Si Supply Could Alter N Uptake and Assimilation of Saplings—A ¹⁵N Tracer Study of Four Subtropical Species

Round 1

Reviewer 1 Report (Previous Reviewer 1)

All of my questions have been addressed by the authors, now the paper can be accepted for publication in Forests.

Author Response

Thank you for the constructive and specific comments from anonymous referees. We have thoroughly revised the manuscript (including highlights, methods, results). We added details according to the comments about “total Si (Si_TOT), soluble and exchangeable Si (Si_SOL) and Biomass were not shown in the results section.” (section 3.1, 4.1). We re-organized the methods, explaining which methods referred to soil and plants according to the comments about “it is hard to distingue which analyses refer to the soil, and which to plants? This is especially regard to Si analyses.” (section 2.2-2.3). Moreover, we have invited a native English speaker to polish the language and provide English language edits.

 

Therefore, here we submit a “Manuscript_tracked changes” version of the manuscript. We believe the manuscript has been significantly improved after the revision. Please see the following responses to the reviewers’ comments.

Reviewer 2 Report (Previous Reviewer 2)

Liu and coworkers resubmitted manuscript 2433393 in an improved form. However, the manuscript still contains significant flaws; therefore I could not recommend it for publishing. See my comments below:

- English has to be significantly improved. It is hard to follow the text. Some sentences are almost unreadable (for instance: )

-    Highlights are not appropriate. The topic of this manuscript Si and N. (The first highlight should be placed at 3rd. ) Highlights 2. and 3. are contradictory each to other. 

Abstract:

Line 36:  total Si, soluble, and exchangeable Si was not shown in the results section.

Line 40: Biomass was not shown in manuscripts.

M&M

The whole M&M section is poorly written. Is it hard to distingue which analyses refer to the soil, and which to plants? This is especially regard to Si analyses.

-Results of total Si in soil were not shown in the manuscript.

-Biomass was not shown in manuscripts

- If the experiment was conducted in 2019. how is it possible now to apply Na2SiO3*9H2O? Something went wrong. Can you provide some explanation? In a previous version of the manuscript, the authors applied Si as H2SiO3.

-       Line (129-130) does not belong here.  It was the soil, not hydroponic experiments. Please delete it.

- How Biomass was measured? On several occasions in the manuscript authors refer to biomass, so these results should be included.

 -Line 166-170. Unnecessary sentence (The Si in the soil can be further classified......)

- Line183-186. It is not clear which samples were determined spectroscopically.

INTRODUCTION

See:  Wu et al 2017. It might be helpful

Wu X, Yu Y, Baerson SR, Song Y, Liang G, Ding C, Niu J, Pan Z and Zeng R (2017) Interactions between Nitrogen and Silicon in Rice and Their Effects on Resistance toward the Brown Planthopper Nilaparvata lugens. Front. Plant Sci. 8:28. doi: 10.3389/fpls.2017.00028

Line:92-94 ( “It was accepted that Si application has the potential..”.). This sentence has no sense. Synergistic effect of what?

Line 96-100. (“Moreover, there were might different responses....”)This section is very hard to understand. See the comment above.

RESULTS

Given that Si-Sol doesn’t vary from previous manuscripts, and different form of Si was applied I find it wary odd. Can you provide some explanation? (Table S1).

line 315-321 (“In this study, there was no significant difference ....” )It still does not explain the lack of differences among treatments.  Authors are suggested to use CaCL2 as an extractant for Si soil from Soil. If plants depleted the Si pool in the soil it would be measurable. The degree of depletion would be depended on plant biomass, as well as, species' ability to acquire Si.  If authors applied different forms of Si in the previous and this version (or Si was prepared in a different manner) it is not expected to gain the same results of Si sol (Supp. Table1). Do you have some explanation? 

What is the initial Si supply?

 

Authors are strongly suggested to perform another soil experiment, more carefully planned, with a selection of appropriate methodology. 

 

Author Response

Response to Reviewer 2:

Liu and coworkers resubmitted manuscript 2433393 in an improved form. However, the manuscript still contains significant flaws; therefore I could not recommend it for publishing. See my comments below:

- English has to be significantly improved. It is hard to follow the text. Some sentences are almost unreadable (for instance: )

Response:

Thank you for reviewing our manuscript and we appreciate the constructive comments which contribute to improving the manuscript. We have revised the manuscript according to the comments, including highlights, methods, and results. We have re-organized the methods, and explain which refer to soil and plants (section 2.2-2.3). Moreover, we have invited a native English speaker to polish the language and provide English language edits. We believe the manuscript has been significantly improved after the revision. Please see the following responses.

