Mechanism of Early Flowering in a Landrace Naked Barley eam8.l Mutant

Round 1

Reviewer 1 Report

I marked in yellow some minor text details that require attention. As a suggestion in the gel after line 150 the 46.40 and 86.23 daltons proteins should be marked.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

We thank you for reviewing this manuscript so carefully. Based on your suggestion, we have amended the relevant sections in the manuscript. The changes are as follows:

Points 1: Some minor text details that require attention

Response 1: Thank you for your suggestion. Three sentences that you marked in the manuscript have been modified.

Line 25: “Although EAM8 gene was expressed at higher levels in Lalu than in other barley lines with longer growth periods, it did not negatively regulate flowering, which further proved that the eam8.l protein was nonfunctional.” was revised to “Although EAM8 gene was expressed at higher level in Lalu than in other barley lines with longer growth period, it did not negatively regulate flowering time. This result further proved that the eam8.l protein was nonfunctional to regulate flowering in barley.”

Line 49: “This results in a very complicated and large regulatory net-work involving a large number of genes.” was revised to “These pathways construct a very complicated and large regulatory network involving a large number of genes.”

Line 156: “Owing to its mutation of the EAM8 gene” was revised to “Owing to the mutation of gene EAM8”

Points 2: As a suggestion in the gel after line 150 the 46.40 and 86.23 daltons proteins should be marked.

Response 2: Thanks again for this suggestion. Firstly, the molecular weight of eam8.l protein in Lalu and EAM8 protein in Dingqing1 were predicted through bioinformatics analysis. So the weight is not entirely accurate. Secondly, the gel (Figure 1A) showed the total protein in leaves of Lalu and Diqing1 and the eam8.l protein and EAM8 protein can’t be picked out in the gel.

All modifications in the manuscript have been highlighted in yellow and the revised manuscript is attached.

Best wishes,

Lei Wang

Author Response File: Author Response.docx

Reviewer 2 Report

I have only some minor remarks:

It seems better to write about plant lines, cultivars or just plants than about “materials”.

The methods section should be described in more detail. It is so scarce, that reproduction of results would be impossible. For example, plant growth conditions should be better described: there is nothing about temperature, illumination or plant growth media. PAC acronym should be explained. Is it paclobutrazol? About work with RNA there is only the name of TaKaRa company, and statistical analysis of data is just solved by “GraphPad prim”?

Is it only one alpha tubulin gene in Hordeum genome? Using only one reference gene seems to be risky, especially when one compares gene expression in different cultivars.

 

Results section should be interpreted more carefully.

In results section the Authors claim that the truncated EAM8 protein is nonfunctional, but what does it mean? What function is or could be lost? Is it only a hypothesis or the Authors have some evidence for such a statement?

I see no evidence that regulation of flowering time by GA and FT1 are independent.  Maybe a regulation model would be helpful?

Author Response

Dear review,

We greatly appreciate your comments on the manuscript. According with your comments, we have carefully revised the relevant part in the manuscript. Below is our description of the revision and answers to related questions.

Point 1: It seems better to write about plant lines, cultivars or just plants than about “materials”.

Response 1: We appreciate it very much for this good suggestion, and we have replaced “materials” with “lines” in “Materials， Methods” and “Results” part. Details are shown in the revised attached manuscript.

Point 2: The methods section should be described in more detail. It is so scarce, that reproduction of results would be impossible. For example, plant growth conditions should be better described: there is nothing about temperature, illumination or plant growth media. PAC acronym should be explained. Is it paclobutrazol? About work with RNA there is only the name of TaKaRa company, and statistical analysis of data is just solved by “GraphPad prim”?

Response 2: Thanks a lot for your good advice, and we add more detail information in methods section.

• We added the description of plant growth conditions like: “All plants were grown in gallon pots (3L) filled with general nutritional soil in standard growth rooms at 25°C with air humidity 53% and all plants were under the same SD photoperiod (8 h light/16 h dark).”
• We added the explaination of PAC acronym:” PAC (paclobutrazol, used to inhibit GA synthesis)”
• The kits used for RNA research in the work are all from TaKaRa company. Now we supplemented the information of kits names in the “Methods”. “Total RNA extraction kits (TaKaRa MiniBEST Plant RNA Extraction Kit), reverse-transcription kits (PrimeScript™ RT reagent Kit with gDNA Eraser), and kits for real-time PCR (RT-qPCR) (TB Green™ Premix Ex Taq™ II) were from TaKaRa Bio Inc.”
• About the statistical analysis, we used the “GraphPad prim” software to perform the data analysis, and the figures were drawn using R software (V 4.0.3). “GraphPad prim” software is a professional statistical analysis software and it was used in many studies (Motulsky, 2017; Zhang et al., 2019; Li et al., 2020).

Point 3: Is it only one alpha tubulin gene in Hordeum genome? Using only one reference gene seems to be risky, especially when one compares gene expression in different cultivars.

