Localization of Aluminum in Epidermal Cells of Mature Tea Leaves
and Kohtaku Suzuki 3
Round 1
Reviewer 1 Report
the work shows some results obtained with a advanced proton microbeam on tea leaf samples. The results obtained are not numerous but certainly of interest. In particular, the distribution of aluminum on the cell wall and the correlation with the distribution of silicon were significant results.
The size of the work is contained, this is certainly a merit. The summary can be extended by adding some information on the experimental apparatus used
Author Response
reviewer 1’s comment,
The summary can be extended by adding some information on the experimental apparatus used
reply
We added the description of
“The results suggest that Al and Si form a complex in epidermal cells of matured tea leaves. Although the micro-PIXE could not identify the forms of the compounds it could reveal their molar ratios, which is critical information for the determination of the forms of the compounds based on quantitative measurements, which could be explored in further investigations.”
in the end of the “Conclusion”.
Reviewer 2 Report
The authors adequately present the analysis of a cross section of a tea leaf by micro-PIXE. Although they clearly observe the distribution of Al and Si in the cell walls in the article, there is no discussion. There is no discussion about the results they have obtained. There is no comparison with the results of other authors, nor references to other authors in the Results and discussion section. They should interpret and describe the significance of their findings.
Author Response
comment
"Although they clearly observe the distribution of Al and Si in the cell walls in the article, there is no discussion. There is no discussion about the results they have obtained. There is no comparison with the results of other authors, nor references to other authors in the Results and discussion section. They should interpret and describe the significance of their findings."
reply
We modified and added descriptions in “Results and Discussion”. The description in the submitted manuscript is:
“It is clearly seen in Fig 4a that aluminum located in upper epidermis cell wall. We can observe four epidermis cells. This result of aluminum distribution coincides with that of EPMA observation which concluded that aluminum localizes in the cell wall. In the present experiment, we observe that silicon is located in the cell wall too. This aluminum and silicon co-localization in the epidermis was found in our previous study, but present result shows their co-localization in a more straightforward manner.”
This part was revised as follows,
“Based on Figure 4a, Al was located in the upper epidermis cell wall. We can observe four epidermis cells. The result of Al distribution is consistent with the observations of Carr et al. based on energy dispersive X-ray microanalysis [8] and Tolra et al. based on low-energy X-ray fluorescence spectro-microscopy [13], which led to the conclusion that Al is localized in the cell wall. In the present study, Si was also localized in the cell wall. Although the Al and Si co-localization in the epidermis was observed in our previous study [3], the present study reveals their co-localization at a subcellular level and at a higher resolution. To the best of our knowledge, this is the first observation of Al and Si co-localization in epidermal cell walls of mature tea leaves. The result suggests that Al and Si form a complex. Some inorganic anions, such as F-, Si+, and sulfate, can create stable complexes with Al and reduce its toxicity [14,15]. Britez et al. observed Al and Si in Faramea marginata, and their results suggested that hydroxyaluminosilicate was formed in the shoot tissues and was a mechanism of Al detoxification [6]. The results of the present study also suggest the formation of hydroxyaluminosilicate in tea leaves is a detoxification mechanism.
Tolra et al. also investigated the distribution of Si and reported that Si was located mainly in mesophyll cells and that the cell walls of the epidermis had much lower Si contents. The reason for the inconsistency between the present study and Tolra et al. is not clear. They used hydroponically cultivated tea plants aged approximately 14 weeks as samples; conversely, we used cuttings of leaves aged about 1 year from a tea garden. The difference in the cultivation conditions and the ages of the tea leaves could be the source of the inconsistency in Si distribution.”
As you can see, discussion and comparison with the results of other authors are added, and we also describe significance of our findings.
According to the reviewer 2’s comment, we also added the comparison with the result of other author for P distribution.
Reviewer 3 Report
The authors show how it is possible to determine the presence of elements in the vegetation. They chose mature tea leaves. The manuscript is interesting, however, there are some revisions to be made before it can be published on Quantum Beam Science.
First of all, it is not possible to use acronyms without the description.
Then, the introduction does not sits well with the results. They assert that the detoxification mechanisms are not yet known, but it is not clear how the only estimate of the elements can serve this purpose. More effort should be made in the discussion part to explain how their data could be useful.
Comments for author File: 
Comments.pdf
Author Response
comment
it is not possible to use acronyms without the description.
reply
Definitions for PIXE, PIGE are presented when we use them at the first time.
comment
the introduction does not sits well with the results. They assert that the detoxification mechanisms are not yet known, but it is not clear how the only estimate of the elements can serve this purpose. More effort should be made in the discussion part to explain how their data could be useful.
reply
We consider the detoxification mechanisms and added the following descriptions in the “Results and Discussion”
“Some inorganic anions, such as F-, Si+, and sulfate, can create stable complexes with Al and reduce its toxicity [14,15]. Britez et al. observed Al and Si in Faramea marginata, and their results suggested that hydroxyaluminosilicate was formed in the shoot tissues and was a mechanism of Al detoxification [6]. The results of the present study also suggest the formation of hydroxyaluminosilicate in tea leaves is a detoxification mechanism.”
Round 2
Reviewer 2 Report
I do not have new comments. This version could be accepted without new changes.
