The Use of Proline in Screening for Tolerance to Drought and Salinity in Common Bean (Phaseolus vulgaris L.) Genotypes
, Monica Boscaiu 4,* and Oscar Vicente 3Round 1
Reviewer 1 Report
The role of proline is a well known fact in stress tolerance. The need for proline estimation and their relation to stress tolerance in the context of common bean needs to be added. In results and discussion section the traits selected based on PCA analysis and correlation needs to highlight certain accessions fro future breeding needs to be added.
Author Response
We are grateful for your constructive suggestions and corrections. It is our belief that the manuscript is substantially improved after making the suggested edits. Additionally we have improved the quality of figures 3 a and b, as in the previous version some symbols were overlapping and the cultivars could not be properly identified.
The role of proline is a well known fact in stress tolerance. The need for proline estimation and their relation to stress tolerance in the context of common bean needs to be added.
Thank you for your suggestion. We have added more information on the significance of the increase in proline concentration as a response to water and salt stress, as well as other types of stresses in plants, in general, and in beans, in particular. More specifically, we highlight contradictory data in the literature indicating both, positive and negative correlations between Pro accumulation and the relative water deficit and salt stress tolerance of different Phaseolus vulgaris cultivars. We have included several new references related to this specific point, after an additional and extensive literature search.
In results and discussion section the traits selected based on PCA analysis and correlation needs to highlight certain accessions fro future breeding needs to be added
Following your suggestion, we have used the PCA analysis to identify some highly tolerant accessions and some highly susceptible ones. We also indicate how the relative position of other accessions along the x-axis (first principal component of the PCA) should allow a rough ranking of their tolerance to water deficit and to salinity. The most tolerant genotypes can be included in future breeding programmes aimed at enhancing the stress tolerance of common beans. Moreover, identifying accessions in the extremes of tolerance can be useful for further studies on the physiological mechanism of tolerance to water deficit and salinity in common bean. Also, they may be used to derive segregating generations where the genomic regions involved in tolerance to both stresses can be identified. These ideas have been introduced in the revised version. We have included a last paragraph in section 3.3. of Results (Principal Component Analysis PCA): ‘Based on the PCA scatter plot in the two-week trial (Fig. 3a), we identified four accessions (7, 5, 13, and 17) with highly negative values for the first component…’. Also, in the ‘Conclusions’ section, we have added the following sentences: ‘The identification of common bean accessions in the extremes of variation for susceptibility and tolerance to water deficit and salinity is of great interest for further studies on the physiological mechanisms of tolerance to both stresses. Also, the development of segregating generations after hybridisation between both types of materials can lead to the identification of genomic regions involved in tolerance to these stresses’.
Reviewer 2 Report
The manuscript entitled “The use of proline in screening for tolerance to drought and salinity in common bean (Phaseolus vulgaris L.) genotypes“ is devoted to an interesting topic. A comprehensive set of morphological parameters, leaf and root water contents as physiological parameters and proline content as biochemical parameter were used to test the drought and salt tolerance of 47 Phaseolus vulgaris genotypes grown under controlled greenhouse conditions. To assess the general responses to applied stress, the authors used a two-way ANOVA, Pearson moment correlations and PCAs. The data set is extensive and could be used in bean breeding programmes to enhance stress tolerance. The originality of the work is not questionable. The manuscript contains all of the components one would expect in a scientific article, but there is still room for improvements (as stated in specific comments).
Specific comments
Materials and methods
In this study 47 P. vulgaris genotypes from Spain (23), Columbia (19) and Cuba (5) were tested for drought and salt tolerance. By which criteria were genotypes divided into two groups and analysed independently? All seedlings were grown and treated at the same time, and just by visual symptoms the authors decided when to stop the stress treatments? Were the wilting and general decline symptoms the same for water and salt stress in all 18 genotypes after two weeks? How did the plants (29 genotypes) look like after an additional week of stress exposure? Did those plants have the same wilting symptoms as the first group?
The sampling methods should also be clarified. The authors stated they used five individual plants (biological replicas) of each genotype per treatment. Which leaves were sampled for the relative water content and proline quantification? It is known that the accumulation of proline under drought could be dependent on leaf age and in some species higher content of proline was found in younger leaves.
This study aimed to determine the overall response of the analysed genotypes to controlled water and salt stress treatments and to propose Pro as a suitable biochemical marker for the rapid selection of bean cultivars with a (relatively) high tolerance to drought or salinity. Why the authors did not apply water and salt stress for the two weeks in all tested genotypes and then performed statistical analysis to point out the most sensitive or more resistant cultivars?