 

-    Highlights are not appropriate. The topic of this manuscript Si and N. (The first highlight should be placed at 3rd. ) Highlights 2. and 3. are contradictory each to other. 

Response: Thank you for your suggestion. We have revised it (tracked changes, line 14-26).

• N uptake and assimilation rates of roots and leaves in Si-accumulating bamboo was higher than that of three other species, whereas we do not show evidence for differences in NH₄⁺ or NO₃⁻ uptake among the four species.
• Silicon (Si) supply did not change the preference for different forms of N among plant saplings and its uptake and assimilation rates among three species, except for Phoebe bournei.
• C concentrations of plant tissues declined with Si supply, due to a partial substitution of organic C compounds by Si.

 

Abstract:

Line 36:  total Si, soluble, and exchangeable Si was not shown in the results section.

Response: Thank you for your suggestion. We have added “total Si (Si_TOT), soluble and exchangeable Si (Si_SOL)” in results (tracked changes, line 274-275; Table S2).

“In addition, there was no significant differences in the concentrations of N (total N, NH₄⁺-N and NO₃^--N), Si_TOT, or Si_SOL in soils among three levels of Si supply (Table S2).”.

 

Line 40: Biomass was not shown in manuscripts.

Response: Thank you for your suggestion. We have revised it, including measurement of biomass in M&M and its results (tracked changes, line 154-165 in M&M; line 243-245 in Results; Table S1).

“We sampled saplings before the ¹⁵N tracer application, and then we collected all biomass from saplings (n = 4) after 1, 15, 75 and 100 days after the ¹⁵N tracer application. All sampled saplings under varying intensities of Si supply and ¹⁵N tracer application were completely randomized for a total of 96 saplings per species.” (M&M).

 

“2.2 Plant samplings and biomass

At the end of the experiment, all saplings were collected, washed and separated into leaves, stems and roots, fully dried (65 °C) for 3 days, then weighed to calculate biomass. All plant samples were ball-milled in preparation for chemical analyses. To determine soil chemical properties, we mixed soil in each plastic pot (about 300-400 g wet weight per sample). Then, soil samples were pooled, air-dried, and ground through a 2 mm sieve. There were a total 384 soil samples.” (M&M).

 

“We found that the root biomass of bamboo was 2-3 times higher than that of other plant species, whereas no significant difference in leaf and stem biomass among species (Table S1).” (Results).

 

“Table S1 The biomass (g) of Phyllostachys pubescens, Phoebe bournei, Schima superba, Cunninghamia lanceolata among three level of Si supply (n = 8). The data represent mean values for biomass. Lowercase letters denote significant differences in biomass among varying silicon supply (one-way ANOVA. P < 0.05).

