Growth, Physiological, Biochemical, and Transcriptional Responses to Drought Stress in Seedlings of Medicago sativa L., Medicago arborea L. and Their Hybrid (Alborea)
, Effie Sarri 1, Μaria Goufa 1, Xristina Vaharidi 1, Alexia Tornesaki 1, Maria Psychogiou 3, Penelope J. Bebeli 1 and Εleni M. Abraham 4,*Round 1
Reviewer 1 Report
Summary:
Medicago arborea is considered the oldest species of the genus and is a drought tolerant species. Therefore, there have been several attempts to introduce favorable M. arborea traits, such as drought tolerance, to M. sativa. For example, hybrids between M. sativa and M. arborea (named Alborea) were produced in the USA and Australia.
The objective of this manuscript is the physiological analysis of the responses of the Medicago parental and hybrid lines to drought. Several parameters (stem elongation rate, seedlings’ height, relative water content, ect …) were assessed and, in general, the hybrid Alborea performs better than its parental lines in optimal water conditions, but the parental species are more drought tolerant than the hybrid in water deficient conditions. In addition, several antioxidant capacity parameters as well as the transcriptional activation of different drought-regulated genes were also studied in the three species under control and drought conditions. Again, the hybrid Alborea exhibited the lowest induction of drought- and stress-regulated genes.
The experiments are well designed and all required controls are included. The statistical analyses are performed adequately. The proposed conclusions are not over-stated, but are supported by the results of the manuscript.
In general, the manuscript is well written and it is a nice description of the physiological and transcriptional responses of the Medicago hybrid Alborea and its parental lines to drought. I can only suggest few minor issues to improve its readiness and for the benefit of the not-specialised readers.
Comments:
Abstract: well written. The objectives are clearly defined, and the main analyses and results described.
1) Introduction: The introduction is quite complete; the general ideas of plant responses to drought and the specific alfalfa or Medicago sativa responses are well reported. In line 88, the category “a.” is missing (or delete “b.” and “c.”).
Materials and methods are well described; they include all required details to reproduce the experiments as well as statistical analysis details.
Results:
2) Abbreviations: please mention that SE stands for “stem elongation rate” in the first sentence of the results (I know it is already mentioned in the Methods, but it’ll help many readers). Similarly, please add what the abbreviations RWC and gas-exchange parameters of Pn, gs, E and WUE stand for in the first sentence of section 3.2. Maybe, a short description of these parameters (not in details as in the Discussion) will help the readers that are not so familiar with them. At the beginning of paragraph 3.3, please explain what ROS, ABTS, SOD, FRAP, POD and MDA stand for. I know that most abbreviations are already mentioned in the Methods, but a short description here (not only in the Discussion) may also help general readers.
3) Figure 1: although the statistical analyses of these results are presented in table 2, I think that adding the standard error or deviation in these graphs will help the readers to appreciate better the value of these data.
4) line 217, the sentence “while for the studied population of M. sativa and M. arborea by only 13% and 5% respectively” lacks the verb.
5) line 285: consider changing “not profound” to minor or small.
6) Figure 3: the expression of the genes is presented as relative expression, but it is not described relative to what. The use of actin as internal housekeeping control gene is only briefly mentioned in the Methods, but it should also be described in the figure legend. In addition, panels are not labeled as “a” nor “b”.
7) Figure 4; the scale at the bottom normally starts from the lowest value on the left to the highest value on the right. Not a big deal, but I think I’ll be easier to interpreter for most readers.
Discussion:
8) line 395, I think the comma should be removed in “Due to the fact, that”
9) It is interesting that AP2/EREB, the gene most responsive to drought, is highly induced in leaves in both parental Medigaco (but not in the hybrid) whereas the opposite occurs in the roots (high induction in the hybrid Alborea but minor induction in the parental lines). Can you comment on these differences? What is its biologically value?
Conclusions:
10) Please avoid colloquial expression such as in line 430 “our hybrid”; it should be Alborea hybrid.
Author Response
Our point-by-point response to the reviewer comment is in the upload file
Reviewer 2 Report
The manuscript describes the drought responses and mechanisms for drought tolerance in the economically important crops M. sativa, M. arborea and the hybrid Alborea. Alfalfa (M. sativa) is an important forage legume while M. arborea is used for forage production in the Mediterranean area.
The authors found that, under drought conditions that the parents M. arborea and M. sativa both showed higher antioxidant capacity and induction of drought-responsive genes suggesting that these species are drought tolerant compared to the hybrid Alborea. The observations that the hybrid has higher stem elongation and photosynthesis rates only under non-drought conditions is important for the use of these forage legumes in Mediterranean areas that are prone to drought.
The manuscript is very well written, particularly the discussion and the results well designed and presented. However, a minor comment is that the manuscript could benefit from being more specific in the results description (see below) particularly describing the differences between M. sativa, M. arborea and Alborea.
Minor comments below.
Check the use of italics is consistently used throughout the manuscript for species names and genes.
METHODS
Line 163. Add the absorption / nm for which the SOD activity was determined.
Line 171. subscript for H2O2
Line 200. MS-Actin-2
RESULTS
Line 221. State that these reductions are in comparison to the controls.
