”.

Tested for first order roots and second order roots, vessel parameters: number, diameter, area, and microscopic observations of the structures present in cross sections can provide valuable information on plant adaptation to stress. However, this requires a very transparent clear overview and comparison of the results obtained with each other. In addition, the structures visible in the pictures should be accurately, exactly visualized and precisely described. In connection with this, the authors must make major changes to the presentation, description and interpretation of the results. In the pictures there are numerous and regularly placed- arranged in one - two layers cortical cells - the opposite of the intercellular spaces localized adjacent to the endodermis - designated as the peri-endodermal layer. Only single ingrowths are visible in other places - this should be noted and interpreted. These structures can indeed fulfill other than mechanical functions. On the other hand, so-called phi-thickenings, which can occur in hypodermal or innercortical layers, are not actually visible in the presented pictures. They could be seen at much higher magnification and using an electron microscope. Both the photos-microscopic images and the Figures with the vessels parameters should be presented in such a way that you can compare the first and second order roots.

Thank you for these comments. We have improved our presentation of the pictures and the descriptions associated with them. We also acknowledge that the availability of an electron microscope would have aided this study, yet was not available to us. 

We added the details of phi-thickening:

The cell-wall ingrowths in the first order lateral roots were detected in the cortical cells under both well-watered and drought stress treatments (Fig. 3). The cell-wall ingrowths were localized at the opposite side of the intercellular spaces adjacent to the endodermis except in under drought at 28 DAE (Fig. 3 b2). The cell-wall ingrowths were found in two positions which were opposite side of the intercellular spaces and cell-cell conjungtion. The 1-2 layers of this cell were found and indicated as peri-endodermal layer in page 14 lines 6-12.  

Cell-wall ingrowths appeared in the cortical cells of the second order lateral root under both conditions (Fig. 8). The 1-2 layers of cell-wall ingrowths were found in peri-endodermal layer in page 20 lines 6-8.

We put the suggestion about the further study on discussion part:

In this study, the 1-2 layers of cell-wall ingrowths were detected in both well-watered and drought stress treatments which were not significantly different for number of cell-wall ingrowths layers. However, the cells could be seen at higher magnification and using an electron microscope in page 22 lines 31-33 and page 23 lines 1-2.

The main reservations refer to the irrelevance of results, the lack of the letter designations for statistical significance, incorrect and incomplete signatures and the lack of explanations regarding the method of staining root preparations. The legibility of all signatures must be improved, starting with Fig.1b, Figures 2 and 3 and Figures 7 and 8 - it is necessary to explain in the title of the graphs how the cells were dyed on red and why staining has occurred in these places. The captions on the remaining charts are too small, unreadable and otherwise not properly placed.

I did free hand cross-section. We strained the samples with diluted Safranin O and took photos without dye removal with either water or the ethanol series.

On the Y axis, it has to be numbers [Fig. 4 & Fig. 9], diameters (μm) [Fig. 5 & Fig. 10], area (μm2) [Fig. 6 & Fig. 11], in the header: normal vessels [a1, b1], biger vessels (size> ...) [a2, b2], smaller vessels (size <...) [a3, b3], the words first order (not 1 order ) order LR and second (not 2 order) order LR should be placed once over six charts and inscriptions 2013 and 2014 on three relevant charts. First order and second order graphs must be compiled in one panel - next to each other or one after the other. It is necessary to apply the statistical significance letters calculated in the ANOVA analysis. Unfortunately, due to the low significance of the results, we cannot talk about differences but about the upward trend of parameters along with DAE. Calculate the correlation coefficients between the parameters (number, diameter, area) and DAE. What guided the authors by selecting plants from different periods (corresponding to different weather conditions) in 2013 and 2014. The convergence of results from 2013 and 2014 should be interpreted despite differences in weather conditions.

We edited the graph of the Figure 4, 5, and 6 with the words first order order LR and Figure 9, 10 and 11 with second order LR.

As reviewer 2 mentioned, and we have added to the manuscript, “Due to the subtleness of the results, our trials had few examples producing a 95% confidence level of differences. The results showed an upward trend of parameters with DAE and the correlation coefficients between the parameters (number, diameter, area) and DAE. The details are below:

Table 1 Mean square from the combined analysis of variance for total vessel numbers, bigger vessel numbers, smaller vessel number, total vessel diameter (um), bigger vessel diameter (um), smaller vessel diameter (um), total vessel area (um²), bigger vessel area (um²), smaller vessel area (um²) of the first order lateral root in 2013 and 2014.

^ns, ^*, ^** = non-significant and significant at P < 0.05 and P < 0.01 probability levels, respectively, durations (7, 14 and 21 days without added water), treatments (well-watered and water stress) and seasons (2013 and 2014) on page 10 lines 1-5.

We added the details about the combined analysis on page 8 lines 9-16.

Combined analysis of variance for total vessel numbers, bigger vessel numbers, Smaller vessel number, Total vessel diameter (um), Bigger vessel diameter (um), Smaller vessel diameter (um), Total vessel area (um²), Bigger vessel area (um²), Smaller vessel area (um²) of the first order lateral root in 2013 and 2014 were show in the Table 1. Significant differences in total vessel numbers, bigger vessel numbers, total vessel area and bigger vessel area were observed in different durations and seasons. The interactions between duration and treatment (D*T) were also significant for total vessel numbers and Smaller vessel area traits. (page 8 lines 9-26)

Table 2 Mean square from the combined analysis of variance for total vessel numbers, bigger vessel numbers, smaller vessel number, total vessel diameter (um), bigger vessel diameter (um), smaller vessel diameter (um), total vessel area (um²), bigger vessel area (um²), smaller vessel area (um²) of the second order lateral root in 2013 and 2014.

^ns, *, ** = non-significant and significant at P < 0.05 and P < 0.01 probability levels, respectively, durations (7, 14 and 21 days without added water), treatments (well-watered and water stress) and seasons (2013 and 2014) on page 17 lines 1-5.

Combined analysis of variance for total vessel numbers, bigger vessel numbers, smaller vessel number, total vessel diameter (um), bigger vessel diameter (um), smaller vessel diameter (um), total vessel area (um²), bigger vessel area (um²), smaller vessel area (um²) of the second order lateral root in 2013 and 2014 were show in the Table 2. Significant differences in duration (D) and treatment (T) were significant (P≤0.01 and P≤0.05) for most traits. Season (S) was significant for total vessel numbers and bigger vessel numbers. The interactions between duration × treatment (D*T) and duration × season (D*S) were also significant for some traits. (page 14 lines 15-22)

Table 3 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the first order lateral root and 21DAE in 2013 and 2014.

Table 4 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the first order lateral root and 28DAE in 2013 and 2014.

Table 5 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the first order lateral root and 35DAE in 2013 and 2014.

Table 6 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the second order lateral root and 21DAE in 2013 and 2014.

 Table 7 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the second order lateral root and 28DAE in 2013 and 2014.

 Table 8 Correlation coefficients between the parameters (number, diameter (um), area (um²)) of the second order lateral root and 28DAE in 2013 and 2014.

Author Response File: Author Response.pdf

Round  2

Reviewer 1 Report

Most of the questions are properly addressed. The manuscript is improved.