Species	1d				15d				75d				100d
	Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2
Leaf
Phyllostachys pubescens	2.6 (0.9)	2.5 (0.5)	2.7 (0.6)		3.5 (0.4)	2.1 (0.5)	2.3 (0.4)		4.9 (1.2)	4.2 (0.7)	4.0 (0.9)		6.0 (0.3)a	4.4 (0.3)b	4.1 (0.2)b
Phoebe bournei	2.6 (0.1)	2.4 (0.2)	2.5 (0.1)		3.3 (0.3)	3.1 (0.4)	2.5 (0.2)		3.5 (0.4)a	3.8 (0.2)a	2.7 (0.2)b		1.8 (0.2)b	2.2 (0.2)b	3.0 (0.3)a
Schima superba	1.9 (0.3)	2.4 (0.5)	2.2 (0.2)		1.9 (0.3)	2.8 (0.3)	2.8 (0.4)		1.0 (0.5)	2.0 (0.6)	1.3 (0.3)		1.2 (0.3)	1.4 (0.5)	2.3 (0.4)
Cunninghamia lanceolata	3.0 (0.2)	2.6 (0.3)	2.8 (0.3)		3.7 (0.4)	2.5 (0.3)	3.2 (0.3)		3.7 (0.3)	3.1 (0.2)	3.7 (0.4)		4.5 (0.7)	3.7 (0.4)	4.4 (0.6)
Root
Phyllostachys pubescens	4.6 (1.4)	5.2 (0.7)	7.5 (2.2)		6.0 (1.0)a	2.6 (0.8)b	3.1 (0.6)b		5.3 (1.1)	4.6 (0.8)	4.7 (0.7)		9.0 (2.0)	5.1 (1.3)	6.2 (0.8)
Phoebe bournei	2.3 (0.3)	2.7 (0.3)	2.8 (0.4)		2.7 (0.2)	2.5 (0.2)	2.6 (0.2)		2.0 (0.2)	1.5 (0.3)	1.8 (0.1)		2.1 (0.3)	2.2 (0.2)	2.2 (0.2)
Schima superba	1.7 (0.2)	1.7 (0.3)	2.3 (0.3)		1.8 (0.4)	2.2 (0.3)	1.9 (0.2)		1.2 (0.2)	1.4 (0.2)	1.3 (0.2)		2.2 (0.2)	1.9 (0.2)	2.0 (0.3)
Cunninghamia lanceolata	2.4 (0.3)	2.3 (0.3)	2.4 (0.3)		3.2 (0.5)	1.9 (0.4)	2.2 (0.4)		2.9 (0.2)a	1.9 (0.3)b	2.1 (0.2)ab		3.2 (0.4)	2.0 (0.3)	2.0 (0.4)
Stem
Phyllostachys pubescens	2.0 (0.8)	2.2 (0.5)	2.1 (0.6)		2.8 (0.3)a	1.4 (0.2)b	1.8 (0.5)ab		2.9 (0.6)	2.6 (0.5)	2.2 (0.4)		3.8 (0.6)a	2.6 (0.1)b	2.1 (0.2)b
Phoebe bournei	1.9 (0.1)	2.2 (0.2)	2.2 (0.2)		2.5 (0.2)	2.3 (0.3)	1.9 (0.1)		2.9 (0.3)	3.4 (0.3)	2.6 (0.3)		3.6 (0.5)	2.7 (0.2)	2.9 (0.3)
Schima superba	1.4 (0.2)	1.4 (0.2)	1.9 (0.2)		1.8 (0.2)	2.0 (0.3)	1.5 (0.1)		1.8 (0.3)	1.8 (0.3)	2.1 (0.1)		2.0 (0.1)	1.9 (0.1)	1.9 (0.2)
Cunninghamia lanceolata	1.4 (0.1)	1.3 (0.2)	1.3 (0.1)		1.6 (0.2)	1.2 (0.2)	1.6 (0.2)		1.7 (0.2)	1.9 (0.2)	1.9 (0.2)		2.1 (0.3)	1.7 (0.2)	2.5 (0.3)

” (Supp. Table1).

 

M&M

The whole M&M section is poorly written. Is it hard to distingue which analyses refer to the soil, and which to plants? This is especially regard to Si analyses.

Response: Thank you for your suggestion. We have revised it. (tracked changes, line 182-200).

“Total Si (Si_TOT) was extracted from soil (0.1g) and plant tissues (0.25g) using sodium hydroxide at 650 °C in a muffle furnace and a hydrochloric acid (1:1) dissolution method (Lu 2000, Reza Ramdan et al. 2022, Pavlovic et al. 2013). To extract soluble and exchangeable Si (Si_SOL / Acid Na-acetate-Si) from soil, we added 30 mL of 1M acid Na-acetate buffer solution at pH 4.0 to 0.75 g soil in a 50 mL centrifuge tube, agitated the sample for 24 h, and centrifuged the sample, representing the most easily absorbed form of Si utilized by plants (Sauer et al. 2006, Klotzbücher et al. 2016). Both digested samples (including extractions for Si_TOT [soil and plant] and Si_SOL [soil]) were then analyzed using spectroscopy with the silicon-molybdenum blue colorimetric method and a molybdate–ascorbic acid procedure at a pH of 1.2~1.3 using an ultraviolet visible spectrophotometer (680nm, UV-5100) (Georgiadis et al. 2013, Song et al. 2013), calibrated with a standard solution (5 mg L^-1 SiO₄^2-).”

 

-Results of total Si in soil were not shown in the manuscript.

Response: Thank you for your suggestion. We have added “total Si (Si_TOT), soluble and exchangeable Si (Si_SOL)” in results (tracked changes, line 274-275; Table S2).

“In addition, there was no significant differences in the concentrations of N (total N, NH₄⁺-N and NO₃^--N), Si_TOT, or Si_SOL in soils among three levels of Si supply (Table S2).”.

 

-Biomass was not shown in manuscripts

Response: Thank you for your suggestion. We have revised it, including measurement of biomass in M&M and its results (tracked changes, line 154-165 in M&M; line 243-245 in Results; Table S1).

“We sampled saplings before the ¹⁵N tracer application, and then we collected all biomass from saplings (n = 4) after 1, 15, 75 and 100 days after the ¹⁵N tracer application. All sampled saplings under varying intensities of Si supply and ¹⁵N tracer application were completely randomized for a total of 96 saplings per species.” (M&M).

