Carbon Isotope Composition and the NDVI as Phenotyping Approaches for Drought Adaptation in Durum Wheat: Beyond Trait Selection
, Maria Newcomb 3, Fadia Chairi 4, Giuseppe Emanuele Condorelli 5, Richard W. Ward 3, Jeffrey W. White 6, Marco Maccaferri 5, Roberto Tuberosa 5, Jose Luis Araus 2,4 and Maria Dolors Serret Molins 4,*Round 1
Reviewer 1 Report
Here, Sanchez-Bragado et al. present a study entitled “Carbon isotope composition and the NDVI as phenotyping approaches for drought adaptation in durum wheat: beyond trait selection”. They performed a large field experiment using 248 elite durum wheat genotypes to evaluate the usefulness of carbon isotope composition and NDVI as tools for field phenotyping.
The introduction is properly presenting the topic and is well-written. As minor comments, I would suggest to describe briefly the effects and importance of drought stress in durum wheat, where the authors mentioned “Drought is a major factor limiting wheat yield”. The authors indicated that 13C discrimination occurs during two steps of CO2: CO2 diffusion and Rubisco carboxylation. How the discrimination takes place in both of these two steps is lacking and would help the reader to understand this relevant trait.
Concerning the methodology, I wonder if the time since the samples were collected until they were dried can affect the isotope analyses. Were the samples harvested using ice/dry ice/liquid nitrogen or maintained at ambient temperature until the drying process? Can the leaf respiration after the harvest affect the results?
How did the authors ensure that the plants were stressed after those weeks without irrigation? Is the change in d13C between control and stressed plants enough to claim for drought symptoms (considering the time gap between these two samplings that can involve also changes in phenology)?
I think the work is well focused on the study of the correlations between biomass, NDVI, phenology and d13C, but I wonder if there is data regarding grain yield in similar growth conditions for this durum wheat panel that can be discussed briefly to see if the correlations that the authors observed for biomass, can be extrapolated for grain yield.
Total leaf nitrogen was not altered by growth conditions and did not correlate strongly with other traits. Maybe this parameter was more influenced by the genotypic variability, could the authors observe strong differences between genotypes inside the durum wheat panel?
The authors interestingly found in a wide panel of durum wheat genotypes that early canopy development is a good plant strategy for biomass production, and likely grain yield, under environments with water limitation. Also, the most productive genotypes presented low water use efficiency. Then, genotypes that can avoid the negative effects of drought in key stages for plant growth will perform better. Moreover, other of the main conclusions is that studies identifying key traits for yield components should avoid the use of genotypes with contrasting phenology. The discussion is very well-written and addressed properly the issues presented in the Results section.
Author Response
Reviewer 1
Here, Sanchez-Bragado et al. present a study entitled “Carbon isotope composition and the NDVI as phenotyping approaches for drought adaptation in durum wheat: beyond trait selection”. They performed a large field experiment using 248 elite durum wheat genotypes to evaluate the usefulness of carbon isotope composition and NDVI as tools for field phenotyping.
The introduction is properly presenting the topic and is well-written. As minor comments, I would suggest to describe briefly the effects and importance of drought stress in durum wheat, where the authors mentioned “Drought is a major factor limiting wheat yield”. The authors indicated that 13C discrimination occurs during two steps of CO2: CO2 diffusion and Rubisco carboxylation. How the discrimination takes place in both of these two steps is lacking and would help the reader to understand this relevant trait.
Our reply:
As the Reviewer suggested we have included some additional information in the Introduction related to the importance and effects of drought stress in durum wheat (see lines 38-40) and as well 13C discrimination caused by CO2 diffusion and carboxylation reaction of Rubisco (please see lines 85-89 of the revised Manuscript version)
Concerning the methodology, I wonder if the time since the samples were collected until they were dried can affect the isotope analyses. Were the samples harvested using ice/dry ice/liquid nitrogen or maintained at ambient temperature until the drying process? Can the leaf respiration after the harvest affect the results?
