Effect of Ultraviolet B Radiation on the Biosynthesis of Carotenoids in Foxtail Millet Grains

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research is important for the study of the regulation of the carotenoid biosynthesis pathway, which are important pigment in many areas of knowledge, including human health.

I suggest analyzing and integrating the results, work on interpretation, and selecting and editing photographs that represent all the results.

I am sending the file with annotations included in the draft

Comments for author File: Comments.pdf

Author Response

Thank you very much for taking the time to review this manuscript.

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1.          In lines 117-121, why did this study choose “HJZ” and “QH” as the materials, how about the UVB tolerance of these two varieties? Was the plant height of these two varieties was same?

2.          In lines 131-132 “the plants in the field were treated with KS-60 UV-shielding film to reduce UV intensity (rUV-B) from heading to the maturity stage”.  Was The shielding film put on the plants or put on the panicle?

3.          In line 243, “All these results were in accordance with the visual observation.” Can you provide some pictures of those two varieties?

4.          In lines 276-278, “53 DEGs were common between HJZ and QH millets and included 15, 2, and 36 up-regulated, down-regulated, and up-and down-regulated genes, respectively.” The “common gene between HJZ and QH millets” what’s mean? And the part about genes “36 up- and down-regulated” what does it mean?

5.          In lines 342-343 and figure 3, is it made by gene fold change between HJZ/QH_CK and HJZ/QH_rUV-B?  The x-ray “HJZ_CK, HJZ_rUV-B, QH_CK, and QH_ rUV-B” what’s mean?

 

6.          In line 374 and line 381, the relative expression levels were calculated using the 2^{−ΔΔ CT} method. So which sample was the calibrator sample?

Author Response

Thank you very much for taking the time to review this manuscript.

Our reply is as follows.

1. In lines 117-121, why did this study choose “HJZ” and “QH” as the materials, how about the UVB tolerance of these two varieties? Was the plant height of these two varieties was same?

HJZ is the Jingu 21 line. Also, both Jingu 21 and QH are widely planted for their high yield and high quality. These two varieties of plants grow normally under UV-B conditions, so they have a certain level of UV-B tolerance. And the plant height of these two varieties is similar.

2. In lines 131-132 “the plants in the field were treated with KS-60 UV-shielding film to reduce UV intensity (rUV-B) from heading to the maturity stage”.  Was The shielding film put on the plants or put on the panicle?

The shielding film was put on the plants. Thank you for pointing this out.

3. In line 243, “All these results were in accordance with the visual observation.” Can you provide some pictures of those two varieties?

The picture of these two varieties is as shown in the attachment.

 

4. In lines 276-278, “53 DEGs were common between HJZ and QH millets and included 15, 2, and 36 up-regulated, down-regulated, and up-and down-regulated genes, respectively.” The “common gene between HJZ and QH millets” what’s mean? And the part about genes “36 up- and down-regulated” what does it mean?

Thank you for pointing this out. Maybe my description was a bit inappropriate. Our results was that “Both HJZ and QH millets contained 53 DEGs, including 15 up-regulated, 2 down-regulated genes and 36 up- or down-regulated genes.” 

5. In lines 342-343 and figure 3, is it made by gene fold change between HJZ/QH_CK and HJZ/QH_rUV-B?  The x-ray “HJZ_CK, HJZ_rUV-B, QH_CK, and QH_ rUV-B” what’s mean?

In lines 342-343 and figure 3, the FPKM (fragments per kilobase per million fragments) value was used to normalize the expression level of each gene. “HJZ_CK” and “HJZ_rUV-B”means the HJZ variety exposed to CK and rUV-B respectively. “QH_CK” and “QH_rUV-B”means the QH variety exposed to CK and rUV-B respectively.

6. In line 374 and line 381, the relative expression levels were calculated using the 2−ΔΔ CTmethod. So which sample was the calibrator sample?

We used the housekeeping gene SiActin in foxtail millet as the reference for each gene of every sample.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This work is very interesting, I would like the importance of this work to be highlighted. I understand that it was thought to increase the carotenoid content, Could you consider the reasons for these results?  I find Figure 1 unclear to me. I suggest being explicit in the titles of the figures. The answers to the suggestions were very simple.

Author Response

Thank you very much for taking the time to review this manuscript again. Please find the detailed responses below.

1. Carotenoids, as the accessory photosynthetic pigments, can be triggered in the photoprotection machinery by UV-B. There are some differences in the tolerance of different plants to UV-B, so a lot of research is needed on the tolerance of foxtail millet to UV-B, so that it can have higher contents of carotenoids and yield when planted in areas with high altitude and strong UV-B.

2. The title of the Figure 1 has modified as that " KEGG enrichment analysis and cluster heat map of differentially expressed genes (DEGs) between HJZ and QH at S4 stage of grain development." And I hope this title can be more explicit.

Reviewer 2 Report

Comments and Suggestions for Authors In my opinion, there are some data analysis errors in the article 1. The Figure 3 was wrong. if they use FPKM(expression value), there wouldn't be a negative value.  2. Figure 4 and Figure 5, used 2^{−ΔΔ CT} method to calculate the relative expression levels. If using this method,  the expression value of every gene needs one sample's expression value was "1" in one figure.   But in those figures, don't have one sample's expression value was "1". So, Figure 4 and Figure 5 were wrong. 
This article needed to recheck results carefully. 

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below.

1. The logarithmic (log) value of FPKM with a base of 2 was used in the Figure 3, so negative values may appear.
2. We used the housekeeping gene SiActin in foxtail millet as the reference and expression value was "1" for each gene of every sample. Thus, none of the samples have an expression value of "1" in the Figure 4 and Figure 5. This representation method is similar to the paper “Yin Y, Yan Z, Guan J, Huo Y, Wang T, Li T, Cui Z, Ma W, Wang X, Chen W. Two interacting basic helix-loop-helix transcription factors control flowering time in rice. Plant Physiol. 2023 May 2;192(1):205-221. doi: 10.1093/plphys/kiad077. PMID: 36756926; PMCID: PMC10152653.”