Blue Light Upregulates Auxin Signaling and Stimulates Root Formation in Irregular Rooting of Rosemary Cuttings

Round 1

Reviewer 1 Report

The ms. By Gil and coauthors studies the contribution of light conditions to adventitious root (AR) formation in Rosmarinus officinalis. I strongly advise the authors to consult an expert English editor to check the writing as I sometimes had difficulties in understanding some of the sentences (e.g., line 14, lines 71-72, ). Please, do also check the typos (e.g., secondary compound accumulation in line 54, ). The Introduction section is well structured and contains useful information although some of the cited references are not correct (5 in line 50, 18 in line 60, ). As regards the experimental design used by the authors, there is some information missing such as the number of stock plants used in the experiment, their age and the collection time of the cuttings, as it is well-known in the AR field that stock plant age and collection season strongly affects rooting in many plant species. Also, because of the high variation found in rooting capacity, 24 cuttings seem to me a low number of samples. Might it be possible to add two additional replicates with 8 cuttings each to confirm their current results? Another major concern is about qRT-PCR analysis, as there is no information provided by the authors on the functional annotation of the studied genes, as well as primer sequences or primer validation. This information is required to assess whether the studied genes are indeed plausible orthologs of TAA1, SUR1, YUC1, etc.

The authors have used shoot growth increase after 2, 4 and 6 weeks on different conditions (3 types of cuttings, 4 light conditions) as a read-out of rooting capacity but this parameter is directly influenced by the studied conditions independently of the rooting capacity. Which is the correlation between shoot and root growth increase on the different conditions? Based on the results found in Fig.1, it is quite clear to me that root growth increase is positively influenced by blue light mainly in basal cuttings, while I don’t see a significant effect of any light treatment in shoot growth increase irrespectively of the type of cutting. As the authors have all the data, they need to perform statistical test to assign the effect of the studied factors (type of cuttings, light conditions, cultivation period) in the studied variables (shoot fresh weigh increase, root fresh weight increase). Also, it is quite difficult to observe in Fig.2 the details described in the Results and Discussion section as regards leaf morphology.

The authors have included some genes related to auxin homeostasis in their study and found differential expression as regards light treatment (FL and BL) and cutting type (apical and basal), however, statistical tests are missing. The auxin biosynthesis pathway has not been previously described in R. officinalis and the auxin precursors/main pathways are not know in this species. Hence the relevance of these results is quite limited.

As regards the NAA treatment, what is the explanation for the observed root fresh weight increase on BL?   

Minor changes:

New results from the group of Prof. Bellini on the effect of light conditions on rooting of woody explants needs to be included (line 208)

“auxin biosynthesis” instead of “auxin signaling” in lines 233 and 236

Author Response

Dear Reviewer #1,

Thank you very much for taking the time to read the manuscript carefully and provide valuable comments along with constructive suggestions. We have prepared detailed answers to each of the comments attached below. Also, the manuscript was revised in a native English editor.

Thanks, again.

Seok Hyun Eom

Author Response File: Author Response.docx

Reviewer 2 Report

The paper entitled “Blue light up regulates auxin signaling and stimulates root formation in irregular rooting of rosemary cuttings” by Chen Same Gil et al. described the effect of blue light on rooting of rosemary cutting. Vegetative propagation by stem cutting is an important method of herb production in agriculture. The results of this paper would provide a useful information and would attract wide range of readers. The experiments and analyses are carefully performed and the conclusions are reasonable.As a conclusion, the paper is sufficient to merit publication in Agronomy. though a minor revision is recommended which needs to include the following points.

(1) I think that it is better to show Figure 2 first, then Figure 1 next.

(2) It might be helpful for readers to show the illustrations or photos of stem cutting. It is a little bit hard to see what is apical, middle and basal. 

(3) Need more explanation about Figure 3B. What do the numbers represent? (Fold change ? Against what?)

Author Response

Dear Reviewer #2,

Thank you very much for taking the time to read the manuscript carefully and provide valuable comments along with constructive suggestions. We have prepared detailed answers to each of the comments below.

Reviewer #2:

The paper entitled “Blue light up regulates auxin signaling and stimulates root formation in irregular rooting of rosemary cuttings” by Chan Saem Gil et al. described the effect of blue light on rooting of rosemary cutting. Vegetative propagation by stem cutting is an important method of herb production in agriculture. The results of this paper would provide a useful information and would attract wide range of readers. The experiments and analyses are carefully performed and the conclusions are reasonable. As a conclusion, the paper is sufficient to merit publication in Agronomy. though a minor revision is recommended which needs to include the following points.

1. I think that it is better to show Figure 2 first, then Figure 1 next.

Response: As you recommended, the order of Figure 1 and 2 is changed.

2. It might be helpful for readers to show the illustrations or photos of stem cutting. It is a little bit hard to see what is apical, middle and basal. 

Response: As your comment, the methodological photos of stem cutting are added in Figure 1.

 Figure 1. (A) Morphological differences in apical, middle, and basal rosemary cuttings irradiated by different light sources—natural sunlight (NSL), fluorescent lamp light (FL), blue light (BL), and red light (RL)—at 4 weeks. (B) Morphological changes of young leaves on apical cuttings.

3. Need more explanation about Figure 3B. What do the numbers represent? (Fold change? Against what?)

Response: The numbers are relative gene expression levels, which are represented by fold change against the expression level of negative control. The negative control consisted of initial rosemary cuttings immediately after cut. As your comment, Figure 3B is detailly explained in line 00-00.

Line 224-226: The relative gene expression levels are presented as fold changes against the gene expression values of initial cuttings immediately after separation from stock plants.

Thank you very much again!

Seok Hyun Eom

Author Response File: Author Response.docx