Our reply:
Samples were collected in the field with no ice. However, they were immediately dried in an oven with enough capacity to dry the samples quickly in order to prevent dark respiration. The quick dry in the large oven was adequate for preventing dark respiration in the leaves sampled in our experiment. Although it has been observed that a delay in the drying process might affect the carbon isotope composition of the flag leaves, all samples were exposed to the same drying processes, with exposure to the same source of variation (even in the event that some dark respiration may have occurred). Moreover, the effect of respiration on the carbon isotope signature may only be relevant when analyzing the water soluble fraction alone (or the photoassimilates directly) (Sanchez-Bragado et al. 2016, Journal of experimental botany; Yousfi et al. Plant, Cell & Environment; Sanchez-Bragado et al. 2019, Plant Physiology) which was not the case in our study where total leaf dry matter was analyzed.
How did the authors ensure that the plants were stressed after those weeks without irrigation? Is the change in d13C between control and stressed plants enough to claim for drought symptoms (considering the time gap between these two samplings that can involve also changes in phenology)?
Our reply:
One indicator of plant water stress in our experiment was the carbon isotope composition, which was statistically enriched two weeks after the last irrigation in the flag leaves and indicated an incipient water stress during plant growth. Carbon isotope composition has been widely used as a plant water status estimator (Richards 1996, Plant Growth Regulation; Condon et al. 2002, Crop Science), including total water accumulated during grain filling (Araus et al. 2003, Crop Science and it is mainly affected by environmental factors rather than phenology, at least during the two weeks that progressive drought was imposed in our experiment. Because the same genotype (and similar plant parts) were sampled before and after the last irrigation, phenological performance was the same for all of the durum wheat panel, thus exposing the genotypes to the same source of variation (absence of water input for the last two weeks).
I think the work is well focused on the study of the correlations between biomass, NDVI, phenology and d13C, but I wonder if there is data regarding grain yield in similar growth conditions for this durum wheat panel that can be discussed briefly to see if the correlations that the authors observed for biomass, can be extrapolated for grain yield.
Our reply:
The main objective of the study was to explore the capacity to predict genotypic variability in green biomass rather than final yield, and the best time to measure such traits in terms of crop phenology and growing conditions. Plant green biomass estimation under field conditions is key as it is labor intensive, and almost impracticable to manage across experiments with hundreds of genotypes that need to be screened, as was the case in the study presented. It is worth considering that increasing plant biomass is a target for present and future breeding because genetic gains in wheat yield have been achieved through increasing traits such as the number of grains per square meter and harvest index (HI), ergo maximizing partitioning to grains, whereas in most cases increases in biomass have not been reported (Fischer et al. 1998; Crop Science). However, under optimal agronomical conditions HI is already not far from its theoretical potential limit of around 0.6 (Austin et al. 1980 Annals of Botany; Slafer et al. 1994, In Genetic improvement of field crops, Inc.: New York). Therefore, to ensure future genetic gains in grain yield, without the limitations imposed by theoretical maximum HI, lines with higher biomass at key growth stages would produce more grains with little change in radiation interception during grain filling (Shearman et al. 2005, Crop Science) and final HI. In this context, breeding for genotypes with high biomass capacity (associated with a high net canopy photosynthetic rate and RUE, even under conditions where some degree of drought stress is present) could have a large impact on genetic gain for yield (Van Den Boogaard. 1996, Australian Journal of Plant Physiology; Villegas et al. 2001, Annals of Botany). The point highlighted by the Reviewer is very pertinent, and it is a step beyond the results presented on this study. Besides that, the phenotypic correlation of δ13C with grain yield has been reported to be stronger than against biomass (Araus et al. 2003, Applied Biology).
Total leaf nitrogen was not altered by growth conditions and did not correlate strongly with other traits. Maybe this parameter was more influenced by the genotypic variability, could the authors observe strong differences between genotypes inside the durum wheat panel?
Our reply:
The range of variation in total nitrogen in the leaf for the control was 3.3% to 5.9% and for stress was 3.5% to 5.9%. The range of variation and the absolute values were very similar in control and stressed plants. In any case, values of 3.3% and beyond correspond to mature but non -senescent flag leaves (e.g. Araus and Tapia, 1986, Plant Physiology; Araus et al. 1997, Plant Cell Environment). Moreover, the small changes in the leaf nitrogen content between the two dates of measurement indicate that flag leaves did not start to senescence (either due to phenology or drought) during the two weeks of the experiment. Under such circumstances, leaf nitrogen content is not expected to be correlated with other traits such as leaf δ13C (Araus et al. 1997, Plant Cell Environment).