Response 3: In barley genome, there is not only one α-tubulin gene. In Schroder’s study (2001), five α-tubulin genes representing at least most members of the gene family were found to be expressed in the barley leaves. In our experiment, we took α–tubulin gene which was not affected by various stress conditions, as an internal reference gene according many previous studies in barley (Suprunova et al., 2004; Ramadan, 2020), and in these studies only the α–tubulin gene was used as a reference gene. When we started our experiment, we tested the expression stability of the α–tubulin gene. We found that α–tubulin gene expression was stable and suitable as a reference gene. Therefore, the results of our work are reliable.

Point 4: Results section should be interpreted more carefully. In results section the Authors claim that the truncated EAM8 protein is nonfunctional, but what does it mean? What function is or could be lost? Is it only a hypothesis or the Authors have some evidence for such a statement?

Response 4: Thanks for this question. EAM8 gene in barley is homologous to the EARLY FLOWERING 3 (ELF3) gene in Arabidopsis. It is responsible for the generation of circadian rhythms and the regulation of photoperiodic flowering. Loss-of-function mutations at gene EARLY MATURITY 8 (EAM8) display arrhythmic expression of circadian clock gene and promote rapid flowering. The information of EAM8 was mentioned in the “Introduction” section.

Our previous study has demonstrated that the G/A mutation at 3257 bp in the intron of eam8.l in Lalu results in a translation frame shift and leads to truncated proteins (Xia et al., 2017). In this study, the results of western blotting also indicated that the protein encoded by eam8.l in Lalu was indeed a truncated incomplete sequence. We speculate that this deficiency may cause the protein to lose function. Now we think deeply about it and found that our resultsonly provide some evidence for this hypothesis. Therefore, our previous description is not strict and precise. We have revised the description of “nonfunctional”. Details are shown in the revised attached manuscript.

Point 5: I see no evidence that regulation of flowering time by GA and FT1 are independent.  Maybe a regulation model would be helpful.

Response 5: Thanks so much for your advice.   

The FT1 transcript levels in leaves under PAC-treated and control Lalu plants at the four-leaf stage were compared. Transcripts of FT1 were not detected in Diqing1 or 86587 leaves regardless of PAC-treatment and control conditions. FT1 gene expression was not affected significantly by PAC-treatment in Lalu (Figure 5). This demonstrated that a reduction in GA biosynthesis in Lalu plants can delay flowering, but this reduction does not affect FT1 expression. A figure showing the regulation model is added as “Figure S5”.

All modifications in the manuscript have been highlighted in yellow and the revised manuscript is attached.

Reference

Li R, Hou Y, Huang J, et al. Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2)[J]. Pharmacological research, 2020, 156: 104761.

Motulsky H J. GraphPad Prism 7.0 statistic guide. GraphPad Software[J]. 2017.

Ramadan A M. Salinity effects on nad3 gene RNA editing of wild barley mitochondria[J]. Molecular Biology Reports, 2020, 47(5): 3857-3865.

Schroder J . α-Tubulin genes are differentially expressed during leaf cell development in barley (Hordeum vulgare L.)[J]. Plant Molecular Biology, 2001, 45(6):723-730.

Suprunova T, Krugman T, Fahima T, et al. Differential expression of dehydrin genes in wild barley, Hordeum spontaneum, associated with resistance to water deficit[J]. Plant, cell & environment, 2004, 27(10): 1297-1308.

Xia T, Zhang L, Xu J, et al. The alternative splicing of EAM8 contributes to early flowering and short-season adaptation in a landrace barley from the Qinghai-Tibetan Plateau[J]. Theoretical and applied genetics, 2017, 130(4): 757-766.

Zhang Q, Liu R X, Chan K W, et al. Exosomal transfer of p-STAT3 promotes acquired 5-FU resistance in colorectal cancer cells[J]. Journal of Experimental & Clinical Cancer Research, 2019, 38(1): 1-14.

Best wishes,

Lei Wang

Author Response File: Author Response.docx

Reviewer 3 Report

The article "Mechanism of Early Flowering in a Landrace Naked Barley eam8.l Mutant" is interesting for the reader. The paper is well-written, and suitable methods are used. Each of the sections is described in detail. The essential parts, results, and discussion are presented in-depth and adequately to the results obtained. This study could provide help for future barley breeding programs.

I have just the following notes:

Line 46 - Arabidopsis thaliana must be in italic.
Line 108 - reference must be with a number.
Line 132 - read the MIQE manual (https://doi.org/10.1373/clinchem.2008.112797) for a precise presentation of terms in an RT-qPCR.

 

Author Response

Dear reviewer,

The co-authors and I would like to thank you for the time and effort spent in reviewing the manuscript. According to your comments, we have revised the manuscript.

Point 1：Line 46 - Arabidopsis thaliana must be in italic.

Response 1: We are very sorry for this mistake and have rectified it. 

Point 2: Line 108 - reference must be with a number.

Response 2: Thanks again for the correction. We have changed the reference here (Boden et al.) to numbers [10].

Point 3：Line 132 - read the MIQE manual (https://doi.org/10.1373/clinchem.2008.112797) for a precise presentation of terms in an RT-qPCR.

Response 3: Thank you very much for pointing out our inaccurate expression. We read the manual carefully and revised the precise presentation for RT- qPCR at Line132 and Line 326.

All modification in the manuscript have been highlighted in yellow and the revised manuscript is attached.

Best wishes,

Lei Wang

Author Response File: Author Response.docx