Results
The results are well written. The number of figures and tables is appropriate.
Line 195: replace “cultivars” with “cultivar” inside the Table 2
Line 204: replace “(Table 1)” with “(Table 2)”
Line 257-260: add abbreviations in Figure 1 caption
Discussion
The first paragraph should be rewritten to be an introductory part of the discussion. In this form, this paragraph is a part of the Introduction. The second paragraph is a concise repetition of the results. The third paragraph is also part of the Introduction in this form. In the intermediate part of the discussion section, the authors should place their findings in a broader context and achieve a balance between interpretation and useful speculation. In general, the discussion section should be rewritten.
Author Response
We are grateful for your constructive suggestions and corrections. It is our belief that the manuscript is substantially improved after making the suggested edits. Additionally we have improved the quality of figures 3 a and b, as in the previous version some symbols were overlapping and the cultivars could not be properly identified.
The manuscript entitled “The use of proline in screening for tolerance to drought and salinity in common bean (Phaseolus vulgaris L.) genotypes“ is devoted to an interesting topic. A comprehensive set of morphological parameters, leaf and root water contents as physiological parameters and proline content as biochemical parameter were used to test the drought and salt tolerance of 47 Phaseolus vulgaris genotypes grown under controlled greenhouse conditions. To assess the general responses to applied stress, the authors used a two-way ANOVA, Pearson moment correlations and PCAs. The data set is extensive and could be used in bean breeding programmes to enhance stress tolerance. The originality of the work is not questionable. The manuscript contains all of the components one would expect in a scientific article, but there is still room for improvements (as stated in specific comments).
Thank you very much for your comments and positive assessment of our work.
Materials and methods
In this study 47 P. vulgaris genotypes from Spain (23), Columbia (19) and Cuba (5) were tested for drought and salt tolerance. By which criteria were genotypes divided into two groups and analysed independently? All seedlings were grown and treated at the same time, and just by visual symptoms the authors decided when to stop the stress treatments? Were the wilting and general decline symptoms the same for water and salt stress in all 18 genotypes after two weeks? How did the plants (29 genotypes) look like after an additional week of stress exposure? Did those plants have the same wilting symptoms as the first group?
The different genotypes were not divided into two groups at the beginning of the treatments, as there was no prior information regarding their relative stress tolerance. Treatments were indeed stopped after two weeks based on visual symptoms (especially wilting of the plants) for those cultivars apparently more sensitive to water deficit. The salt stress treatments of these cultivars were stopped at the same time, even tough, as indicated in the manuscript, under the conditions used in our experiments salt stress appeared to be less damaging for the plants than water stress. Therefore, the symptoms of the selected salt-treated plants after two weeks treatment were generally milder than those of the water-stressed counterparts. For the remaining, apparently more tolerant 29 genotypes, the symptoms after the additional week of treatment were, in general, similar to those observed after two weeks for the first group of cultivars. In any case, it is important to point out that there is a significant variability within each group of cultivars (as revealed by the results shown in the manuscript, for example by the PCA scatterplots) and that the classification of a given genotype in one or the other group was partly subjective. Since the two groups of plants were analysed independently, we cannot exclude that a given cultivar of the first group could be more tolerant to salt or water stress than some cultivar(s) of the second group, or vice versa. The decision to work with two independent groups of plants, taken on the spot, tried to avoid losing a significant part of the genotypes due to plant death if the treatments were prolonged for all of them, and to exclude the possibility that part of the most tolerant cultivars could not be distinguished if all treatments were stopped too early.
Not all seedlings were grown at the same time, to facilitate handling of the plants and, mostly, collection of the material at the end of the treatments. Treatments were performed gradually with groups of four or five cultivars and starting with a difference of two or three days between groups. It is important to mention that the conditions in the greenhouse did not vary during the whole period.
The sampling methods should also be clarified. The authors stated they used five individual plants (biological replicas) of each genotype per treatment. Which leaves were sampled for the relative water content and proline quantification? It is known that the accumulation of proline under drought could be dependent on leaf age and in some species higher content of proline was found in younger leaves.
At the end of the treatments we harvested the whole plants, collecting separately roots, stems and leaves. Therefore, for each individual plant, a pool of all leaves was used for determination of water content percentage and Pro quantification. An additional sentence has been included in section ‘2.2. Plant Growth and Stress Treatments’, describing in more detail the sampling procedure.