The authors interestingly found in a wide panel of durum wheat genotypes that early canopy ; development is a good plant strategy for biomass production, and likely grain yield, under environments with water limitation. Also, the most productive genotypes presented low water use efficiency. Then, genotypes that can avoid the negative effects of drought in key stages for plant growth will perform better. Moreover, other of the main conclusions is that studies identifying key traits for yield components should avoid the use of genotypes with contrasting phenology. The discussion is very well-written and addressed properly the issues presented in the Results section.
We appreciate the positive comments of the Referee.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The study idea is very interesting and needed but I have some concerns that the authors did not mention or discuss in the manuscript
- The study has been conducted for only one season which makes it very difficult to draw a conclusive conclusion. normally fieldwork should be conducted in multiple locations or for multiple seasons.
- Authors compared the lines at two different stages (anthesis and mid grain filling), at anthesis the plants were under normal condition but at grain filling the plants were under stress and they consider the differences between them as control and stress. They measured the NDVI and 13C composition. I found this comparison very hard as the plants were at different physiological stages and also environmental conditions that might be reflected in the measured parameters. Authors should highlight these facts and discuss how their conclusions will not be affected or to what extent it can be valid
Author Response
Reviewer 2
The study idea is very interesting and needed but I have some concerns that the authors did not mention or discuss in the manuscript
- The study has been conducted for only one season which makes it very difficult to draw a conclusive conclusion. normally fieldwork should be conducted in multiple locations or for multiple seasons.
Our reply:
We agree with the Reviewer that the study was conducted over one year alone. However, it was performed in a large set of genotypes under very well managed conditions, which makes the conclusions achieved less dependent on a multi-seasons approach. Moreover, and more importantly, one of the main objectives of the work was not to develop a breeding study per se, but to illustrate how crop phenology, in terms the crop stage at the time of measurement (in this case vegetation indices and δ13C) and a large range of genotypic variability in time to anthesis, may affect phenotyping results.
- Authors compared the lines at two different stages (anthesis and mid grain filling), at anthesis the plants were under normal condition but at grain filling the plants were under stress and they consider the differences between them as control and stress. They measured the NDVI and 13C composition. I found this comparison very hard as the plants were at different physiological stages and also environmental conditions that might be reflected in the measured parameters. Authors should highlight these facts and discuss how their conclusions will not be affected or to what extent it can be valid
Our reply:
As stated in the previous point, one of the objectives of this study was to illustrate how the performance of phenotypic traits may be affected by phenology. To address this objective, we established two different premises for evaluation: (i) the crop stage when measurements were conducted (in the case of NDVI) and (ii) the genotypic differences in anthesis time (in the case of δ13C and the NDVI measured around anthesis). A second objective was to assess how water status (in this case how the induction of progressive water stress during gain filling) may affect the performance of phenotyping (in this case δ13C and NDVI). As pointed out in one of the replies to the first Reviewer, the nitrogen content during grain filling indicated that at least relative to measurements at anthesis, the flag leaves were not undergoing senescence. This was in spite of the fact that the decrease in the NDVI predicted senescence of the lower part of the canopy, which was due to the phenological stage (grain filling), and that this occurred simultaneously with the development of water stress (increase in δ13C).
Author Response File: Author Response.pdf
Reviewer 3 Report
Broad comment:
The title itself is going to attract many readers as the research is highly related to the current priorities. The manuscript includes ample information. However, there are areas for improvement. Improvement is needed mainly on concise writing. I strongly recommend thorough English editing.
Major comments:
Abstract: One sentence that indicates carbon isotope discrimination and NDVI are the physiological traits associated with drought adaptation. (After the first sentence).
Section 1: Include some information about NDVI (RE) and compare it with NDVI (SE).
Section 2.1: This section contains ample information about the germplasm and experimental conditions. However, information is scattered. It should be written more succinctly.
Section 2.2: Why there is either/or in the first sentence when the SE and RE both have been used? Avoid using elsewhere-you can use a better way to cite the information. Why NDVI SE and RE are used? Was there any intention to compare the results obtained using these two different parameters?
Section 2.3: Based on the order in the content within the text, the title may look better as: Carbon isotope analyses and dry biomass evaluation.
Section 2.4: I suggest the content related to carbon isotope analyses in section 2.3 to be included in section 2.4.
Section 3.1: There is a repetition of some information from materials and methods. Citation of Table 2 is not at the appropriate place.
Section 3.2: Avoid the use of text more suitable for the discussion section in the result section. For example lines 284-286.