Table 2. Give a description of the drought stress dates in the Methods under section 2.3
Line 215. It would be clearer for the reader to be specific about the difference between the species by naming them specifically rather than using” between entries”
Figure 1a. It is worth mentioning that while Alborea still the tallest (cm) even under drought stress.
Figure 1b. Is the difference between the two parent species significant? State in the text.
Line 238. The Pn of Alborea is reduced after the first week of drought stress but this is still significantly higher than that of the parents at this stage. Please amend this paragraph to reflect this.
Line 258. “POD activity and protein content increased significantly among the entries under drought treatment.” Please be specific, which species? Alborea has the highest POD activity increase?
Line 276. “The transcript abundance of all genes except ZFN was higher under drought stress (Fig 3a&b)” Please be specific is this for all species or only the parent species?
Figure 3. Describe the gene that are down regulated and which species in the results not only those that are upregulated.
In Figure 3 is gene expression relative to the controls? Also add the housekeeping gene used to the figure legend.
Line 279. “On average, all genes examined were preferentially up-regulated 278 in leaves compared to roots, with the exception of P5CS1” Is this true ? what about SIMKK ?
DISCUSSION
Line 343 “This can be advantageous under short periods of severe drought stress but could have a negative impact when drought stress is imposed for long periods”. Yes, would it be interesting to see how long
Line 345. As above the Pn of Alborea is reduced after the first week of drought stress but this is still significantly higher than that of the parents at this stage. Please amend to reflect this.
Line 357. “ a substantial increase was measured for POD. “ which species ?
Line 389. Down regulation of transcripts (particularly in the hybrid) is not mentioned in the results but is discussed in the discussion section (line 390). Please amend this for the genes that show down-regulation.
Line 409. Is it significant/worth mention that this was found in rice and that you have evidence for its importance in a dicot species?
Line 419. Add species name for Chili.
Line 436. “Several other studies have indicated that hybrids with increased drought tolerance also exhibited lesser changes of Pn and gs in water deficit”. Seems contradictory to Line 429, “On the other hand, the increased growth of hybrids and their tolerance to stressful environments act in an antagonistic manner.” amend to reflect this is not always the case.
Line 448. “………..tolerant that their hybrid to drought”
Author Response
Our point-by-point response to the reviewer commonts is in the upload file.
Author Response File: 
Author Response.docx
Reviewer 3 Report
The manuscript represents a very interesting study comparing vigour and drought sensitivity of the Alborea hybrid to its parents. These kind of studies are of high importance to agricultural applications of hybrids in adverse environmental conditions. The manuscript is very well written and organized and I have only a few questions and minor issues.
Major questions:
- How were the target genes selected? Why exactly these genes? Is the selection based on previous RNAseq or microarray analysis? In general, information about the choice of the genes, their functions, the pathways they take part in, and the reasons behind focusing exactly on their expression is missing in the introduction. There is information about the gene function only in the discussion.
- What about other members of the gene families? Why exactly the expression of the selected genes was followed? Why are they chosen over other members of their families? What if the changes in expression of one family member are compensated by another?
- Why were genes encoding SOD and POD not included in the set of genes for the transcriptional measurements? It would have been nice if the expression of genes encoding the POD and SOD enzymes was followed to reveal more about the level of regulation of their activity: it would show if the enzymes are upregulated already at the transcriptional levels or only their activities increase a in drought conditions.
- Line 136-137: Are the conditions used for the gas-exchange measurements in alfalfa well established? If so, please add a reference.
- Are the same plants used for the physiological, transcriptional and biochemical measurements? In the section describing the growth conditions, only shoots are mentioned to be harvested and the expression levels are also measured in roots? Are these the roots of the same plants?
Minor issues:
- In the abstract, as well in the the introduction, the abbreviated names of the genes are not explained. The first time an abbreviation is mentioned, it should be explained.
- Line 33-34: "Under drought conditions, the studied population of M. arborea showed less reduction of relative water content, all gas exchange parameters, less lipid peroxidation and more antioxidant capacity" Less reduction and less lipid peroxidation compared to what?To Alborea?
- Please correct CO2 to CO2 all over the text. The same for FeCl3 (line 148)
- Table 1: all abbreviations should be explained in a legend or a footnote
- Line 206: Please change anova to ANOVA and make it consistent throughout the whole text.
- Table 2: Are the values after the ± standard errors or standard deviations? Please explain in a legend or a footnote
- Table 2: Please also explain all abbreviations in a legend or a footnote. The same for all tables and figures.
- Figure 1: Error bars are missing. Are the presented data means? If so, error bars should be added and explained in the legend. Please explain the type of data in the legend.
- Line 237: "Pn of the entries did not significantly differ", but in the figure, Al is labeled with C and the other two entries are labeled with B, which means that they differ significantly.
- Line 241: "gradually was decreased". The correct phrasing is : "was gradually decreased"
- Figure 2, panel d: M. sa at D1 has no letter above the bar, indicating the statistics. Is this a typo or there is another reason?
- Figure 3: Labeling of the panels is missing the figure, but present in the legend (a and b).
- Line 437: "compared to drought-sensitive hybrids and up-regulated ROS scavengers", the ROS scavengers make no sense in this sentence.
Author Response
Our point-by-point response to the reviewer comments is in the upload file.
Author Response File: Author Response.docx
