Morphological Features and Biomass Partitioning of Lucerne Plants (Medicago sativa L.) Subjected to Water Stress
Round 1
Reviewer 1 Report
Few concerns have been addressed.
Author Response
Reviewer 1
Few concerns have been addressed.
Response
Minor changes have been made to the Introduction and Methods and major changes to the Results and Discussion section see response to Reviewer 3.
Reviewer 1
English language and style are fine/minor spell check required.
Response
Thank you for this comment. A final spell check has been conducted and the paper edited by the co-author born and educated in the origin of the English language.
Reviewer 2 Report
The revised manuscript is much improved and is suitable for Agronomy.Author Response
Reviewer 2
Moderate English changes required.
Response
The whole paper has been subject to review by the co-author born and educated in the origin of the English language.
Reviewer 2
The revised manuscript is much improved and suitable for Agronomy.
Response
Thank you for this positive comment.
Reviewer 3 Report
The manuscript describes a pot experiment in which the effects of graduated different soil dryness on morphological parameters of alfalfa plants at different stages of development are investigated. A morphological adaptation of the plants to the water deficit was demonstrated. This was most evident in the latest examined developmental phase of the plants (flowering) and under lowest water supply.
In the introduction the problem is worked out in an excellent way and with the integration of numerous references. In the chapter "Materials and Methods", the experimental procedure is described in sufficient detail and the statistical analyses are presented clearly and precisely. The results are clearly presented in standardized graphics. The discussion, however, is not yet satisfactory: In the first part (including Table 1), only the results are summarized repeatedly. This part should be shortened and attached to the chapter "Results". In contrast to the introduction (27 citations), the discussion contains only 6 citations. Reference [28] was not found in the text, why is it not shown on line 260? The discussion should be revised. Important aspects such as the transferability of pot experiments to field conditions are not addressed. Comparable studies are not sufficiently included in the considerations.
For example:
M Abid et al. (2015): Variation in phenological parameters of Alfalfa Medicago sativa response to water stress Int.J.Curr.Microbiol.App.Sci 4: 532-540 or
Drought Strategies for Alfalfa (ANR Publication 8522, July 2015, http://anrcatalog.ucanr.edu
The results could be used to model the relationship between irrigation and productivity and derive an optimal marginal irrigation rate for practical purposes. This would greatly enhance the novelty value of the study.
Author Response
Reviewer 3
The manuscript describes a pot experiment in which the effects of graduated different soil dryness on morphological parameters of alfalfa plants at different stages of development are investigated. A morphological adaptation of the plants to the water deficit was demonstrated. This was most evident in the latest examined developmental phase of the plants (flowering) and under lowest water supply.
In the introduction the problem is worked out in an excellent way and with the integration of numerous references. In the chapter "Materials and Methods", the experimental procedure is described in sufficient detail and the statistical analyses are presented clearly and precisely. The results are clearly presented in standardized graphics.The manuscript describes a pot experiment in which the effects of graduated different soil dryness on morphological parameters of alfalfa plants at different stages of development are investigated. A morphological adaptation of the plants to the water deficit was demonstrated. This was most evident in the latest examined developmental phase of the plants (flowering) and under lowest water supply.
In the introduction the problem is worked out in an excellent way and with the integration of numerous references. In the chapter "Materials and Methods", the experimental procedure is described in sufficient detail and the statistical analyses are presented clearly and precisely. The results are clearly presented in standardized graphics.The discussion, however, is not yet satisfactory: In the first part (including Table 1), only the results are summarized repeatedly. This part should be shortened and attached to the chapter "Results".
Response:
Thank you for this excellent summary and positive feedback.
Reviewer 3
The discussion, however, is not yet satisfactory: In the first part (including Table 1), only the results are summarized repeatedly. This part should be shortened and attached to the chapter "Results".
Response
Agreed. The first part of the Discussion has now been placed in the Results along with Figure 4 and Table 1. We trust that this aspect of the revised paper is acceptable.
