Barley Plants Overexpressing Ferrochelatases (HvFC1 and HvFC2) Show Improved Photosynthetic Rates and Have Reduced Photo-Oxidative Damage under Drought Stress than Non-Transgenic Controls

Round 1

Reviewer 1 Report

The manuscript deals with the investigation of the roles of two Ferrochelatase genes in barley under drought and oxidative stress. The Authors performed a series of growth experiments / physiological measurements using cv. Golden Promise, the most efficient genotype for genetic transformation in barley, and the tigrina-d12 mutant of cv. Bonus. Overall, procedures and methodologies are appropriate, as well as the results are clearly presented. The topic is relevant for the barley/wheat scientific community, and as such, I think that the manuscript is of interest to the audience of MDPI-Agronomy. However, in recommending its publication, I suggest the Authors to take into consideration the following comments:

- The Authors should stress a bit more the novelty of their findings respect to the existing literature, in particular as far as the involvement of both FCs instead of one is concerned.

- Are FC1 and FC2 the only two Ferrochelatase genes in barley? Are these two genes harboured by the same chromosomal region? Does this region correspond to some phenotypic effect revealed by previous QTL/MTA studies?

- L102-107: Give some detail on the growth chamber (greenhouse?) used for the experiments. Also, indicate whether any positional effect was detected and how pots/replicates were spatially arranged in the chamber.

- L128 “was considered to be equivalent…”: Do you have a reference supporting this statement?

- Make uniform the unit for pressure measurement between text (MPa) and Figures 1 and S1 (bar). I recommend using Pascal as it is standard for SI.

- L160-161: Was this identification made on standard agarose gel?

- L161 and L201 “Description of… are described in…”: Please, avoid repetition.

- L162 and L201: With respect to Table S1, clarify that this table summarizes all primers used in your work.

- L325: Move the caption immediately under Figure 4.

Author Response

Reviewer 1 comments

 

• The Authors should stress a bit more the novelty of their findings respect to the existing literature, in particular as far as the involvement of both FCs instead of one is concerned.

1. The introduction of the manuscript was revised with existing literature to highlight the intriguing controversy regarding distinct roles of FC isoforms in photosynthesis and drought stress tolerance. Even though previous studies have proposed that FC2-derived heme pool is solely used for photosynthesis, whereas heme produced by FCI is implicated in enhancing stress tolerance, our study provide a body of evidence to suggest that both FC isoforms are able to improve photosynthetic performance and protect plants from photo-oxidative damage under drought conditions. Please see lines 90-131.

• Are FC1 and FC2 the only two Ferrochelatase genes in barley? Are these two genes harboured by the same chromosomal region? Does this region correspond to some phenotypic effect revealed by previous QTL/MTA studies?

1. Our bioinformatics analysis revealed that there are only two FC genes in barley. A detailed experimental procedure on identification of HvFC sequences have been included in a separate manuscript, which is concurrently under peer review in the Agronomy Journal [35]. Two identified HvFC genomic sequences were used in a BLASTx search in IPK Barley BLAST server (http://webblast.ipk-gatersleben.de/barley) and it was found that HvFC1 and HvFC2 are localized to two different chromosomes (5H and 1H respectively). Based on publicly available information we have not as yet identified any obviously associated agronomic trait data to these loci.

• In L102-107: Give some detail on the growth chamber (greenhouse?) used for the experiments. Also, indicate whether any positional effect was detected and how pots/replicates were spatially arranged in the chamber.

1. All the growth room conditions have been mentioned in the methodology. However, in order to provide more clarity, the text was slightly modified. Please see lines 148-150.

• In L128 “was considered to be equivalent…”: Do you have a reference supporting this statement?

1. A relevant reference is included. Please refer to line 177.

• Make uniform the unit for pressure measurement between text (MPa) and Figures 1 and S1 (bar). I recommend using Pascal as it is standard for SI.

1. This has been amended. Please refer to Figure 1 and S1.

• In L160-161: Was this identification made on standard agarose gel?

1. As mentioned in line 206, PCR products before and after digestion were analyzed in 2% agarose gel.

• In L161 and L201 “Description of… are described in…”: Please, avoid repetition.

1. “Description of” was removed to avoid repetition.

• In L162 and L201: With respect to Table S1, clarify that this table summarizes all primers used in your work.

1. We have reviewed the Table and can confirm all primers used in this paper have been listed in Table S1.

• In L325: Move the caption immediately under Figure 4.

1. Figure 4 was moved accordingly.

Reviewer 2 Report

In this MS author discussed about Barley plants that overexpressing Ferrochelatases (HvFC1 and HvFC2) show improved photosynthetic rates and have reduced photo-oxidative damage under drought stress than non-transgenic controls. Over all manuscript is Ok but I have my concerns.

1. Figure 1, 2, 4, 7, 8 are poorly represented. Word are hidden behind the images. it seems that MS is submitted in hurry. For example 0 hidden figure 1, null hidden in Figure 2, same with other figures.
2. Author claimed to have 3 independent lines in MS but Figure 1 represent only one line each gene. Its not scientifically correct to show only one line. Which generation author have used for experiment and are those line are stable please mention everything.
3. There is no information how the construct is made? Make one map of construct and how many independent lines you have got? Show proof with genomic DNA PCR and how much the expression level increase. Show qRT-PCR.
4. Figure 4, Figure 7c and Figure 8c in qRT-PCT data where is wild type expression.
5. Table one add how much is PCR product length is?
6. Figure 2 is not consistent Lane 1 is zoomed where as lane 2 and 3 is different. Imaging should be uniform. Also there is no fresh weight or green weight measured after the drought stress.
7. Analyse some drought stress marker gene analysis and make some mechanical pathway to indicate your genes.