 

“2.2 Plant samplings and biomass

At the end of the experiment, all saplings were collected, washed and separated into leaves, stems and roots, fully dried (65 °C) for 3 days, then weighed to calculate biomass. All plant samples were ball-milled in preparation for chemical analyses. To determine soil chemical properties, we mixed soil in each plastic pot (about 300-400 g wet weight per sample). Then, soil samples were pooled, air-dried, and ground through a 2 mm sieve. There were a total 384 soil samples.” (M&M).

 

“We found that the root biomass of bamboo was 2-3 times higher than that of other plant species, whereas no significant difference in leaf and stem biomass among species (Table S1).” (Results).

 

“Table S1 The biomass (g) of Phyllostachys pubescens, Phoebe bournei, Schima superba, Cunninghamia lanceolata among three level of Si supply (n = 8). The data represent mean values for biomass. Lowercase letters denote significant differences in biomass among varying silicon supply (one-way ANOVA. P < 0.05).

Species	1d				15d				75d				100d
	Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2		Si + 0	Si + 7 g/m2	Si + 14 g/m2
Leaf
Phyllostachys pubescens	2.6 (0.9)	2.5 (0.5)	2.7 (0.6)		3.5 (0.4)	2.1 (0.5)	2.3 (0.4)		4.9 (1.2)	4.2 (0.7)	4.0 (0.9)		6.0 (0.3)a	4.4 (0.3)b	4.1 (0.2)b
Phoebe bournei	2.6 (0.1)	2.4 (0.2)	2.5 (0.1)		3.3 (0.3)	3.1 (0.4)	2.5 (0.2)		3.5 (0.4)a	3.8 (0.2)a	2.7 (0.2)b		1.8 (0.2)b	2.2 (0.2)b	3.0 (0.3)a
Schima superba	1.9 (0.3)	2.4 (0.5)	2.2 (0.2)		1.9 (0.3)	2.8 (0.3)	2.8 (0.4)		1.0 (0.5)	2.0 (0.6)	1.3 (0.3)		1.2 (0.3)	1.4 (0.5)	2.3 (0.4)
Cunninghamia lanceolata	3.0 (0.2)	2.6 (0.3)	2.8 (0.3)		3.7 (0.4)	2.5 (0.3)	3.2 (0.3)		3.7 (0.3)	3.1 (0.2)	3.7 (0.4)		4.5 (0.7)	3.7 (0.4)	4.4 (0.6)
Root
Phyllostachys pubescens	4.6 (1.4)	5.2 (0.7)	7.5 (2.2)		6.0 (1.0)a	2.6 (0.8)b	3.1 (0.6)b		5.3 (1.1)	4.6 (0.8)	4.7 (0.7)		9.0 (2.0)	5.1 (1.3)	6.2 (0.8)
Phoebe bournei	2.3 (0.3)	2.7 (0.3)	2.8 (0.4)		2.7 (0.2)	2.5 (0.2)	2.6 (0.2)		2.0 (0.2)	1.5 (0.3)	1.8 (0.1)		2.1 (0.3)	2.2 (0.2)	2.2 (0.2)
Schima superba	1.7 (0.2)	1.7 (0.3)	2.3 (0.3)		1.8 (0.4)	2.2 (0.3)	1.9 (0.2)		1.2 (0.2)	1.4 (0.2)	1.3 (0.2)		2.2 (0.2)	1.9 (0.2)	2.0 (0.3)
Cunninghamia lanceolata	2.4 (0.3)	2.3 (0.3)	2.4 (0.3)		3.2 (0.5)	1.9 (0.4)	2.2 (0.4)		2.9 (0.2)a	1.9 (0.3)b	2.1 (0.2)ab		3.2 (0.4)	2.0 (0.3)	2.0 (0.4)
Stem
Phyllostachys pubescens	2.0 (0.8)	2.2 (0.5)	2.1 (0.6)		2.8 (0.3)a	1.4 (0.2)b	1.8 (0.5)ab		2.9 (0.6)	2.6 (0.5)	2.2 (0.4)		3.8 (0.6)a	2.6 (0.1)b	2.1 (0.2)b
Phoebe bournei	1.9 (0.1)	2.2 (0.2)	2.2 (0.2)		2.5 (0.2)	2.3 (0.3)	1.9 (0.1)		2.9 (0.3)	3.4 (0.3)	2.6 (0.3)		3.6 (0.5)	2.7 (0.2)	2.9 (0.3)
Schima superba	1.4 (0.2)	1.4 (0.2)	1.9 (0.2)		1.8 (0.2)	2.0 (0.3)	1.5 (0.1)		1.8 (0.3)	1.8 (0.3)	2.1 (0.1)		2.0 (0.1)	1.9 (0.1)	1.9 (0.2)
Cunninghamia lanceolata	1.4 (0.1)	1.3 (0.2)	1.3 (0.1)		1.6 (0.2)	1.2 (0.2)	1.6 (0.2)		1.7 (0.2)	1.9 (0.2)	1.9 (0.2)		2.1 (0.3)	1.7 (0.2)	2.5 (0.3)

” (Supp. Table1).