Reviewer 3
Reference [28] was not found in the text, why is it not shown on line 260?
Response:
Thank you for pointing out this oversight. We double checked all the References carefully and found that there are two reference ones in the list so we have reordered and revised the References accordingly. We have also replaced two references and added an additional reference.
Comparable studies are not sufficiently included in the considerations.
For example:
M Abid et al. (2015): Variation in phenological parameters of Alfalfa Medicago sativa response to water stress Int.J.Curr.Microbiol.App.Sci 4: 532-540 or
Drought Strategies for Alfalfa (ANR Publication 8522, July 2015, http://anrcatalog.ucanr.edu
Response:
Many thanks for your suggestion. We have referred to the ANR Publication 8522 and quoted from it in the first paragraph of the Discussion (see below), but the paper by Abid et al. 2015 is concerned with seed production in lucerne and is not applicable to this study which was conducted in the vegetative phase. The first paragraph of the Discussion now reads:
"Water deficits are well known to reduce the growth of most crops and pastures (e.g., 3, 8, 10, 25, 26). We chose to study the effect of water shortage on the early growth of lucerne as it has been stated that ”seedling alfalfa (alfalfa during its first 2 to 5 months of growth) is not as drought tolerant as is established alfalfa (lucerne), so moisture stress at this time should be avoided. If soil moisture is inadequate during the establishment phase, excessive plant mortality and stand loss can occur” [26]. However, in arid and semiarid areas in which lucerne is grown, drought stress cannot always be avoided during establishment and early vegetative growth. In this study, we show that water deficits in the vegetative stage reduce the production of aboveground forage, largely as a result of a reduction in leaf biomass and leaf area due to the production of smaller leaves, particularly under severe stress (45% PC). The water stress treatments also reduced the length of the taproot, but increased lateral root development and, in the moderate stress (65% PC) treatment increased the root biomass. The increase in dry weight of the roots in the moderate and severe water stress treatments with development was proportionally greater than in the leaves, resulting in the R/S ratio increasing with increasing water deficit."
Reviewer 3
The results could be used to model the relationship between irrigation and productivity and derive an optimal marginal irrigation rate for practical purposes. This would greatly enhance the novelty value of the study.
Response
We disagree. While the study is relevant to irrigation, we consider a much more comprehensive study with different levels of irrigation is required to use the data in modelling the relationship and productivity. We have modified the final paragraph of the Discussion to emphasise what we consider relevant implications of the study:
"The different water stress levels applied in our experiment induced different growth responses in lucerne, indicating that the severity of water stress and the development of water stress must be taken into consideration when developing a management strategy for lucerne. The observation that taproot length was reduced by the reduction in water availability suggests that if irrigation is available, application of water to prevent a reduction in taproot growth would be beneficial, as anecdotal evidence suggests that the root system should be 1 to 1.5 m depth in the soil before the onset of severe stress [26]. If irrigation is not available, withholding grazing or harvesting in the late vegetative stage should be avoided to maintain biomass and reserves for regrowth when the water shortage is relieved."
Round 2
Reviewer 3 Report
My comments were mainly taken into account.
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
The topic addressed in this manuscript could present some potential interest to the readers. However, I have several concerns regarding the experimental design, data presentation, conclusions and applicability of findings.
Starting from the given keywords, the word transcription doesn't apply to this study at all.
The authors aimed to evaluate the effect of water deficits on the growth and biomass distribution in different tissues of Lucerne. What is the applicability of these findings? In the conclusion the author has suggested to consider the outcomes of this study while designing an irrigation strategy for Lucerne to save water but the results as well as discussion aren't clearly supporting this idea.
The experimental design is seriously flawed, for each growth stage/time point the authors have harvested three plants per treatment from single pot (line 93-94) that doesn't follow the claim of completely randomized design. In all the graphs, the error bars aren't shown, probably because reading from just 3 plants would present horribly large error bars.
