Differences in Characteristics of Photosynthesis and Nitrogen Utilization in Leaves of the Black Locust (Robinia pseudoacacia L.) According to Leaf Position

Round 1

Reviewer 1 Report

Dear authors,

The manuscript “Differences in Characteristics of Photosynthesis and Nitrogen Utilization Characteristics in Leaves of the Black Locust (Robinia pseudoacacia L.) According to Leaf Position” is well planned and written. I believe that it can be improved with minor revision. Therefore, I suggest revisions as following:

• explain clearly the sentence at lines 336-338: “On a leaf area basis, sun leaves have significantly higher Chl concentration than shade leaves, although the Chl concentration on a dry mass basis gives the opposite result (data not shown)”;
• discuss the sentence at line 354: “Moreover, sun leaves have a much higher maximum stomatal conductance (gs) than shade leaves.” For example, how do sun leaves control higher transpiration due to high irradiance and temperature?
• explain clearly the sentence at lines 392-393: “The shade leaves did not appear to produce RuBisCo in low irradiation conditions”, do they not produce RuBisCo or have low RuBisCo concentrations?
• How can be defined the roles of sun and shade leaves in the light use efficiency of canopy?  

Author Response

Thank you very much for your proper suggestion and comments on our manuscript.

1) Comment on the line 336-338 (344-346)

Thank you for your comment. As your comment, it is difficult to understand the explanation of Chl concentration per leaf dry mass without showing the data. In this study, all parameters were analyzed per leaf area, but some previous studies have considered chlorophyll concentration per leaf dry mass. Chlorophyll concentration in leaves can give opposite results depending on whether the comparison is made by leaf dry mass or by leaf area.

It was not clear for readers. As your suggestion, we changed the sentence as follows :

“On a leaf area basis, sun leaves have significantly higher Chl concentration than shade leave. Some previous study, however, the Chl concentration on a dry mass basis gives the opposite result [64]. It is suggested that, because the leaf structure of the sun and shade leaves is different.”

2) Comment on the line 354 (365)

   Thank you for your comment. As your comment, it is insufficient to explain leaf position, high irradiance, leaf temperature and stomatal conductance.

As your suggestion, we changed the sentence as follows :

“Moreover, sun leaves have a much higher maximum stomatal conductance (g_s) than shade leaves (Figure 4). This may indicate that they can open their stomata much more than shade leaves. Leaf temperature is determined by ambient temperature and light intensity, but also under control of leaf physical traits and transpiration (e.g. stomatal conductance). When leaves are exposed to sunlight, they can avoid high light damage by reducing the amount of radiation they receive via heat convection and transpiration.”

3) Comment on the line 392-393 (409)

Thank you very much for your pointing out. As you pointed out, we changed the description of this sentence as follows :

“The shade leaves did not produce RuBisCo in low irradiance conditions and are left as remaing N. As a result, the RuBisCo concentration in shade leaves was significantly lower than sun leaves.”

4) Comment on the role of sun and shade leaves

   Thank you very much for your very important point. We were also focused on this point. Leaves in different positions of the canopy of R. pseudoacacia probably use different types and intensities of solar radiation for photosynthesis. To this end, leaves at various positions in the R. pseudoacacia canopy would respond by varying their leaf structure and N utilization patterns.

Thank you very much for your proper suggestion and comments on our manuscript.

Reviewer 2 Report

Overall, this manuscript provides and interesting comparison of shade and sun leaves in Robinia pseudoacacia , focusing especially on divergent nitrogen partitioning strategies in these two leaf types. The authors demonstrate reduced photosynthetic capacity, but a higher allocation to easily remobilizable proteins in shade leaves.

Must of the justification points to the understanding the strategies that make R. pseudoacacia more competitive than native species, but because there are no species comparisons in this work nor in any other work cited, this makes drawing conclusions about the contribution of the observed traits to the fitness of this species questionable. The traits observed are interesting, but the connection with competition is not clear. There are also some weaknesses in analysis, and in the presentation of statistical analysis and experimental (or survey) design that make assessment of the statistical approach difficult.  

104-117 The numbers of trees are not stated, nor the pattern of leaf sampling, this makes assessing the analysis difficult.

Lines 220-222 do not describe the kinds of analysis preformed nor the approach to performing them.  What kinds of procedures (SAS) or commands (R) were used to implement what kinds of models? In the table legends it says ANOVAs were used, but what was the model, were there

Line 230: This type of association  and Figure 2 comes with no quantification.  A regression could be used to test these associations. 

Minor comments:

Line 392: seems to have some mistake, as interpretation of this sentence would indicate that shade leaves become soluble proteins.

The term adaptation is used frequently throughout, but acclimation is the correct term.  Adaptation is an evolutionary process in populations while acclimation is the adjustment of an organism to a set of conditions.

Irradiance should be used instead of irradiation.

Author Response

Thank you very much for your proper suggestion and comments on our manuscript.

As your suggestion, it is necessary to understand that R. pseudoacacia has a strategic response compared to native species. For that reason, this study focused on the N-utilization pattern and leaf structure in sun and shade leaves to clarify the factors that make R. pseudoacacia competitive.

1) Comment on the line 104-117

   Thank you very much for your pointing out. As you pointed out, we rewrote the description of this sentence more clearly for our readers.

2) Comment on the line 220-222

   Thank you very much for your comments. SAS procedure and R command were described per the reviewer’s suggestion. (Lines 229-232).

3) Comment on the Fig. 2.

  As your suggestion, Fig. 2 was not clear for readers. As your suggestion, we split the Fig. 2 into (a) N vs. RuBisCo and (b) N vs. Chl concentrations. Regression results were reported on the figures as well. Captions, captions, results, and discussion were rewritten correspondingly. Thank you for your helpful suggestion.

4) Comment on the line 392

   Thank you very much for your pointing out. These paragraphs are difficult to understand. We changed the sentence of these paragraphs as follows ;

“The explanation is that R. pseudoacacia leaves grown in low irradiance conditions allocate N less rapidly to RuBisCo (N_R; 49.9 to 21.5%) than to other photosynthetic proteins, such as N_E (8.3 to 7.0 %) and N_L (6.0 to 5.1 %).”

“These results indicate that less N is allocated to RuBisCo in shade leaves. The shade leaves did not produce RuBisCo in low irradiance conditions and are left as remaining N. As a result, the RuBisCo concentration in shade leaves was significantly lower than sun leaves.”

5) Comment on the adaptation

  Thank you so much for your pointing out. We changed adaptation to acclimation in the document.

6) Comment on the irradiance

  Thank you so much for your pointing out. We changed irradiance to irradiation in the document.

Thank you very much for your proper suggestion and comments on our manuscript.

Author Response File: Author Response.docx