 

- If the experiment was conducted in 2019. how is it possible now to apply Na2SiO3*9H2O? Something went wrong. Can you provide some explanation? In a previous version of the manuscript, the authors applied Si as H2SiO3.

Response: We are sorry that there was not more of an explanation for this change. We intended to express that Si was applied as silicate (SiO₃^2-) in the first version of the manuscript. However, we mistakenly explained that it was applied as H₂SiO₃ due to a linguistic error. Thanks for your attention to detail.

 

-       Line (129-130) does not belong here.  It was the soil, not hydroponic experiments. Please delete it.

Response: Thank you for your suggestion. We have removed it (tracked changes, line 144-145).

 

- How Biomass was measured? On several occasions in the manuscript authors refer to biomass, so these results should be included.

Response: Thank you for your suggestion. We have revised it, including measurement of biomass in M&M and its results (tracked changes, line 154-165 in M&M; line 243-245 in Results; Table S1).

“We sampled saplings before the ¹⁵N tracer application, and then we collected all biomass from saplings (n = 4) after 1, 15, 75 and 100 days after the ¹⁵N tracer application. All sampled saplings under varying intensities of Si supply and ¹⁵N tracer application were completely randomized for a total of 96 saplings per species.” (M&M).

 

“2.2 Plant samplings and biomass

At the end of the experiment, all saplings were collected, washed and separated into leaves, stems and roots, fully dried (65 °C) for 3 days, then weighed to calculate biomass. All plant samples were ball-milled in preparation for chemical analyses. To determine soil chemical properties, we mixed soil in each plastic pot (about 300-400 g wet weight per sample). Then, soil samples were pooled, air-dried, and ground through a 2 mm sieve. There were a total 384 soil samples.” (M&M).

 

“We found that the root biomass of bamboo was 2-3 times higher than that of other plant species, whereas no significant difference in leaf and stem biomass among species (Table S1).” (Results).

 

 -Line 166-170. Unnecessary sentence (The Si in the soil can be further classified......)

Response: Thank you for your suggestion. We have removed it (tracked changes, line 184-189).

 

- Line183-186. It is not clear which samples were determined spectroscopically.

Response: Thank you for your suggestion. We have revised it. (tracked changes, line 182-200).

“Total Si (Si_TOT) was extracted from soil (0.1g) and plant tissues (0.25g) using sodium hydroxide at 650 °C in a muffle furnace and a hydrochloric acid (1:1) dissolution method (Lu 2000, Reza Ramdan et al. 2022, Pavlovic et al. 2013). To extract soluble and exchangeable Si (Si_SOL / Acid Na-acetate-Si) from soil, we added 30 mL of 1M acid Na-acetate buffer solution at pH 4.0 to 0.75 g soil in a 50 mL centrifuge tube, agitated the sample for 24 h, and centrifuged the sample, representing the most easily absorbed form of Si utilized by plants (Sauer et al. 2006, Klotzbücher et al. 2016). Both digested samples (including extractions for Si_TOT [soil and plant] and Si_SOL [soil]) were then analyzed using spectroscopy with the silicon-molybdenum blue colorimetric method and a molybdate–ascorbic acid procedure at a pH of 1.2~1.3 using an ultraviolet visible spectrophotometer (680nm, UV-5100) (Georgiadis et al. 2013, Song et al. 2013), calibrated with a standard solution (5 mg L^-1 SiO₄^2-).”

 

INTRODUCTION

See:  Wu et al 2017. It might be helpful

Wu X, Yu Y, Baerson SR, Song Y, Liang G, Ding C, Niu J, Pan Z and Zeng R (2017) Interactions between Nitrogen and Silicon in Rice and Their Effects on Resistance toward the Brown Planthopper Nilaparvata lugens. Front. Plant Sci. 8:28. doi: 10.3389/fpls.2017.00028

Line:92-94 ( “It was accepted that Si application has the potential..”.). This sentence has no sense. Synergistic effect of what?

Response: Thank you for your suggestion. We have read and revised it (tracked changes, line 102-107).

“Si application has the potential to alter N assimilation rates in some Si-accumulating plants, such as rice and wheat (Schaller et al. 2012, Wu et al 2017, Carvalho et al. 2022). This also means that plant uptake and utilization of different forms of inorganic N could be affected by altered available Si pools following bamboo expansion.”

 

Line 96-100. (“Moreover, there were might different responses....”)This section is very hard to understand. See the comment above.