Figures presenting graphs are not of good quality. Figure panel labels are overwriting the bars and are intermingled with the other labels. Figure legends are improper. For example, in figure 4 the author has used TA, TB and TC labels but their description is missing in the legend. On the other hand figure 3 legend has the description but none of these labels are used in the figure.
Reviewer 2 Report
Originality/Novelty: Is the question original and well defined? Do the results provide an advance in current knowledge?The objective of the study was clearly stated. The results of the study advance knowledge of growth and biomass accumulation during various growth stages in Lucerne during water-limited conditions. Could result in additional studies with Lucerne or with other agriculture species.
Significance: Are the results interpreted appropriately? Are they significant? Are all conclusions justified and supported by the results? Are hypotheses and speculations carefully identified as such?The significance of results are interpreted correctly. There is justification for the study but no specific hypotheses are stated.
Scientific soundness: is the study correctly designed and technically sound? Are the analyses performed with the highest technical standards? Are the data robust enough to draw the conclusions? Are the methods, tools, software, and reagents described with sufficient details to allow another researcher to reproduce the results?The authors’ used a randomized complete block design but did not include it in the analysis.
Interest to the Readers: Are the conclusions interesting for the readership of the Journal? Will the paper attract a wide readership, or be of interest only to a limited number of people? (Please see the Aims and Scope of the journal)The paper should attract a wide enough group of readers given the aims and scope of the journal.
Overall merit: Is there an overall benefit to publishing this work? Does the work provide an advance towards the current knowledge? Do the authors have addressed an important long-standing questions with smart experiments?While the current manuscript would have greatly benefited from physiological measurements to support the authors’ findings, few studies consider resource allocation in the context of developmental stage and climatological issues such as drought, which is important especially with species utilized for agriculture. For this reason, there is benefit in publishing this work.
English Level: Is the English language appropriate and understandable?YES
Please provide an overall recommendation for the publication of the manuscript as follows:
Accept in present form: The paper is accepted without any further changes.
Accept after Minor Revisions: The paper is in principle accepted after revision based on the reviewer’s comments. Authors are given five days for minor revisions.
Accept after minor revisions.
Reconsider after Major Revisions: The acceptance of the manuscript would depend on the revisions. The author needs to provide a point by point response or provide a rebuttal if some of the reviewer’s comments cannot be revised. Usually, only one round of major revisions is allowed. Authors will be asked to resubmit the revised paper within ten days and the revised version will be returned to the reviewer for further comments.
Reject: the article has serious flaws, makes no original contribution, and the paper is rejected with no offer of resubmission to the journal.
REVIEW REPORT
Luo Y-Z., Li G., Yan G., Liu H., Turner N.C. Morphological features and biomass partitioning of Lucerne plants (Medicago sativa L.) under water stress
A brief summary (one short paragraph) outlining the aim of the paper and its main contributions.In the present study, the authors are investigating the impact of drought stress and allocation strategy (i.e. vegetative growth, leaf features, and biomass) during early developmental stages of Medicago sativa L. cv. Xinjiangdaye. This species/line was introduced to the region 150 years ago with little research available to determine traits contributing to the success of this species under arid-semiarid conditions (annual rainfall is 250-400 mm). In general, the authors found that drought impacted biomass accumulation and the distribution of resources above- and below-ground. Growth and biomass was also influenced by water limitation based on plant developmental stage. The intensity and timing of water stress should be considered when developing irrigation and water use strategies.
Broad comments highlighting areas of strength and weakness. These comments should be specific enough for authors to be able to respond. The authors use 85%, 65%, and 45% PC for full water supply, moderate water supply, and severe water supply. How were these values selected? How does the reader know that 45% is severe water stress for this species and in this region? The authors utilized a randomized complete block design, but there is no mention in the statistical analyses of how blocks were included in the analysis. Also, and fundamental of ANOVA analyses, did the data meet the assumptions of ANOVA (i.e. normal and equal variance) or were the data transformed for analyses. If so, then say so. Define focal stages in the text. What is the difference between seedling, branching, squaring, and flowering stages? Specific comments referring to line numbers, table or figures. Reviewers need not comment on formatting issues that do not obscure the meaning of the paper, as these will be addressed by editors.Lines 36-38: Excellent point!
