Altering Tetrapyrrole Biosynthesis by Overexpressing Ferrochelatases (Fc1 and Fc2) Improves Photosynthetic Efficiency in Transgenic Barley
, Peter Langridge 1,3,* and Ryan Whitford 1Round 1
Reviewer 1 Report
Tetrapyrroles in particular heme plays central roles in multiple metabolic processes in both plants and animals. However, the influence of modified FC expression on photosynthesis in cearal crop plants has not been addressed before. In this context, this study provides the first try to answer this question.
Intriguingly, the anthors indeed provided evidence that suggest that overexpression of each FC isoform in barley is able to improve photosynthetic efficiency. I am wondering whether this effect will alter macrophenotype of the transgenic barly plants, such as biomass, plant height, etc.
Have authors tried to meansure heme contents in these transgenic lines? Whether the improved photosynthetic efficiency is associated with altered heme levels?
Could authors clearify the qPCR protocol and what's the reference genes used e.g. in Fig. 3？
It is difficult for me to decide whether HvFC1 is localised in mitochordia or not only based on the image in figure 4. It is better to proivde more control images, such as the chlorophyll fluorescence image which can further confirm the localisation of HvFC1 in plastids. It is recommended to transitly express a mitochordia-localised protein, which is fused with GFP, in an onion epidermal cell.
Could authors explain why FC antibody can recognise both HvFC isoforms in the figure 5c?
Minor points:
Line 60, check the citation [15-17.4?]
Check the author name of Reference #29
Line 182, authors mentioned the usage of antibody for cytochrome b6, but I did not see any western blot for cytochrome b6.
In figure 1, use Ferrochelatase or Fe-chelatase
In figure 5, could authors provide the summary senetence for this figure?
Another paper dealing with the differential function of two FC isoforms may be cited. (Doi: 10.1111/pce.13448)
Author Response
Reviewer 1 comments
1. Are there any differences in yield components between FC overexpressing transgenic lines and control?
As mentioned in Table S1, we measured height, number of leaves and tillers, plus shoot and root dry weight of three different independent transgenic lines per construct observing no statistically significant difference between them. However, we do recognize that these are indicators for biomass as opposed to yield parameters per se. Since biomass assessment was destructive, and the plants were grown under controlled growth conditions, measurements of yield components have been found to have little field-based yield relevance. Therefore, we did not collect this data. Follow-up studies were planned for assessing yield under field conditions.
2. Have authors tried to measure heme contents in these transgenic lines? Whether the improved photosynthetic efficiency is associated with altered heme levels?
As mentioned in lines 502-509, we attempted to identify whether FC overexpressing transgenic lines produce more heme relative to controls by measuring both total and free heme pools. However, heme content couldn’t be accurately quantified using the acid acetone extraction method due to its rapid conversion into ProtoIX by demetalation. Future investigation will focus on establishing a more accurate heme quantification method.
3. Could authors clarify the qPCR protocol and what's the reference genes used e.g. in Fig. 3?
In our study, four internal control genes (GAPDH, HSP70, cyclophilin and tubulin) were used and a rigorous normalization strategy was employed to accurately quantify the transcript abundance of target genes, as described in Burton et al., 2004 [40]. The normalization factor derived from the geometric mean of most stably expressed control genes was calculated using the geNorm program and the normalized transcript abundance of target genes was calculated by dividing the raw expression data by the NF value. Lines 169-172 were amended appropriately.
4. Provide a control image such as the chlorophyll fluorescence image to confirm the localization of HvFC1 in plastids.
The revised manuscript now contains the images with red florescent signals from plastid localized AtHEMA2 as an appropriate control, it colocalizes with HvFC1. Please refer to amended Figure 4. The figure caption and lines 197, 276-277 were also modified.
5. Could authors explain why FC antibody can recognise both HvFC isoforms in the figure 5c?
The polyclonal FC antibody used [48] is non-selective. Therefore, in the immunoblot assay, this anti-ferrochelatase antibody binds to both FC isoforms, whether they be native or transgenic. Consequently, so detectable bands in Figure 5C relative to controls represents the total FC expression for each independent transformation event. To provide more clarity, lines 313-314 were amended.
6. Line 60, check the citation [15-17.4?]
Typographical error was corrected.
7. Check the author name of Reference #29?
Author names were amended accordingly.
8. Line 182, authors mentioned the usage of antibody for cytochrome b6, but I did not see any western blot for cytochrome b6.
We attempted to identify whether HvFC overexpressing transgenics contain a higher level of Cytochrome b6 than controls by using an immunoblot assay with cytochrome b6 antibodies. However, as mentioned in 487-491, we do not believe we were able to successfully detect this protein. We speculate that the antibody used may have cross-reacted with the large subunit of Rubisco. Our future studies will focus on addressing this issue.
9. In figure 1, use Ferrochelatase or Fe-chelatase
This has been amended. Please see line 74.
10. In figure 5, could authors provide the summary sentence for this figure?
Summary statement was added to the Figure 5. Please see line 339.
11. Another paper dealing with the differential function of two FC isoforms may be cited. (Doi: 10.1111/pce.13448)
The relevant reference has been included. Please refer to line 102, 465-466.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript " Altering tetrapyrrole biosynthesis by overexpressing Ferrochelatases (FC1 and FC2), improves photosynthetic efficiency in transgenic barley" by Nagahatenna et al. describes the characterization of two Ferrochelatase (FC) isomers and their role in photosynthesis. Overall, the manuscript is well written.
- lines 433-434 The authors say " However, it is possible that HvFC1 is targeted to mitochondria in non-photosynthetic tissues, such as the root where it is also expressed" Do authors have any evidence or reference related to it or it is just speculation?
- Fig. 5 b - Null line shows almost the same expression of FC as present in WT. The FC expression should be very low in the null mutants. Any explanation?
- In fig.4, the authors need to show "only GFP" and brightfield images.
Author Response
Reviewer 2 comments
- Lines 433-434 the authors say "However, it is possible that HvFC1 is targeted to mitochondria in non-photosynthetic tissues, such as the root where it is also expressed" Do authors have any evidence or reference related to it or it is just speculation?
In our study, we could not detect HvFC1 in mitochondria and unfortunately, we did not examine non-photosynthetic tissues. However, previous studies have demonstrated that AtFC1 is targeted to both chloroplast and mitochondria. As HvFC1 is highly expressed in barley roots, we therefore speculate that this isoform may localize in root mitochondria. The sentence 466 has been amended to clarify that this is a speculative statement.
- Fig. 5 b - Null line shows almost the same expression of FC as present in WT. The FC expression should be very low in the null mutants. Any explanation?
These are not loss-of-function mutations for FC described here, but null refers to segregants shown not to have the transgene (or null for the transgene). This is typical methodology in the field that accounts for possible effects of somaclonal variation from tissue culture when analyzing tissue culture derived transgenics lines. Therefore, the identification of a similar expression to WT, indicates that the observed transgenic differences are not a consequence of the tissue culture process. Figure 5 caption has been amended to further clarify the definition of null in this context.
- In fig.4, the authors need to show "only GFP" and brightfield images.
The revised manuscript now contains additional images such as bright field image, red florescent signals from plastid localized AtHEMA2 (control), GFP fluorescent signals from HvFC1-GFP and merged image to represent the colocalization of HvFC1 with AtHEMA2. Please refer to amended Figure 4. The figure caption and lines 276-277 were also accordingly modified.