Response: Thank you for your suggestion. We have rewritten it (tracked changes, line 107-113).

“Moreover, plant utilization of inorganic N under altered available Si in soils may differ between bamboo and other trees due to their active, passive or exclusive mechanisms with respect to Si uptake (Ma and Yamaji 2006, Cornelis et al. 2010, Ma and Yamaji 2015).”

 

RESULTS

Given that Si-Sol doesn’t vary from previous manuscripts, and different form of Si was applied I find it wary odd. Can you provide some explanation? (Table S1).

Response: We are sorry that there was not more of an explanation for this change. We intended to express that Si was applied as silicate (SiO₃^2-) in the first version of the manuscript. However, we mistakenly explained that it was applied as H₂SiO₃ due to a linguistic error. Thanks for your attention to detail.

 

line 315-321 (“In this study, there was no significant difference ....” )It still does not explain the lack of differences among treatments.  Authors are suggested to use CaCL2 as an extractant for Si soil from Soil. If plants depleted the Si pool in the soil it would be measurable. The degree of depletion would be depended on plant biomass, as well as, species' ability to acquire Si.  If authors applied different forms of Si in the previous and this version (or Si was prepared in a different manner) it is not expected to gain the same results of Si sol (Supp. Table1). Do you have some explanation? 

Response: Thank you for your suggestion. Indeed, we just intended to estimate the consequences of Si supply on N (three forms of inorganic N) uptake through a ¹⁵N tracer in this study, especially comparing an Si-accumulating bamboo to other plants. Here, we pay more attention to plant tissues rather than specific soil properties. In addition, we have explained more about “there was no significant difference in the concentration of Si_SOL or Si_TOT in soil among three levels of Si supply” (tracked changes, line 321-331).

Thank you for your suggestion again, and we will take it into consideration and perform another soil experiment in the future that focuses more on the specific soil properties.

“In this study, there was no significant difference in the concentration of Si_SOL or Si_TOT in soils among three levels of Si supply. This is in part due to the fact that silicic acid was readily and quickly taken up by by roots of plants actively via specific transporters or passively through transpiration (Mandlik et al. 2020). Second, amorphous Si is unstable and easily transformed to silicic acid and keeps a dynamic balance with other forms of Si in soil (Hallmark et al. 1983, Savant et al. 1999), contributing to relatively unchanged concentrations of Si_SOL. The concentration of Si_TOT was minimally changed by Si supply in this study, because total Si was an abundant element in the soil, mostly determined by crystalline Si and influenced by parent material and rock weathering (Yang et al. 2020, Liu et al. 2022).”

 

What is the initial Si supply?

Response: Thank you for your suggestion. We have made several changes in the manuscript (tracked change, line 45-50; 379-382).

“Moreover, Si supply did not change the uptake preference for N forms or overall uptake and assimilation rates in most species; however, high concentration of Si supply slightly increased NO₃^--N uptake and assimilation rates in fine roots and leaves of P. bournei, particularly immediately following the addition of Si.”

 

“One pattern we found was that under high concentrations of Si supply we saw slightly higher NO₃^--N uptake and assimilation rates for roots and leaves, early in the experiment.”

 

Authors are strongly suggested to perform another soil experiment, more carefully planned, with a selection of appropriate methodology. 

Thank you for your suggestion again, and we will take it into consideration and perform another soil experiment in the future that focus more on the specific soil properties.

Author Response File: Author Response.docx

Reviewer 3 Report (Previous Reviewer 3)

I have looked through the PDFs received by mail. I find the MS to look much better that the first version, so, it may be recommended for a publication.

 

In the course of technical editing, I hope, small grammar flaws still present in the MS (e.g., lines 91, 100) will be corrected, as well as formatting errors (see. lines 272-286).

Author Response

Thank you for reviewing our manuscript and we appreciate the comments which contribute to improving the manuscript. We have invited a native English speaker to polish the language and provide English language edits, including modify some grammar flaws and formatting errors. We have made several changes in the manuscript (all discussion, tracked change, line 303-396). We believe the manuscript has been significantly improved after the revision. Please see the following responses.

Round 2

Reviewer 2 Report (Previous Reviewer 2)

Dear Authors,

I recommended publishing the submitted manuscript in presented form, given that you addressed almost all my remarks.    

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Moso bamboo is an important forest resource due to its versatility, but the rapid expansion of this species has caused several problems. Meanwhile, the bamboo expansion also altered the nutrient cycle and stoichiometry of the ecosystem. In addition, moso bamboo is a Si accumulator. Hence, the manuscript ‘High Si supply alter N uptake and assimilation of four saplings — a ¹⁵N tracing study’ by Liu et al is very interesting and important.The authors did a lot of work, and the methodology used is adequate for the objectives of the study. The results are of interest and support the conclusions. That being said, the manuscript has the potential to be accepted. However, there are still some major issues that need to be addressed before the paper could be accepted as follows.