This study aimed to determine the overall response of the analysed genotypes to controlled water and salt stress treatments and to propose Pro as a suitable biochemical marker for the rapid selection of bean cultivars with a (relatively) high tolerance to drought or salinity. Why the authors did not apply water and salt stress for the two weeks in all tested genotypes and then performed statistical analysis to point out the most sensitive or more resistant cultivars?
We completely agree that it would have been simpler to apply the salt and water stress treatment for only two weeks to all tested genotypes. However, as mentioned above, we could have missed some important information, not being able to detect differences within the group of most tolerant cultivars if the treatments would have been stopped too early. What is important is that, despite some quantitative differences, the general conclusions of the work apply to the two groups of plants, analysed independently; particularly, the strong negative correlation of Pro accumulation and the relative stress tolerance of the tested cultivars. Also, identification of the most stress-tolerant genotypes is of great importance for plant breeders, which represents a practical aspect of our work that probably was not sufficiently highlighted in the previous version of the manuscript. In the revised version, as suggested by the first reviewer, we identified the most sensitive cultivars (among those exposed to two weeks treatments) and the most tolerant ones (among those from the three weeks treatment) based on the scattered plots of the PCAs.
Results
The results are well written. The number of figures and tables is appropriate.
Thank you very much for your comment
Line 195: replace “cultivars” with “cultivar” inside the Table 2
Corrected, as indicated
Line 204: replace “(Table 1)” with “(Table 2)”
Sorry for the mistake, which has been corrected
Line 257-260: add abbreviations in Figure 1 caption
Abbreviations added, as requested
Discussion
The first paragraph should be rewritten to be an introductory part of the discussion. In this form, this paragraph is a part of the Introduction.
The second paragraph is a concise repetition of the results. The third paragraph is also part of the Introduction in this form. In the intermediate part of the discussion section, the authors should place their findings in a broader context and achieve a balance between interpretation and useful speculation. In general, the discussion section should be rewritten.
The ‘Discussion’ section has been extensively modified. The first paragraph was deleted and partially transferred to ‘Introduction’, and part of the introduction that was redundant with the discussion was also eliminated. The second paragraph, now the introduction to the discussion, has been substantially reduced, deleting information redundant with that already mentioned in ‘Material and Methods’ or ‘Results’. The core of the ‘Discussion’ in the revised version focuses on the roles of Pro in the responses to stress in plants, in general, and in common beans, in particular, and on the possibility to use it as a biomarker for the selection of (relatively) stress tolerant cultivars and its practical importance in plant breeding. To better summarise the major findings of the work, a ‘Conclusions’ section has been introduced.
Reviewer 3 Report
The authors of this MS aimed to compare different bean genotypes under water deficit or salt stress. They used beans from Spain, Cuba and Colombia.
The MS contains a lot of data, with tons of statistical analysis. Unfortunately, I was not able to totally understand the circumstances that the authors used in their experiments. It is not clear how the plants were treated, it is said that the individual plants were planted in a 1,6 L pot, then the pots were placed in a tray, and the treatments were done in these trays. The plants were irrigated twice a week with 1.5 L deionised water (w/o NaCl), but it is not shown how much plants were in those trays, or at least how big was that tray at least. This is a piece of very important information. The other thing, which is not clear for me, is how can the Authors compare two different experimental designs. I think that the experiments should have been completed after two weeks, then every parameter would be comparable, which would make the situation much easier. The other thing I miss is the comparison of widely used genotypes with the selected ones.
The last thing I would like to mention is that I miss a clear conclusion, a future perspective from the discussion. The only thing that is highlighted is that proline can be used as a good stress marker, but is a well-known fact for decades now, so I think it is not enough.
Author Response
We are grateful for your constructive suggestions and corrections. It is our belief that the manuscript is substantially improved after making the suggested edits. Additionally we have improved the quality of figures 3 a and b, as in the previous version some symbols were overlapping and the cultivars could not be properly identified.
The authors of this MS aimed to compare different bean genotypes under water deficit or salt stress. They used beans from Spain, Cuba and Colombia.
The MS contains a lot of data, with tons of statistical analysis. Unfortunately, I was not able to totally understand the circumstances that the authors used in their experiments. It is not clear how the plants were treated, it is said that the individual plants were planted in a 1,6 L pot, then the pots were placed in a tray, and the treatments were done in these trays. The plants were irrigated twice a week with 1.5 L deionised water (w/o NaCl), but it is not shown how much plants were in those trays, or at least how big was that tray at least. This is a piece of very important information.