Author Response

Reviewer 2

 

• Figure 1, 2, 4, 7, 8 are poorly represented. Word are hidden behind the images. it seems that MS is submitted in hurry. For example 0 hidden figure 1, null hidden in Figure 2, same with other figures.

All the figures have been modified accordingly.

• Author claimed to have 3 independent lines in MS but Figure 1 represent only one line each gene. It’s not scientifically correct to show only one line. Which generation author have used for experiment and are those line are stable please mention everything.

We believe that the reviewer is refereeing to the Figure 3. As mentioned in the manuscript, we stated the observation that all the independent transgenic lines ectopically overexpressing either HvFC1 or HvFC2 did not wilt even 8 days after water withholding whereas controls wilted progressively. Considering the phenotypes of all transgenics were consistent, only one representative transgenic line per construct was used in the Figure 3. In order to provide more clarity, lines 297 and 312 were revised accordingly.

The revised manuscript now mentions that the transgenic lines used in this study were stable transformants from T₂ generation. Please see line 140 and 142.

• There is no information how the construct is made? Make one map of construct and how many independent lines you have got? Show proof with genomic DNA PCR and how much the expression level increase. Show qRT-PCR.

We understand Reviewer 2’s concern, regarding the need for information concerning constructs and subsequent evaluation of the T₀ expression. As cited in the materials and methods (line 139-144), a detailed experimental procedure on the identification of two HvFC in barley, cDNA cloning and plasmid construction, barley transformation, and analysis of overexpression lines have been incorporated in a separate manuscript, which is concurrently under peer-review in the Agronomy journal [35]. The manuscript also contains a schematic illustration of the vector used for barley transformation and demonstrates evidence for enhanced transcript in addition to protein levels of HvFCs in selected T₂ overexpression lines relative to wild-type and null controls.

• Figure 4, Figure 7c and Figure 8c in qRT-PCT data where is wild type expression.

In the 2^-ΔΔCT method, which was used to calculate the relative expression of target genes, wild-type control was used as the calibrator. Therefore, fold change in target gene expression was normalized to the wild-type control as well as to endogenous reference genes as stated in the Methodology. Therefore, wild-type expression is not represented in figures. Lines 254-256 were revised to provide additional clarity.

• Table one adds how much is PCR product length is?

Table S1 was corrected with the appropriate amplicon length.

• Figure 2 is not consistent Lane 1 is zoomed whereas lane 2 and 3 is different. Imaging should be uniform. Also, there is no fresh weight or green weight measured after the drought stress.

We agree with the Reviewer 2’s point that imaging should be as consistent as practically possible. However, for the purpose of scientific interpretation, key phenotypic differences between lines in response to dehydration are evident in images in lanes 2 and 3 (wilting of controls relative to transgenics under drought stress then recovery). Figure 3 was unaltered. Unfortunately, biomass differences were not recorded post drought stress.

• Analyze some drought stress marker gene analysis and make some mechanical pathway to indicate your genes.

As shown in Figure 5 & 8, we attempted to demonstrate the transcriptional network likely involved drought stress responsiveness by assessing Catalase and superoxide dismutase. We believe a more comprehensive transcript profiling to be more appropriate as a follow-up study in itself. Towards placing our genes in a network context, we present a schematic representation of the tetrapyrrole biosynthesis pathway. Please refer to Figure 1.

Round 2

Reviewer 2 Report

I can not see the Figure in newly submitted MS Except figure 5. Also the lines changes author mentioned were not exactly there. Please see carefully the line number before mentioning in the reply.

Also I do not believed that with in 2 generation the line would be stable. It would require atleast 3-4 generation. Add vector name and construct in figure If you are giving information in public domain you should mention everything. You can mention again in next publication.

If your all OX lines did not wilt where is proof you should produce data having atleast 2 line of each genes. Also How did you know the lines are stable after 2nd generation? (Southern data ?)

Author Response

Reviewer 2 comments

 

1. Missing Figures in the MS and incorrect line numbers

We are not sure why the reviewer has found differences in line number and also the loss of a figure. In the MS word document submitted, all figures are visible and line numbers were accurate. We assume that the reviewer was using the PDF fine and this may have been reformatted when converting to PDF. We have made sure that all the figures are clearly visible in the files submitted and line numbers are accurate and we are only submitting the Word version with track changes to help avoid confusion.

 

2. Concern about the stability of overexpression lines selected for the study

Lines 139-149 were revised to provide detailed information on identification of overexpression lines. As mentioned in the revised manuscript, all 29 T₀ independent transformation events generated were evaluated for transgene integrity, copy number and expression. Single-copy transformation events were selected and T₁ and T₂ generations were further analyzed to confirm the presence of transgene expression. Our experience, and from data published in the literature, single copy transgenic lines tend to be quite stable.  Most instability occurs when multiple and partial copies of the transgene are inserted.

Although we have provided additional information on the stability of the transgenic lines, we do not fully understand the reviewer’s concerns.  We have used transgenic lines to examine the impact of ectopic expression as a tool to explore the role of the ferrochelatases in drought responses.  For this purpose, demonstration of ectopic expression is important.  Indeed, transient gene expression is a well-established and frequently used technique for this type of study.  Transgene stability is important if large quantities of homogeneous seed are required for field trials or commercial release, but this is not the case for the experiments described in this paper.

 

3. Add vector name and construct map

The revised manuscript now contains the vector names (Line 140) and the construct map has been included as a supplementary figure.