Although this manuscript is written well, there are many typing errors and syntactic errors, which are listed in detail later. Table 2 is unfriendly to the audience. In addition, I suggest the author to revised the results because the current description of the results is a little prolix.

There are some obscure in the data analysis. The authors did not state whether the data in conform to the premise of ANOVA.

In terms of reality, this work doesn't present the differences between moso bamboo and other species when they are planted together. However, this work provides us with a foundation understanding of the effects of Si supply on N uptake and assimilation.

 

Page 5, 6, Table 2: there were no significant difference in uptake and assimilation rates? I suggest you list the stand errors of the data. The author should check this in the table carefully. For example, the N¹⁵ enrichment (NH₄¹⁵NO₃) in the root of Phyllostachys pubescens is higher than that of the rest species, which was different from the performance of leaf N¹⁵ enrichment and uptake or assimilation rates. Please explain it in results and discussion.

Abstract:

Page 1, paragraph 1, line 1-2: “that some trees … Si accumulation”.This cause is somewhat confusing. I suggest to revise “that some trees were replaced by bamboo, due to plant communities control the quantity of phytoliths and Si accumulation.”

Page 1, paragraph 1, line 8: The generic names should not be abbreviated when they first appear.

Page 1, paragraph 1, line 11-12: “abundantbiomass” was inappropriate adjectives, please revise it.

 Introduction

Page 1, paragraph 2, line 3: Revise 'by' to 'with'.

Page 2, paragraph 3, line 8-10, 16-18: “To explore the influences … bamboo and other trees” and “which helps us predict uptake and utilize … following bamboo expansion.” Please revise 'utilize' to 'utilization '.

Materials and methods

Page 2, paragraph 4, line 4-5: “which were the dominant species associated with bamboo expansion in subtropical forests.” — 'were' should be deleted

 

Page 3, paragraph 1, line 6: “The ¹⁵N labeling was conducted prior to first sampling.”

Page 3, paragraph 1, line 13: “We sampled seedlings (leaf, stems, and roots) at Prior to the ¹⁵N tracer application,”

Page 4, paragraph 2, line 4: “Prior to analysis, all digested samples were then determined using…”

— Revise 'prior to' to 'before'.

 Page 3, paragraph 1, line 10: Revise 'in details' to 'in detail'.

Results
Page 4, paragraph 4: The describe of this result should be more concise.

Page 4, paragraph 4, line 2, 3: “with C content ranged from 0.38% to 0.44%, N content ranged from 0.38% to 0.44%,”. —Revise 'ranged' to 'ranging'.

Page 4, paragraph 4, line 7: “stem and leaf was ranged from 2 to 3.5 g, 1.8 to 5.2 g and 1.7 to 2.6 g per saplings.” — 'was' should be deleted and Revise 'saplings' to 'sapling'.

 Page 5, Table 1 header: “P. pubescens, P. bournei, S. superba and C. lanceolata” should be Italic

Discussion

Page 9, paragraph 1, line 15:“including phytholiths, are energetically cheaper to form…” — Revise 'phytholiths' to 'phytoliths'.

Page 9, paragraph 3, line 2:“suggested a NO₃^-preference of…” — Revise 'suggested' to 'suggesting '.

Conclusion

Page 10, paragraph 1, line 1-3: “Si is indispensable for…organic C compounds by Si” revised it to “Si is indispensable for high Si-accumulating bamboo, in which C concentration of plant tissue declined with Si supply, due to partial substitution of organic C compounds by Si.”

References

The cited paper 22 and 23 seems the same one.Page 10-12: The scientific names of plant in the references should be italic. Please check your references carefully. Such as citation 1, 12, 20, 24, 31, 37…

Reviewer 2 Report

The presented manuscript deal with interesting and actual scientific topics, about Si influences on the N-nutrition of 4 forest species.  Although results could be potentially interesting and of both practical and scientific importance, due to poorly executed experiments, the interpretation of results is highly speculative, and the conclusion is not supported by presented data. Authors speculated that high Si supply influenced N uptake and assimilation. However, despite the high rate of Si application, there was no increase in Si availability in soil, nor any differences in Si supply in soil in any treatments.  

