Rice Shoot 13C Abundance at Maximum Tillering Stage Is Well Suited to Distinguish Differences in Water Use Efficiency for Water-Saving Rice Technologies
, Lin Guo 2, Meiju Liu 2 and Shan Lin 2
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Reviewer Comment
· Some sentences are lengthy and complex, which may hinder comprehension. For example, "We demonstrated for the first time that shoot δ13C of rice at the maximum tillering stage could better characterize the water use efficiency at the whole growth stage..." could be simplified for clarity.
· Avoid phrases like "may because" and replace them with "possibly because" or "likely due to" to enhance precision.
· In discussing the implications of higher δ13C values due to solar radiation, expand on how this knowledge might influence rice cultivation practices, particularly in regions facing changing climate conditions.
· The conclusions section could benefit from a stronger summary of the practical applications of findings. For instance, consider discussing how farmers or agricultural policymakers could use δ13C measurements to optimize irrigation strategies.
· Suggest areas for further research based on findings. For example, investigating δ13C across more diverse environmental conditions or different rice cultivars could provide additional insights.
Suggestions for Materials and Methods section improvement
· Consider rephrasing for conciseness. For instance, the sentence describing the size and separation of plots could be streamlined: "Each plot (90 m²) was separated by impermeable film and cement walls."
· Clarify the number of replications upfront: "The experiment consisted of nine treatments arranged in a randomized block design with three replications."
· For soil water content measurements, clarify if these measurements occurred at specific times relative to the growth stages or continuously throughout the season.
· In the sample processing subsection, briefly mention why specific temperatures and times were chosen for drying and grinding samples, linking them to best practices in isotope analysis.
· When discussing the statistical analyses, include a brief description of the significance levels (e.g., why certain levels were chosen) and how they relate to the hypotheses being tested.
Comments for author File: 
Comments.pdf
Author Response
1#Reviewer Comment
- Some sentences are lengthy and complex, which may hinder comprehension. For example, "We demonstrated for the first time that shoot δ13C of rice at the maximum tillering stage could better characterize the water use efficiency at the whole growth stage..." could be simplified for clarity.
>> Thank you for pointing this out. We agree with this comment. We have simplified these sentences. (Line 241-243 and Line 291-293)
- Avoid phrases like "may because" and replace them with "possibly because" or "likely due to" to enhance precision.
>> Thank you for pointing this out. We agree with this comment. We have replaced it with“may because” (Line 243)
- In discussing the implications of higher δ13C values due to solar radiation, expand on how this knowledge might influence rice cultivation practices, particularly in regions facing changing climate conditions.
>> Thank you for pointing this out. We have supplement in (Line 275-276)
- The conclusions section could benefit from a stronger summary of the practical applications of findings. For instance, consider discussing how farmers or agricultural policymakers could use δ13C measurements to optimize irrigation strategies.
>> Thank you for pointing this out. We agree with this comment. We supplemented in (Line 294-296)
- Suggest areas for further research based on findings. For example, investigating δ13C across more diverse environmental conditions or different rice cultivars could provide additional insights.
>> Thank you for pointing this out. We agree with this comment. We supplemented in (Line 298-300)
Suggestions for Materials and Methods section improvement
- Consider rephrasing for conciseness. For instance, the sentence describing the size and separation of plots could be streamlined: "Each plot (90 m²) was separated by impermeable film and cement walls."
>> Thank you for pointing this out. We agree with this comment. We have revised the rephrasing (Line 103).
- Clarify the number of replications upfront: "The experiment consisted of nine treatments arranged in a randomized block design with three replications."
>> Thank you for pointing this out. We agree with this comment. We have clarified the number of replications upfront (Line 104-105).
- For soil water content measurements, clarify if these measurements occurred at specific times relative to the growth stages or continuously throughout the season.
>> Thank you for pointing this out. We agree with this comment. We have add with“during the whole growth period” (Line 128)
- In the sample processing subsection, briefly mention why specific temperatures and times were chosen for drying and grinding samples, linking them to best practices in isotope analysis.
>> Thank you for pointing this out. We agree with this comment. We have add with“to avoid nutrient loss” (Line 140)
- When discussing the statistical analyses, include a brief description of the significance levels (e.g., why certain levels were chosen) and how they relate to the hypotheses being tested.
>> Thank you for pointing this out. We agree with this comment. We have revised in (Line 157-161)
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript presents an interesting study on the assessment of irrigation methods using d13C, concluding that this technique is quite robust for this purpose, presenting very clear associations with crop-level WUE. Unlike other cereals, this method has not been extensively applied in rice, so in this regard the manuscript is novel. The manuscript is generally well structured, but some sections need to be better explained, including additional methodological details, a more consistent presentation of results, and a deeper discussion of some results.
SPECIFIC COMMENTS
Figures. Order the figures as they are cited in the text. For example, Figure 7 should be Figure 1 as it is cited in the beginning.
Figure 1 and Table 1 (and methods). Why this comparison and anova is only presented for 2014? From later figures d13C analyses were also done in 2013. Was the sampling design the same? If not, this is not clear in the methods.