Section 4.1: Discuss in a few lines about why NDVI (SE) and NDVI (RE) both were used? In a diversity panel, not all the genotypes undergo leaf rolling and not to the same extent, and also phenological stages of the crops are also diverse. In this context, does this discussion indicate that NDVI does not yield good predictions in a diversity panel especially under drought conditions? There should be a conclusive or a summary sentence at the end of this section.
Section 4.2: Summarize the discussion in one or two sentences.
Section 4.3: The discussion has a lot of information it should be more concise for the readers not to be distracted. Also, summarize the message at the end.
Conclusion: The conclusion section needs more work. It should be shortened. Make it more specific. Some of the points that have been presented in the conclusion section can be taken to the discussion section. For example: line 486-489, 491-493 etc.
The text contains substantial information. However, this information could be presented more precisely. There is a repetition of information and words.
Minor comments:
Line 35: find a better option for “herbaceous crop by extent”
Line 44: I would prefer not to use “proven”.
Line 49: Does “but also under Mediterranean conditions” in the whole sentence mean that the Mediterranean environment does not have a high yielding environment?
Line 87: “or even” may sound better with “to” or “and”.
Line 107: “phenotyping”: should it be “phenotypic”?
Lines 109-112: Take this sentence out from this paragraph. Maybe it can be fitted somewhere in the earlier paragraphs.
Line 121: Accommodate the name of the panel in the previous sentence such that there is a better connection between the two sentences.
Line 124: Remove “as explained elsewhere”.
Line 124: No need to repeat 248 accessions.
Line 141: The “gray bars” should be “cyan bars”
Line 141-142: No need to repeat “accumulated”
Line 259: Is it NDVI (RE) or NDVI (SE)?
Line 260: Is it 12th or 13th February?
Line 374: Write full form of VPD.
Table 1 and Table 2: Indicate in the caption what AVG stands for in the table.
Figure 2: In the caption write the full form of DM similar to other abbreviations.
Figure 3: In the caption write the full form of DM similar to other abbreviations. I prefer to have a classical Pearson correlation table rather than the figures. These figures look fancy though.
Figure 6: Linear regression of the relationship of the TOTAL N (%)?
Author Response
Reviewer 3
Broad comment:
The title itself is going to attract many readers as the research is highly related to the current priorities. The manuscript includes ample information. However, there are areas for improvement. Improvement is needed mainly on concise writing. I strongly recommend thorough English editing.
The submitted manuscript had been English-edited by a native (Australian) editor. We have submitted the revised version for his perusal again.
Major comments:
Abstract: One sentence that indicates carbon isotope discrimination and NDVI are the physiological traits associated with drought adaptation. (After the first sentence).
Our reply:
Done. Please see words added to line 16 of the abstract in the revised version of the Manuscript.
Section 1: Include some information about NDVI (RE) and compare it with NDVI (SE).
Our reply:
As suggested by the Reviewer, some information about the NDVI (RE) and NDVI (SE) has been included in Section 1. Please see lines 63-66 of the revised Manuscript. Moreover, the Materials and Methods section has been expanded.
Section 2.1: This section contains ample information about the germplasm and experimental conditions. However, information is scattered. It should be written more succinctly.
Our reply:
Information has been streamlined, moving the part about water conditions to the end of the section. In addition, the text has been revised to make the sentences more concise.
Section 2.2: Why there is either/or in the first sentence when the SE and RE both have been used? Avoid using elsewhere-you can use a better way to cite the information. Why NDVI SE and RE are used? Was there any intention to compare the results obtained using these two different parameters?
Our reply:
The first point of the Reviewer is correct, as both SE and RE sensors have been used, so either/or has been deleted from the first sentence of the main text. Also, the term 'elsewhere' has been replaced. The purpose of using cameras with slightly different band centers and bandwidths was to compare the capacity for phenotyping of the NDVIs generated. Please see the additional information and sentence modification at lines 162-165 in the revised Manuscript.
Section 2.3: Based on the order in the content within the text, the title may look better as: Carbon isotope analyses and dry biomass evaluation.
Our reply:
As mentioned by the Reviewer on the following point (Section 2.4), “carbon isotope composition” has been included in section 2.4, and then the term “carbon isotope analysis” has been deleted from the title of Section 2.3.
Section 2.4: I suggest the content related to carbon isotope analyses in section 2.3 to be included in section 2.4.