Unfortunately, major flaws are considering methodology.  Si was applied in the soil at two different rates; however, Si availability in the soil was not increased compared to control soil without Si application. The concentration of available Si in the soil does not vary in any treatment (Table S1, Supplemental Materials). Therefore, it is almost impossible to discuss the effect of Si, (or high Si supply) on any soil or plant properties, including N nutrition. Furthermore, Si content in plant material seems to be random (Table 1), independent from Si content in the soil. There is no clear trend in Si content in plant tissues (root, leaf, and stem) in regard to Si supply in the soil.  It is quite surprising?  Is there any explanation for this? There are two possibilities: either Si concentration in plant tissues was not determined well, or applied Si was not plant-available.

Silicon was applied as H2SiO3, so there is a question if this form of Si can be plant available.  Results presented in the manuscript (Si concentration in soil and plant tissue) suggest that this form of Si was not plant-available.  Given that Si(OH)4  (see Coscun et at 2019) is the only plant-available form of silicon, different kinds of Si compounds should be used as Si fertilizers (for instance K-Si, Na-Si, or freshly prepared Si acid).

The M&M section lacks some important explanations:

1.            in M&M (section 2.2 section)  authors mention that soil was tested for concentration of Si, and concentration of soluble Si. What is the difference? To which Si pool "concentration of Si" refers.  How it was determined? Is it the total Si concentration?  There is no table or figures which show it.

2.            It is not clear how Si content in plant material was determined. Microwave digestion with only H2SO4 and H2O2 is not good enough to extract total Si from plant material.  Usually, HF or a strongly alkaline solution is used to extract Si from plant tissue.  For instance, see:  Pavlovic et al 2013; Rivai et al 2022.

3.            M&M section should contain more relevant references.

In Table 1 there are no units of the concentration of the elements in different tissues. Is it ppm, %...?

REF: Coskun, D., Deshmukh, R., Sonah, H., Menzies, J. G., Reynolds, O., Ma, J. F., Kronzucker, H. J., & Bélanger, R. R. (2019). The controversies of silicon's role in plant biology. New Phytologist, 221(1), 67-85. https://doi.org/10.1111/nph.15343

Rivai RR, Miyamoto T, Awano T, Yoshinaga A, Chen S, Sugiyama J, Tobimatsu Y, Umezawa T, Kobayashi M. Limiting silicon supply alters lignin content and structures of sorghum seedling cell walls. Plant Sci. 2022 Aug;321:111325. doi: 10.1016/j.plantsci.2022.111325. Epub 2022 May 16. PMID: 35696925.

Pavlovic, J., Samardzic, J., Maksimović, V., Timotijevic, G., Stevic, N., Laursen, K. H., Hansen, T. H., Husted, S., Schjoerring, J. K., Liang, Y., & Nikolic, M. (2013). Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast. New Phytologist, 198(4), 1096-1107. https://doi.org/10.1111/nph.12213

 

Reviewer 3 Report

This manuscript (MS) presents results of an interesting experiment with increased Si supply and 15N-labelling. From the point of the scientific content, the MS certainly deserves publishing in Forests.

However, the present condition of the MS is below the standards of a scientific journal. Although the language is mostly comprehensible, the abundant grammar flaws make it difficult to read the text. Also, the authors did not provide the line numbers, which makes it hard to point to the flaws.

General comments

p. 9: It looks like a bit of a contradiction. Probably, some more explanation is required:

"there were lower concentration of C and N of P. pubescens and P. bournei with Si supply (Si + 7 or Si + 14 g/m2) than that of control (Si + 0)" 

"found that Si supply (Si + 14 g/m2) significantly increased 15N enrichment in tissues of P. bournei" 

Minor comments (only some examples are given due to a great number of the cases)

p. 1:

"High Si supply alter N uptake" -> alters?

"Si availability would be fluctuated by bamboo expansion", "which fluctuate biogeochemical Si cycle" -> unusual use of 'fluctuate'

"To explore the influences of Si availability on uptake and assimilation rates of root and leaf for different forms of inorganic N between bamboo and other trees." -> incomplete sentence

"seedlings of P. pubescens, P. bournei, S. superba and C. lanceolata" -> it's a standard to provide the names of authors at the first appearance of Latin names

"(total N, NH4+ - N and NO3-- N) and its 15N enrichment)" -> some unnecessary bracket

"compare" -> compared?

"whereas Silicon (Si) supply didn’t change the" -> unnecessary use of a bold font

p.2:

"improve" -> improves?

"compare" -> compared?

"particularly available Si" -> something lacks between 'particularly' and available'

"there were might different responses" -> grammar problem

"utilize" -> utilizing, utilization?

"whether differ between" -> something lacks between 'whether' and 'differ'

"we planted one-year seedlings were into the plastic pots" -> unnecessary 'were'?

Editing of the language is required.