Lines 65-66: Based on the annual rainfall (250-400 mm), the region is semi-arid by definition. However, as stated, it sounds as if M. sativa Xinjiangdaye is successful in water-limited conditions, but not under arid-semiarid conditions. The authors need to clarify this point?
Lines 76-77: What was the frequency of nutrient solution addition? Was this a one-time application? How did the authors determine the type of nutrients to be added?
Figure 1, Figure 2, Figure 3 & beyond: Add/include standard error bars!
Lines 192-211: Redundant and has already been stated in lines 152-171.
Lines 270-272: Why would plants with fewer leaves and smaller leaves have lower water-use efficiency if photosynthesis is maintained? Also, have other studies of Lucerne measured photosynthesis under drought conditions? It would be nice to briefly refer to results of photosynthesis/physiology studies that support the authors’ findings. If not, then perhaps future studies are needed.
Reviewer 3 Report
General comments:
This is a fairly straight-forward examination of water limitation effects on lucerne growth, development, and yield. It is a container growth design with the individual container as the replicate. Hence it suffers from many of the same pitfalls as any other such container grown experiment. However, the biological strain applied to the system was very well managed. Thus, the strain was slowly applied and maintained over time, unlike many container experiments particularly those typically done by molecular biologists in which a sudden drought stress is applied. It was unfortunate that this was not pointed out in the discussion. Many of the results were unsurprising. Limited water limited growth. Of particular interest to this reader was that lateral roots were increased in response. Again, this result and its importance was not addressed in the discussion. This was a very well executed experiment with considerable effort expended both in the writing and the field work. I think the authors should address these and point to any other strengths of the work and of the importance of such results in the Discussion section. The work was "undersold" in this reader's opinion. Too, the Discussion section is a great place to acknowledge weaknesses and limitations in the design or to rationalize what was done, if the authors like. The manuscript is highly applied, which is consistent with a highly applied field such as Agronomy. It is suitable for the journal and of interest to the readers.
Stats:
If the results of a particular set of analyses are not presented, then the methods should probably not be mentioned. Eliminate description of the correlation procedure in the stats methods description (unless I missed results of such in the results section or somewhere). Also, I understand that statisticians want us common folks to 1) first use an anova to ask the whether there are any differences worth pursuing and then 2) employ means separation to identify where the differences might lie. But I argue that plants do grow and that this is known. Thus, it is not unsurprising to find that there is a difference in there. Moreover, the results of the anova which I assume was a one way analysis given the way means separation was done, were not presented or referenced. Too, mention of using anova begs the question of whether it was a single- or or multi-way analysis, which would have been considerable effort and perhaps beyond the purview of the work. At the very least the stats section needs clarified.
I think stats referenced in Figs 1-3 were inappropriately applied. What is the importance of looking at the significance of a response across growth stages? The data are not discussed in this way. The data are discussed as differences across treatments WITHIN growth stages. Hence, the data should be analyzed in this way. This would be a more meaningful and simpler way to treat and present the data. The authors might even be able to get away with a multiway t-test with Tukey or Bonferroni correction within each treatment. Perhaps this would minimize or avoid the strict requirement for normally distributed data, since n=4* and a t-distribution is used? Could the authors either provide a rationale for which stats were applied in the Discussion, or redo this? The data should be reanalyzed and means separated as the differences are discussed in the text.
*That is, I think n=4 containers. In the stats section, the authors might want to add the text "The individual container was treated as the replicable unit" (or however that would be said to avoid potential confusion by the reader). Replicable? Replicate-able?