Our reply:
Done. Please see response from section 2.3
Section 3.1: There is a repetition of some information from materials and methods. Citation of Table 2 is not at the appropriate place.
Our reply:
As suggested by the Reviewer some information that was already present in the Materials and Methods section has been removed and first citation of Table 2 fixed.
Section 3.2: Avoid the use of text more suitable for the discussion section in the result section. For example lines 284-286.
Our reply:
As suggested by the Reviewer, these three lines have been removed from the Results section of the revised manuscript. In fact, this reasoning was already included in the Discussion section.
Section 4.1: Discuss in a few lines about why NDVI (SE) and NDVI (RE) both were used?
Our reply:
The Reviewer’s point is correct, as no discussion was included in the text comparing the RE and SE sensors. This is worthwhile considering, although the different band centers and bandwidths of the cameras did not significantly affect the NDVI values, suggesting that both kinds of sensors provide similar information in terms of the normalized difference vegetation index (NDVI). Following the suggestion of the Reviewer, a few lines have been included in the Discussion section (please see lines 371-378 of the revised Manuscript).
In a diversity panel, not all the genotypes undergo leaf rolling and not to the same extent, and also phenological stages of the crops are also diverse. In this context, does this discussion indicate that NDVI does not yield good predictions in a diversity panel especially under drought conditions? There should be a conclusive or a summary sentence at the end of this section.
Our reply:
In this section, the idea was to discuss the average values of the studied parameters under control and drought conditions, and as well as the physiological basis of the sensitivity of these parameters as drought increased. No correlations against biomass are described in this section. However, following the suggestions of the Reviewer, one summary sentence about the NDVI has been included at the end of the section. Please see lines 397-401 of the revised Manuscript.
Section 4.2: Summarize the discussion in one or two sentences.
Our reply:
Following the suggestions of the Reviewer, one summary sentence has been included at the end of the section. Please see lines 449-453 of the revised Manuscript.
Section 4.3: The discussion has a lot of information it should be more concise for the readers not to be distracted. Also, summarize the message at the end.
Our reply:
As suggested by the Reviewer, this section has been shortened and the summary part at the end rewritten.
Conclusion: The conclusion section needs more work. It should be shortened. Make it more specific. Some of the points that have been presented in the conclusion section can be taken to the discussion section. For example: line 486-489, 491-493 etc. The text contains substantial information. However, this information could be presented more precisely. There is a repetition of information and words.
Our reply:
As suggested by the Reviewer, the Conclusion section was shortened and some points presented in the Conclusion have been relocated to the Discussion after identifying and removing repeated information.
Minor comments:
Line 35: find a better option for “herbaceous crop by extent”
Done
Line 44: I would prefer not to use “proven”.
Done
Line 49: Does “but also under Mediterranean conditions” in the whole sentence mean that the Mediterranean environment does not have a high yielding environment?
Sentence has been corrected.
Line 87: “or even” may sound better with “to” or “and”.
Corrected.
Line 107: “phenotyping”: should it be “phenotypic”?
Corrected.
Lines 109-112: Take this sentence out from this paragraph. Maybe it can be fitted somewhere in the earlier paragraphs.
Corrected, and moved to the beginning of the paragraph, please see lines 113-115.
Line 121: Accommodate the name of the panel in the previous sentence such that there is a better connection between the two sentences.
Done, please see line 129.
Line 124: Remove “as explained elsewhere”.
Done.
Line 124: No need to repeat 248 accessions.
Done.
Line 141: The “gray bars” should be “cyan bars”
Corrected.
Line 141-142: No need to repeat “accumulated”
Done.
Line 259: Is it NDVI (RE) or NDVI (SE)?
Corrected: it is SE.
Line 260: Is it 12th or 13th February?
Corrected: 13th February.
Line 374: Write full form of VPD.
Done.
Table 1 and Table 2: Indicate in the caption what AVG stands for in the table.
Done.
Figure 2: In the caption write the full form of DM similar to other abbreviations.
Done.
Figure 3: In the caption write the full form of DM similar to other abbreviations. I prefer to have a classical Pearson correlation table rather than the figures. These figures look fancy though.
Our reply:
We appreciate the suggestion of the Reviewer but we think that given the sign of the positive and negative correlations, a figure might be more visual and may help the reader to interpret easily the sign and significance of the correlations.
Figure 6: Linear regression of the relationship of the TOTAL N (%)?
Yes, corrected.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
All are fine
Author Response
Thank you