Figure 4 (and methods). The standard samples are not described in the methods. I guess these are repeated samples of a given material (e.g. flour), not the reference material used for calibration, but it is not specified. The methods for isotope analyses should be better described, indicating which reference material and standard samples were used.
Fig. 3, and discussion L.269-272. Although this could contribute, there are several alternative explanations that could be partly responsible. 1) the most important, you are relating d13C (WUEi=A/gs) with yield-WUE (grain yield/water received). There are several scaling issues in between: from d13C to leaf level WUEi (A/gs), from WUEi to leaf-level WUE (A/E), and from leaf-level WUE to plant level and finally grain yield, which also involves the harvest index. Unless you performed measures of whole-plant biomass, eventual differences in harvest index between years cannot be accounted. 2) With higher radiation you may also expect higher photosynthesis and hence higher A/gs. In that case we would expect higher biomass in 2013, although not necessarily reflected in grain yield. 3) For the air temperature and humidity, increasing radiation would change the thermal balance of the leaf, increasing leaf temperature and thus increasing leaf-level VPD (even if atmospheric VPD is not higher). If this were the case, we would have higher water use in 2013, and for the same A/gs (=d13C) we would have lower A/E in 2013. To disentangle the different options, it would be useful to look separately at water use and grain yield (the two terms in WUE) and, if available, aboveground biomass.
L.57. “differences in carbon isotope composition (δ13C) of crop stability” I would say “crops” or “plant material”, you can use isotopes to asses “crop stability”, but is it not what has isotopes.
L.59 “The relationship between stable carbon isotope composition and water use efficiency has been widely used in isotopic analyses in plant physiological ecology (Hultine and Marshall, 2000).” I would remove here “in isotope analyses”, you don’t use the relationship in the analyses.
L.69 Important to note here that it is intrinsic water use efficiency (A/gs) not “real” water use efficiency (A/E) wich also depends on evaporative demand (VPD).
L.74-77 The second sentence seems to contradict the first one, what was done exactly in previous works and what is missing? Were those e.g. potted plants?
L.95 I would say ten-days mean rainfall is not very easy to interpret, is it the mean of daily accumulated precipitation, mm day-1?
L.101 “(2) GCRPSsat - The raised beds hold without a visible water layer and the furrows between the raised beds were filled with water so that soil water content was kept saturation from transplanting to two weeks before harvest; ” (3) GCRPS80% - It has the same water regime with GCRPSsat before the middle-tillering stage these two definitions are quite difficult to follow if not particularly familiar with the cultivation set-up. A scheme would be really helpful.
L.119. From L101 above and this one, It is not fully clear what is the situation of GCRPSsat after tillering, is it kept at saturation or filled with water as a paddy system? Intuitively I understand GCRPSsat is wetter than GCRPS80%.
L.131. Does “plant shoot” refers to the whole aboveground biomass (including all stems and leaves) or only the stems without leaves? Please specify with e.g. “whole plant shoot”
Comments on the Quality of English LanguageFor clarity, an additional revision of the language style is also advisable. It is also particularly important to carefully revise the appropiate use of isotope terminology and the definition of key concepts such as (intrinsic) water use efficiency. Some examples:
L. 38 I would remove “the parameter” as it is repeated later in the sentence
L. 49 “were imposed”
L.62. “Discriminates against…” who?
L.67. “balance between the rates of inward CO2 diffusion (Farquhar et al., 1989)” something is missing here, “balance between … and …” I guess you mean assimilation?
L.70 “Plant tissue δ13C, in particular C3 plants, can” change order
L.80. “carbon isotopes”
L98 “consisted”
Author Response
The manuscript presents an interesting study on the assessment of irrigation methods using d13C, concluding that this technique is quite robust for this purpose, presenting very clear associations with crop-level WUE. Unlike other cereals, this method has not been extensively applied in rice, so in this regard the manuscript is novel. The manuscript is generally well structured, but some sections need to be better explained, including additional methodological details, a more consistent presentation of results, and a deeper discussion of some results.
SPECIFIC COMMENTS
Figures. Order the figures as they are cited in the text. For example, Figure 7 should be Figure 1 as it is cited in the beginning.
>>Agree. I, accordingly, reordered the figures.
Figure 1 and Table 1 (and methods). Why this comparison and anova is only presented for 2014? From later figures d13C analyses were also done in 2013. Was the sampling design the same? If not, this is not clear in the methods.
>>The sampling design were the same.
Figure 4 (and methods). The standard samples are not described in the methods. I guess these are repeated samples of a given material (e.g. flour), not the reference material used for calibration, but it is not specified. The methods for isotope analyses should be better described, indicating which reference material and standard samples were used.
>>Yes. Thank you for pointing this out. We agree with this comment. We have supplemented the information.