Figures were of poor quality and must be improved before publication. It appeared that Sigmaplot or some other graphical routine was used so improvement can be made with little problem.
Suggestions for improvement follow:
In Figs 1-3 the panels are identified with letters in the busy corner of the graph. This should be in the negative space in the upper left. Figure 2 is an excellent example of this mistake. Place the panel letters in the upper left corner within the rectilinear borders of the graph. The legend will easily fit beneath this. The border in Fig 2 is gray. Why not black? Why are the fonts in panels A and B different sizes? Tick marks should always point to the values. This way the origin has a tick mark.The ticks defining Growth Stage pointing inward are excellent.
Look at Fig 1B. The legend is wonky. Why is the font on the significance values larger than the numerical values? The label on the ordinate (y-axis) is wonky, too. A space should be inserted in the units:
(m2 leaf-1) not (m2leaf-1)
In Fig 1B the background of the text box in labeling it panel B is opaque so that it cuts off the significance letter. The text box should have been resized or should have had a "no color" or "transparent" text box to save space. There are no error bars, which is not a problem statistically but it conveys little information about variability, plus the caption leads us to expect them. Individual panel border lines were heavier than the axes and tick marks. Border marks should be simple crisp and black. When there is no need for color, color should not be used. Using color on the bars is good. But the colors should be chosen to imply treatment and have easily understood meaning. Blue as high water level is good. But maybe swap out red and green. The dry hot red color is more consistent with drought, No? (Just a suggestion).
What is the value of comparing means of each treatment across growth stages? Why not simply compare within each growth stage?
Figure 4 is great. VERY well done. Beautiful grouping and labeling on the x-axis. But note that here there is no WW (well watered), MS (Moderate Stress), and SS (Severe Stress). Instead, the authors opted for Treatments A thru C. Indeed, throughout the manuscript they are labelled two different ways. I suggest picking one method of designating and labeling the treatments and sticking to it. Personally, I find WW more informative than TA. I suggest change everything in the manuscript to WW, MS, and SS. The y-axis label of 120% can be eliminated. One way to do this in Sigmaplot and maintain the balance of the figure is to define the scale as going from 0 to 119.99. This will make the 120% tick "go away". If the tick is required or preferred then edit it out using an image editor or place a text box over the label? Too, most people like the outward facing tick, but an argument can be made for inward facing in that it saves space for tick labels. I am an old traditionalist here. Either way I guess. But seriously, the other Figs need a lot of work. The authors might consider putting error bars on the graph, but by now their eyes are glazed over from all the suggestions. If it is too much to ask just say so.
I recognize that good graphs are NOT easy to make. They take time. Lots of time. It is much appreciated that graphs were used rather than just dumping data into a table. But we should all try to make them as clear as possible. The point of making them is to communicate information. Graphs are not a place to simply regurgitate all of the data.
Minor comments:
Why are lines 290-299 in the discussion? Aren't they more properly results? Too, the Discussion section is the first place the results in Fig 4 are mentioned. Results should be presented in the Results section. Please separate results from discussion of them. Or the authors could explain why this cannot be done.
Growth is undeniably the most unambiguous measure of biological strain resulting from water limitation. It is also the relevant measure given that shoot biomass is directly related to yield in this crop plant. The authors should review the concept of Relative Growth (see Methods and Table 1). The methods state RGR was used (which would be interesting to see) but Table 1 presents absolute growth rates. RGR is not addressed in the text either. Maybe pick one or provide both?
In the future, the authors might consider looking at node accretion rate in response to water limitation in the experimental design stage. This can be a non-destructive measurement, though this represents considerable work.
Finally:
After looking over this review it seems rather negatively opinionated and lengthy. Please understand that this reviewer is attempting to provide constructive comments throughout, rather than simply point out flaws and leave it at that. It is sincerely hoped that the corresponding authors will accept the suggestions in the vein in which they are offered.