We apologise for not providing enough information on the experimental procedures in the original version of the manuscript; this has been corrected in the revised version. The size of the tray was 55 x 40 cm, and the number of pots per tray was 10. Therefore, each tray contained the plants of two cultivars (five replicas of each) subjected to the same treatment.
The other thing, which is not clear for me, is how can the Authors compare two different experimental designs. I think that the experiments should have been completed after two weeks, then every parameter would be comparable, which would make the situation much easier.
The same question was raised by the second reviewer. We completely agree that it would have been easier to apply the salt and water stress treatment for only two weeks to all cultivars. However, we could have missed some important information, not being able to detect differences between the most tolerant genotypes, if they were not significantly affected after two weeks of treatment. Nevertheless, we did not compare directly the two groups of cultivars subjected to different stress conditions, which is not possible, as rightly indicated by the reviewer. As mentioned in the text and shown in figures and tables, all analyses (two-way ANOVA, Pearson correlations, PCAs) were performed independently for each group. What is important is that, despite some quantitative differences, the general responses to the stress treatments of the plants from both groups follow the same patterns, and the conclusions of the work, particularly the strong negative correlation of Pro accumulation and the relative stress tolerance of the tested cultivars, also apply to the two groups of genotypes. Obviously, because they are not directly compared, we cannot exclude some overlapping between the two groups of ‘more sensitive’ and ‘more tolerant’ genotypes: a given cultivar of the first group could be more tolerant to water deficit, or to salinity, than one or more cultivars of the second group, and vice versa.
The other thing I miss is the comparison of widely used genotypes with the selected ones.
As in most reports by other authors, we used commercial varieties in a previous work on common bean (Al Hassan et al. 2016 and Morosan et al. 2017, both cited in the text), in which we established that, in response to salt stress or water deficit, Pro contents were lowest in the most tolerant cultivar, whereas it accumulated at the highest level in the most sensitive. This was the starting point of the present work, carried out in collaboration with personnel from the seed bank of COMAV, who provided the seeds of Spanish local landraces poorly known, and which we have characterised in a recent publication (Arteaga et al. 2019, Agriculture). Colombian seeds were kindly supplied by Dr. Beebe form CIAT, and our co-author Dr Yabor, who participated in this work during her stay in our laboratory, provided the Cuban material. By using seeds with different provenances, many belonging to local landraces, we tried to enlarge the knowledge on genotypes virtually unknown regarding their responses to stress, but that can represent an important source of material for breeders. In fact, it seems logical to assume that the most stress-tolerant genotypes will not be found amongst commercial bean cultivars, but rather amongst those landraces, less productive in terms of yield but adapted to local, often stressful field conditions.
The last thing I would like to mention is that I miss a clear conclusion, a future perspective from the discussion. The only thing that is highlighted is that proline can be used as a good stress marker, but is a well-known fact for decades now, so I think it is not enough.
Pro is indeed a good stress marker, in the sense that Pro accumulation in response to different abiotic stressors is well-established in many plant species. What is still controversial is the role of Pro in the mechanisms of stress tolerance in a given species or group of related species, and whether Pro accumulation is positively or negatively correlated with the relative degree of tolerance when comparing related taxa. In the revised version, we address this question in more detail, highlighting contradictory data in the literature indicating both, positive and negative correlations between Pro accumulation and the relative water deficit and salt stress tolerance of different Phaseolus vulgaris cultivars. We have included several new references related to this specific point, after an additional and extensive literature search. In both cases, most published work was based on the analysis of a few genotypes, often only one stress-tolerant and one stress-susceptible. Our extensive study on a large number of cultivars, and the extensive statistical analysis of the data, clearly indicate that there is an unequivocal association of higher Pro contents with stronger growth inhibition; that is, with a higher sensitivity to stress of the bean cultivars. Therefore, our results support the use of Pro as a biochemical marker for the large-scale screening of bean cultivars, specifically to exclude the most sensitive, those accumulating Pro to higher concentrations in response to water or salt stress. We also included a more practical conclusion. The identification of common bean accessions in the extremes of variation for susceptibility and tolerance to water deficit and salinity (as we included in the new version) is of great interest for further studies of physiological mechanisms of tolerance to both stresses. Also, the development of segregating generations after hybridisation between both types of materials can lead to the identification of genomic regions involved in tolerance to these stresses.
Round 2
Reviewer 1 Report
The authors carried out the suggestions and edits provided in the revised version.
Reviewer 3 Report
Thank you for your detailed answers.