Fig. 3, and discussion L.269-272. Although this could contribute, there are several alternative explanations that could be partly responsible. 1) the most important, you are relating d13C (WUEi=A/gs) with yield-WUE (grain yield/water received). There are several scaling issues in between: from d13C to leaf level WUEi (A/gs), from WUEi to leaf-level WUE (A/E), and from leaf-level WUE to plant level and finally grain yield, which also involves the harvest index. Unless you performed measures of whole-plant biomass, eventual differences in harvest index between years cannot be accounted. 2) With higher radiation you may also expect higher photosynthesis and hence higher A/gs. In that case we would expect higher biomass in 2013, although not necessarily reflected in grain yield. 3) For the air temperature and humidity, increasing radiation would change the thermal balance of the leaf, increasing leaf temperature and thus increasing leaf-level VPD (even if atmospheric VPD is not higher). If this were the case, we would have higher water use in 2013, and for the same A/gs (=d13C) we would have lower A/E in 2013. To disentangle the different options, it would be useful to look separately at water use and grain yield (the two terms in WUE) and, if available, aboveground biomass.
>>In our manuscript，irrigation water use efficiency (WUEi, kg grain m-3 water) was calculated as the grain yield per amount of irrigation water . We supplemented the information ”With higher radiation higher photosynthesis and hence higher intrinsic WUE. In that case we would expect higher biomass in 2013, although not necessarily reflected in grain yield.” In line 275
L.57. “differences in carbon isotope composition (δ13C) of crop stability” I would say “crops” or “plant material”, you can use isotopes to asses “crop stability”, but is it not what has isotopes.
>>Yes. Thank you for pointing this out. We agree with this comment. We have revised this point (Line 57).
L.59 “The relationship between stable carbon isotope composition and water use efficiency has been widely used in isotopic analyses in plant physiological ecology (Hultine and Marshall, 2000).” I would remove here “in isotope analyses”, you don’t use the relationship in the analyses.
>>Yes. Thank you for pointing this out. We agree with this comment. We have removed “in isotope analyses”(Line 59-60).
L.69 Important to note here that it is intrinsic water use efficiency (A/gs) not “real” water use efficiency (A/E) wich also depends on evaporative demand (VPD).
>>Agree. Accordingly, we have revised the intrinsic water use efficiency in Line 67
L.74-77 The second sentence seems to contradict the first one, what was done exactly in previous works and what is missing? Were those e.g. potted plants?
>>Yes. Thank you for pointing this out. We agree with this comment. We have revised：δ13C values of the plants at the maximum tillering stage and the newly fully developed leaves were significantly and positively correlated with water use efficiency (Tao et al., 2015; Liu et al., 2013). However, this application on different tissues in rice plant has not been reported in the field of rice water use efficiency. （L.74-77）
L.95 I would say ten-days mean rainfall is not very easy to interpret, is it the mean of daily accumulated precipitation, mm day-1?
>>Yes. Thank you for pointing this out. We agree with this comment. We have revised the description “the average of ten-days for daily rainfall” . （L.96）
L.101 “(2) GCRPSsat - The raised beds hold without a visible water layer and the furrows between the raised beds were filled with water so that soil water content was kept saturation from transplanting to two weeks before harvest; ” (3) GCRPS80% - It has the same water regime with GCRPSsat before the middle-tillering stage these two definitions are quite difficult to follow if not particularly familiar with the cultivation set-up. A scheme would be really helpful.
>>Yes. Thank you for pointing this out. We agree with this comment. We have supplemented the scheme of different system in Fig 2.
L.119. From L101 above and this one, It is not fully clear what is the situation of GCRPSsat after tillering, is it kept at saturation or filled with water as a paddy system? Intuitively I understand GCRPSsat is wetter than GCRPS80%.
>> Thank you for pointing this out. We agree with this comment. We have revised the description “In both GCRPSsat and GCRPS80%, there was no standing water layer on the soil surface without a standing water layer during the growing season” (Line 125-128)
L.131. Does “plant shoot” refers to the whole aboveground biomass (including all stems and leaves) or only the stems without leaves? Please specify with e.g. “whole plant shoot”
>>Agree. Accordingly, we have revised the description in Line 139
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI thank the authors for considering my suggestions to improve the manuscript. The introduction has been improved and the methods are now clearer. I still however have some concern on the presentation of the results. In the reply the authors stated that the 2013 and 2014 years followed the same design, but still it is not clear why only data for 2014 is presented in Figs. 3 and 4, and the ANOVA in table 1. To be consistent, the same analyses should be presented for the two years of study. Otherwise please clarify in the response and manuscript if there are any reasons why this cannot be done for 2013.
Author Response
Thank you very much for taking the time to review this manuscript. Thank you for pointing this out. Our manuscript was based on a site-specific experiment with same design, and we found different experimental years mainly showed the similar trend among water treatments for 13C and WUE in previous research (Liu et al., 2013; Tao et al., 2015; Zhang et al., 2016ï¼›Jin et al., 2016ï¼›Zhang et al., 2017). In this manuscript, firstly, for the comparison for the growth stage and plant parts among water managements, to be more concise and clear, we only showed the data in 2014 in Figs. 3 and 4, and the ANOVA in table 1; the data of the second experimental year 2014 would be more precise. Secondly, we described the overall difference between two years showed Figs. 6, 7 and 8.
